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References

  1. Ford AC, Yuan Y, Moayyedi P. 2020. Helicobacter pylori eradication therapy to prevent gastric cancer: systematic review and metaanalysis. Gut 69: 2113-2121.
    Pubmed CrossRef
  2. Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, et al. 2021. Global Cancer Statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J. Clin. 71: 209-249.
    Pubmed CrossRef
  3. Karimi P, Islami F, Anandasabapathy S, Freedman ND, Kamangar F. 2014. Gastric cancer: descriptive epidemiology, risk factors, screening, and prevention. Cancer Epidemiol. Biomarkers Prev. 23: 700-713.
    Pubmed PMC CrossRef
  4. Song Z, Wu Y, Yang J, Yang D, Fang X. 2017. Progress in the treatment of advanced gastric cancer. Tumour Biol. 39: 1010428317714626.
    Pubmed CrossRef
  5. Schistosomes, liver flukes and Helicobacter pylori. IARC Working Group on the Evaluation of Carcinogenic Risks to Humans. Lyon, 7-14 June 1994. IARC Monogr. Eval. Carcinog. Risks Hum. 61: 1-241.
    Pubmed PMC
  6. Gonzalez CA, Megraud F, Buissonniere A, Lujan Barroso L, Agudo A, Duell EJ, et al. 2012. Helicobacter pylori infection assessed by ELISA and by immunoblot and noncardia gastric cancer risk in a prospective study: the Eurgast-EPIC project. Ann. Oncol. 23: 1320-1324.
    Pubmed CrossRef
  7. Hooi JKY, Lai WY, Ng WK, Suen MMY, Underwood FE, Tanyingoh D, et al. 2017. Global prevalence of Helicobacter pylori infection: Systematic review and meta-analysis. Gastroenterology 153: 420-429.
    Pubmed CrossRef
  8. Cho J, Prashar A, Jones NL, Moss SF. 2021. Helicobacter pylori infection. Gastroenterol. Clin. North Am. 50: 261-282.
    Pubmed PMC CrossRef
  9. Wang F, Meng W, Wang B, Qiao L. 2014. Helicobacter pylori-induced gastric inflammation and gastric cancer. Cancer Lett. 345: 196-202.
    Pubmed CrossRef
  10. Hochegger H, Takeda S, Hunt T. 2008. Cyclin-dependent kinases and cell-cycle transitions: does one fit all? Nat. Rev. Mol. Cell Biol. 9: 910-916.
    Pubmed CrossRef
  11. Enders GH. 2012. Mammalian interphase cdks: dispensable master regulators of the cell cycle. Genes Cancer 3: 614-618.
    Pubmed PMC CrossRef
  12. Gao X, Leone GW, Wang H. 2020. Cyclin D-CDK4/6 functions in cancer. Adv. Cancer Res. 148: 147-169.
    Pubmed CrossRef
  13. Tadesse S, Caldon EC, Tilley W, Wang S. 2019. Cyclin-eependent kinase 2 inhibitors in cancer therapy: An update. J. Med. Chem. 62: 4233-4251.
    Pubmed CrossRef
  14. Kastan MB, Bartek J. 2004. Cell-cycle checkpoints and cancer. Nature 432: 316-323.
    Pubmed CrossRef
  15. Ford HL, Pardee AB. 1999. Cancer and the cell cycle. J. Cell Biochem. Suppl 32-33: 166-172.
    Pubmed CrossRef
  16. Sherr CJ. 1996. Cancer cell cycles. Science 274: 1672-1677.
    Pubmed CrossRef
  17. Fong LY, Nguyen VT, Farber JL, Huebner K, Magee PN. 2000. Early deregulation of the the p16ink4a-cyclin D1/cyclin-dependent kinase 4-retinoblastoma pathway in cell proliferation-driven esophageal tumorigenesis in zinc-deficient rats. Cancer Res. 60: 4589-4595.
    Pubmed
  18. Guha R, Yue B, Dong J, Banerjee A, Serrero G. 2021. Anti-progranulin/GP88 antibody AG01 inhibits triple negative breast cancer cell proliferation and migration. Breast Cancer Res. Treat. 186: 637-653.
    Pubmed PMC CrossRef
  19. Zhao J, Li X, Liu J, Jiang W, Wen D, Xue H. 2018. Effect of progranulin on migration and invasion of human colon cancer cells. J. Coll. Physicians Surg. Pak. 28: 607-611.
    Pubmed CrossRef
  20. Walsh CE, Hitchcock PF. 2017. Progranulin regulates neurogenesis in the developing vertebrate retina. Dev. Neurobiol. 77: 1114-1129.
    Pubmed PMC CrossRef
  21. He Z, Ong CH, Halper J, Bateman A. 2003. Progranulin is a mediator of the wound response. Nat. Med. 9: 225-229.
    Pubmed CrossRef
  22. Wei J, Zhang L, Ding Y, Liu R, Guo Y, Hettinghouse A, et al. 2020. Progranulin promotes diabetic fracture healing in mice with type 1 diabetes. Ann. NY Acad. Sci. 1460: 43-56.
    Pubmed PMC CrossRef
  23. Xu B, Chen X, Ding Y, Chen C, Liu T, Zhang H. 2020. Abnormal angiogenesis of placenta in progranulindeficient mice. Mol. Med. Rep. 22: 3482-3492.
    CrossRef
  24. Lu J, Huang J, Shan M, Hu X, Guo W, Xie W, et al. 2021. Progranulin ameliorates lung inflammation in an LPS-induced acute lung injury mouse model by modulating macrophage polarization and the MAPK pathway. Ann. Clin. Lab. Sci. 51: 220-230.
    Pubmed
  25. Li H, Zhang Z, Feng D, Xu L, Li F, Liu J, et al. 2020. PGRN exerts inflammatory effects via SIRT1-NF-kappaB in adipose insulin resistance. J. Mol. Endocrinol. 64: 181-193.
    Pubmed CrossRef
  26. Feng JQ, Guo FJ, Jiang BC, Zhang Y, Frenkel S, Wang DW, et al. 2010. Granulin epithelin precursor: a bone morphogenic protein 2-inducible growth factor that activates Erk1/2 signaling and JunB transcription factor in chondrogenesis. FASEB J. 24: 1879-1892.
    Pubmed PMC CrossRef
  27. Abdulrahman A, Eckstein M, Jung R, Guzman J, Weigelt K, Serrero G, et al. 2019. Expression of GP88 (Progranulin) protein is an independent prognostic factor in prostate cancer patients. Cancers (Basel). 11: 2029.
    Pubmed PMC CrossRef
  28. Feng T, Zheng L, Liu F, Xu X, Mao S, Wang X, et al. 2016. Growth factor progranulin promotes tumorigenesis of cervical cancer via PI3K/Akt/mTOR signaling pathway. Oncotarget 7: 58381-58395.
    Pubmed PMC CrossRef
  29. Kimura A, Takemura M, Serrero G, Yoshikura N, Hayashi Y, Saito K, et al. 2018. Higher levels of progranulin in cerebrospinal fluid of patients with lymphoma and carcinoma with CNS metastasis. J. Neurooncol. 137: 455-462.
    Pubmed CrossRef
  30. Yang D, Wang LL, Dong TT, Shen YH, Guo XS, Liu CY, et al. 2015. Progranulin promotes colorectal cancer proliferation and angiogenesis through TNFR2/Akt and ERK signaling pathways. Am. J. Cancer Res. 5: 3085-3097.
    Pubmed PMC
  31. Buraschi S, Neill T, Xu SQ, Palladino C, Belfiore A, Iozzo RV, et al. 2020. Progranulin/EphA2 axis: A novel oncogenic mechanism in bladder cancer. Matrix Biol. 93: 10-24.
    Pubmed PMC CrossRef
  32. Yabe K, Yamamoto Y, Takemura M, Hara T, Tsurumi H, Serrero G, et al. 2021. Progranulin depletion inhibits proliferation via the transforming growth factor beta/SMAD family member 2 signaling axis in Kasumi-1 cells. Heliyon 7: e05849.
    Pubmed PMC CrossRef
  33. Fang W, Zhou T, Shi H, Yao M, Zhang D, Qian H, et al. 2021. Progranulin induces immune escape in breast cancer via up-regulating PD-L1 expression on tumor-associated macrophages (TAMs) and promoting CD8(+) T cell exclusion. J. Exp. Clin. Cancer Res. 40: 4.
    Pubmed PMC CrossRef
  34. Zhou C, Huang Y, Wu J, Wei Y, Chen X, Lin Z, et al. 2021. A narrative review of multiple mechanisms of progranulin in cancer: a potential target for anti-cancer therapy. Transl. Cancer Res. 10: 4207-4216.
    Pubmed PMC CrossRef
  35. Liu B, Li X, Sun F, Tong X, Bai Y, Jin K, et al. 2019. HP-CagA+ regulates the expression of CDK4/CyclinD1 via reg3 to change cell cycle and promote cell proliferation. Int. J. Mol. Sci. 21: 224.
    Pubmed PMC CrossRef
  36. Wang H, Sun Y, Liu S, Yu H, Li W, Zeng J, et al. 2011. Upregulation of progranulin by Helicobacter pylori in human gastric epithelial cells via p38MAPK and MEK1/2 signaling pathway: role in epithelial cell proliferation and migration. FEMS Immunol. Med. Microbiol. 63: 82-92.
    Pubmed CrossRef
  37. Livak KJ, Schmittgen TD. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 25: 402-408.
    Pubmed CrossRef
  38. Tang W, Lu Y, Tian QY, Zhang Y, Guo FJ, Liu GY, et al. 2011. The growth factor progranulin binds to TNF receptors and is therapeutic against inflammatory arthritis in mice. Science 332: 478-484.
    Pubmed PMC CrossRef
  39. Arechavaleta-Velasco F, Perez-Juarez CE, Gerton GL, Diaz-Cueto L. 2017. Progranulin and its biological effects in cancer. Med. Oncol. 34: 194.
    Pubmed PMC CrossRef
  40. Jian J, Li G, Hettinghouse A, Liu C. 2018. Progranulin: A key player in autoimmune diseases. Cytokine 101: 48-55.
    Pubmed PMC CrossRef
  41. Demorrow S. 2013. Progranulin: a novel regulator of gastrointestinal cancer progression. Transl. Gastrointest. Cancer 2: 145-151.
    Pubmed PMC CrossRef
  42. Dong T, Yang D, Li R, Zhang L, Zhao H, Shen Y, et al. 2016. PGRN promotes migration and invasion of epithelial ovarian cancer cells through an epithelial mesenchymal transition program and the activation of cancer associated fibroblasts. Exp. Mol. Pathol. 100: 17-25.
    Pubmed CrossRef
  43. Klupp F, Kahlert C, Franz C, Halama N, Schleussner N, Wirsik NM, et al. 2021. Granulin: an invasive and survival-determining marker in colorectal cancer patients. Int. J. Mol. Sci. 22: 6436.
    Pubmed PMC CrossRef
  44. Wang W, Hayashi J, Kim WE, Serrero G. 2003. PC cell-derived growth factor (granulin precursor) expression and action in human multiple myeloma. Clin. Cancer Res. 9: 2221-2228.
    Pubmed
  45. Lu R, Serrero G. 2000. Inhibition of PC cell-derived growth factor (PCDGF, epithelin/granulin precursor) expression by antisense PCDGF cDNA transfection inhibits tumorigenicity of the human breast carcinoma cell line MDA-MB-468. Proc. Natl. Acad. Sci. USA 97: 3993-3998.
    Pubmed PMC CrossRef
  46. Serrero G, Hawkins DM, Yue B, Ioffe O, Bejarano P, Phillips JT, et al. 2012. Progranulin (GP88) tumor tissue expression is associated with increased risk of recurrence in breast cancer patients diagnosed with estrogen receptor positive invasive ductal carcinoma. Breast Cancer Res. 14: R26.
    Pubmed PMC CrossRef
  47. Ho JC, Ip YC, Cheung ST, Lee YT, Chan KF, Wong SY, et al. 2008. Granulin-epithelin precursor as a therapeutic target for hepatocellular carcinoma. Hepatology 47: 1524-1532.
    Pubmed CrossRef
  48. Wu JY, Lee YC, Graham DY. 2019. The eradication of Helicobacter pylori to prevent gastric cancer: a critical appraisal. Expert Rev. Gastroenterol. Hepatol. 13: 17-24.
    Pubmed PMC CrossRef
  49. Zhou Z, Ye G, Peng J, He B, Xu S, Fan W, et al. 2021. Expression of Wnt3, beta-catenin and MMP-7 in gastric cancer and precancerous lesions and their correlations with Helicobacter pylori infection. Zhong Nan Da Xue Xue Bao Yi Xue Ban 46: 575-582.
  50. Molaei F, Forghanifard MM, Fahim Y, Abbaszadegan MR. 2018. Molecular signaling in tumorigenesis of gastric cancer. Iran Biomed. J. 22: 217-230.
    Pubmed PMC CrossRef
  51. Padda J, Khalid K, Cooper AC, Jean-Charles G. 2021. Association between Helicobacter pylori and gastric carcinoma. Cureus 13: e15165.
    Pubmed PMC CrossRef
  52. Kato MV, Sato H, Anzai H, Nagayoshi M, Ikawa Y. 1997. Up-regulation of cell cycle-associated genes by p53 in apoptosis of an erythroleukemic cell line. Leukemia 11 Suppl 3: 389-392.
    Pubmed
  53. Woodward TA, Klingler PD, Genko PV, Wolfe JT. 2000. Barrett's esophagus, apoptosis and cell cycle regulation: correlation of p53 with Bax, Bcl-2 and p21 protein expression. Anticancer Res. 20: 2427-2432.
    Pubmed
  54. Chinzei N, Hayashi S, Ueha T, Fujishiro T, Kanzaki N, Hashimoto S, et al. 2015. P21 deficiency delays regeneration of skeletal muscular tissue. PLoS One 10: e0125765.
    Pubmed PMC CrossRef
  55. Megraud F, Bessede E, Varon C. 2015. Helicobacter pylori infection and gastric carcinoma. Clin. Microbiol. Infect. 21: 984-990.
    Pubmed CrossRef
  56. Evan GI, Vousden KH. 2001. Proliferation, cell cycle and apoptosis in cancer. Nature 411: 342-348.
    Pubmed CrossRef
  57. Kar S. 2016. Unraveling cell-cycle dynamics in cancer. Cell Syst. 2: 8-10.
    Pubmed CrossRef
  58. Pack LR, Daigh LH, Meyer T. 2019. Putting the brakes on the cell cycle: mechanisms of cellular growth arrest. Curr. Opin. Cell Biol. 60: 106-113.
    Pubmed PMC CrossRef
  59. Campbell GJ, Hands EL, Van de Pette M. 2020. The role of CDKs and CDKIs in murine development. Int. J. Mol. Sci. 21: 5343.
    Pubmed PMC CrossRef
  60. Canepa ET, Scassa ME, Ceruti JM, Marazita MC, Carcagno AL, Sirkin PF, et al. 2007. INK4 proteins, a family of mammalian CDK inhibitors with novel biological functions. IUBMB Life 59: 419-426.
    Pubmed CrossRef
  61. Jackson RJ, Adnane J, Coppola D, Cantor A, Sebti SM, Pledger WJ. 2002. Loss of the cell cycle inhibitors p21(Cip1) and p27(Kip1) enhances tumorigenesis in knockout mouse models. Oncogene 21: 8486-8497.
    Pubmed CrossRef
  62. Ahmed A, Smoot D, Littleton G, Tackey R, Walters CS, Kashanchi F, et al. 2000. Helicobacter pylori inhibits gastric cell cycle progression. Microbes Infect. 2: 1159-1169.
    Pubmed CrossRef
  63. Li N, Xie C, Lu NH. 2016. p53, a potential predictor of Helicobacter pylori infection-associated gastric carcinogenesis? Oncotarget 7: 66276-66286.
    Pubmed PMC CrossRef
  64. Sherr CJ. 1995. D-type cyclins. Trends Biochem. Sci. 20: 187-190.
    Pubmed CrossRef
  65. Shirin H, Sordillo EM, Oh SH, Yamamoto H, Delohery T, Weinstein IB, et al. 1999. Helicobacter pylori inhibits the G1 to S transition in AGS gastric epithelial cells. Cancer Res. 59: 2277-2281.
    Pubmed
  66. Scotti C, Sommi P, Pasquetto MV, Cappelletti D, Stivala S, Mignosi P, et al. 2010. Cell-cycle inhibition by Helicobacter pylori Lasparaginase. PLoS One 5: e13892.
    Pubmed PMC CrossRef
  67. Cover TL, Krishna US, Israel DA, Peek RM Jr. 2003. Induction of gastric epithelial cell apoptosis by Helicobacter pylori vacuolating cytotoxin. Cancer Res. 63: 951-957.
    Pubmed
  68. Kim KM, Lee SG, Kim JM, Kim DS, Song JY, Kang HL, et al. 2010. Helicobacter pylori gamma-glutamyltranspeptidase induces cell cycle arrest at the G1-S phase transition. J. Microbiol. 48: 372-377.
    Pubmed CrossRef
  69. Li H, Liang D, Hu N, Dai X, He J, Zhuang H, et al. 2019. Helicobacter pylori inhibited cell proliferation in human periodontal ligament fibroblasts through the Cdc25C/CDK1/cyclinB1 signaling cascade. J. Periodontal. Implant Sci. 49: 138-147.
    Pubmed PMC CrossRef
  70. Alquezar C, Esteras N, Bartolome F, Merino JJ, Alzualde A, Lopez de Munain A, et al. 2012. Alteration in cell cycle-related proteins in lymphoblasts from carriers of the c.709-1G>A PGRN mutation associated with FTLD-TDP dementia. Neurobiol. Aging 33: 429.e427-420.
    Pubmed CrossRef
  71. Alquezar C, Esteras N, de la Encarnacion A, Alzualde A, Moreno F, Lopez de Munain A, et al. 2014. PGRN haploinsufficiency increased Wnt5a signaling in peripheral cells from frontotemporal lobar degeneration-progranulin mutation carriers. Neurobiol. Aging 35: 886-898.
    Pubmed CrossRef
  72. Teo ZL, Versaci S, Dushyanthen S, Caramia F, Savas P, Mintoff CP, et al. 2017. Combined CDK4/6 and PI3Kalpha inhibition is synergistic and immunogenic in triple-negative breast cancer. Cancer Res. 77: 6340-6352.
    Pubmed CrossRef
  73. Kollmann K, Briand C, Bellutti F, Schicher N, Blunder S, Zojer M, et al. 2019. The interplay of CDK4 and CDK6 in melanoma. Oncotarget 10: 1346-1359.
    Pubmed PMC CrossRef
  74. Lu Y, Zheng L, Zhang W, Feng T, Liu J, Wang X, et al. 2014. Growth factor progranulin contributes to cervical cancer cell proliferation and transformation in vivo and in vitro. Gynecol. Oncol. 134: 364-371.
    Pubmed CrossRef
  75. He Z, Ismail A, Kriazhev L, Sadvakassova G, Bateman A. 2002. Progranulin (PC-cell-derived growth factor/acrogranin) regulates invasion and cell survival. Cancer Res. 62: 5590-5596.
    Pubmed
  76. Peek RM Jr, Wirth HP, Moss SF, Yang M, Abdalla AM, Tham KT, et al. 2000. Helicobacter pylori alters gastric epithelial cell cycle events and gastrin secretion in Mongolian gerbils. Gastroenterology 118: 48-59.
    Pubmed CrossRef

Article

Research article

J. Microbiol. Biotechnol. 2022; 32(7): 844-854

Published online July 28, 2022 https://doi.org/10.4014/jmb.2203.03053

Copyright © The Korean Society for Microbiology and Biotechnology.

Helicobacter pylori-Induced Progranulin Promotes the Progression of the Gastric Epithelial Cell Cycle by Regulating CDK4

Zongjiao Ren1†, Jiayi Li1†, Xianhong Du1,2, Wenjing Shi3, Fulai Guan4, Xiaochen Wang1, Linjing Wang5, and Hongyan Wang1,2*

1Department of Pathogenic Microbiology, Basic Medical College, Weifang Medical University, Weifang 261053, Shandong, P.R. China
2Key Lab for Immunology in Universities of Shandong Province, Basic Medical College, Weifang Medical University, Weifang 261053, Shandong, P.R. China
3Department of Gynecology, Weifang Medical University Affiliated Hospital, Weifang 261000, Shandong, P.R. China
4Laboratory of Morphology, Weifang Medical University, Weifang 261053, Shandong, P.R. China
5Clinical Medical College, Weifang Medical University, Weifang 261053, Shandong, P.R. China

Correspondence to:Hongyan Wang,     sdwfwhy@163.com

These authors contributed equally to this work.

Received: March 28, 2022; Revised: June 11, 2022; Accepted: June 16, 2022

Abstract

Helicobacter pylori, a group 1 carcinogen, colonizes the stomach and affects the development of stomach diseases. Progranulin (PGRN) is an autocrine growth factor that regulates multiple cellular processes and plays a tumorigenic role in many tissues. Nevertheless, the mechanism of action of PGRN in gastric cancer caused by H. pylori infection remains unclear. Here, we investigated the role of PGRN in cell cycle progression and the cell proliferation induced by H. pylori infection. We found that the increased PGRN was positively associated with CDK4 expression in gastric cancer tissue. PGRN was upregulated by H. pylori infection, thereby promoting cell proliferation, and that enhanced level of proliferation was reduced by PGRN inhibitor. CDK4, a target gene of PGRN, is a cyclin-dependent kinase that binds to cyclin D to promote cell cycle progression, which was upregulated by H. pylori infection. We also showed that knockdown of CDK4 reduced the higher cell cycle progression caused by upregulated PGRN. Moreover, when the PI3K/Akt signaling pathway (which is promoted by PGRN) was blocked, the upregulation of CDK4 mediated by PGRN was reduced. These results reveal the potential mechanism by which PGRN plays a major role through CDK4 in the pathological mechanism of H. pylori infection.

Keywords: Helicobacter pylori, PGRN, CDK4, gastric epithelial cells, cell cycle

Introduction

Gastric cancer is the fourth-leading cause of cancer mortality, with over 700,000 deaths each year. The incidence of gastric cancer is highest in Eastern Europe, Eastern Asia, and South America [1, 2]. Due to a low rate of early detection, most gastric cancer patients are generally diagnosed at a late stage, and the overall five-year survival rate is about 20% [3, 4]. H. pylori infection is a key pathogenic cause for gastric carcinoma and it has been recognized as a group 1 carcinogen by the WHO [5]. H. pylori infects more than half of the global population, and nearly all noncardiac gastric cancers are attributed to this bacterium [6, 7]. A variety of virulence factors produced by H. pylori can cause a chronic inflammatory response in the gastric mucosa, which then develops into gastric or duodenal ulcers, atrophic gastritis, gastric cancer, or gastric mucosa-associated lymphoid tissue lymphoma [8, 9]. Although major progress has been made in the diagnosis and treatment of H. pylori in recent years, the pathogenesis of H. pylori-induced gastric cancer is still unclear.

The cell cycle is a complicated and elaborate regulatory process influenced by multiple factors both inside and outside the cell, where cells generate two daughter cells through a series of replication, division, and growth events under regulating multiple cyclins. Among them, key cycle transitions are driven by different cyclin-dependent kinases (CDKs) and their activated cyclin subunits [10]. CDKs related to cell cycle interphase activation in mammals mainly include CDK4 and CDK6 in G1 phase and CDK2 near the beginning of S phase [11]. The CDK4/6 binds to cyclin D and drives the cell-cycle transition from G1 to S by phosphorylates retinoblastoma protein (RB)[12]. CDK2 is a core cell-cycle regulator that facilitates the transition from S to G2 in the late G1 stage by binding to cyclins E and A, and continuing to phosphorylate RB and release E2F transcription factors (E2Fs) [13]. Indeed, the cell-cycle checkpoints of cancer are often associated with DNA damage and genetic defects [14, 15]. Disorders in the cell cycle disrupt normal mitosis, often causing uncontrolled proliferation, leading to cancer development [16]. In the early G1-S checkpoint of rat esophageal cancer caused by zinc deficiency, the expression of cyclin D1, CDK4, and RB increases, the p16INK4a cycle D1/cycle-dependent kinase 4 RB pathway is dysregulated, and this is closely associated with cell proliferation [17]. Therefore, the study of cell cycle changes is an important entry point to study cell proliferation.

Progranulin (PGRN), is a growth factor consisting of 593 amino-acid residues, and is also called granulin-epithelin precursor, proepithelin, acroglanin, or GP88. PGRN plays a crucial role in miscellaneous physiological processes involving cell development, cell cycle progression, wound healing, repair and formation of blood vessels and tissues, inflammation, and the growth of bone and cartilage [18-26]. As an important regulatory factor in tumors, PGRN is strongly expressed in various tumors, including cervical cancer, prostate cancer, bladder cancer, colorectal cancer, and lymphoma, and is associated with overall survival [27-31]. Studies have shown that inhibition of PGRN can inhibit tumor growth. For example, PGRN repression inhibits the proliferation of hematopoietic cancer cells [32]. Blocking PGRN can suppress the proliferation and migration of triple-negative breast cancer cells [18]. Furthermore, studies have demonstrated that PGRN regulates the expression of tumor-associated macrophage PD-1, promotes CD8+ T cell rejection, and induces breast cancer immune escape [33]. Thus, the high expression of PGRN is closely associated with the progression of malignancies [34]. However, the mechanism by which PGRN induces cellular responses in H. pylori infected cells remains unclear.

Studies have shown that the virulence factor Cag A of H. pylori can promote cell proliferation by affecting cell cycle progression [35]. We have previously reported that PGRN is upregulated by H. pylori through the p38MAPK and MEK1/2 signaling pathways, and then promotes the migration and proliferation of gastric epithelial cells [36]. However, the role of PGRN in H. pylori-induced cell cycle progression remains unclear, and the potential mechanisms are still to be illustrated. In this research, we found not only that PGRN and CDK4 were both overexpressed in gastric cancer, but there was also a positive correlation between them. The upregulation of PGRN induced by H. pylori increased the cell cycle progression and the proliferation of gastric epithelial cells. As a target gene of PGRN, CDK4 participated in the regulation of the cell cycle. This study is the first to investigate the function and mechanism of PGRN and CDK4 in cell cycle progression and proliferation induced by H. pylori in assays performed in vitro.

Materials and Methods

Tissue Samples

One hundred gastric cancer tissue and adjacent normal tissue samples were provided by Weifang Peoplés Hospital and the Affiliated Hospital of Weifang Medical University through gastroscopy and gastric cancer surgery, respectively. The age, gender, and relevant clinical data of all subjects were collected as approved by the Ethics Committee of Weifang Medical University (2022YX045). There was no statistical difference in the tissue sources of each group in age, gender, TNM stage, and other related indicators.

Cell Culture and Reagents

BGC-823 gastric cancer cells were grown in RPMI 1640 (Gibco, USA) supplemented with 10% newborn bovine serum (Gibco) in a CO2 incubator at 37°C containing 5% CO2. The PI3K/Akt inhibitor LY294002, the nuclear factor-κB (NF-κB) inhibitor BAY11-7082, and the MAPK inhibitor UO126 were from Cell Signaling Technology (USA). The three signal pathway inhibitors were dissolved in dimethyl sulfoxide (DMSO, China) solution.

H. pylori Culture

H. pylori strain 26695 was maintained in our laboratory. Bacteria were incubated in Brucella broth with 5% fetal bovine serum at 37°C under microaerophilic conditions containing 10% CO2, 5% O2 and 85% N2. Depending on the experimental requirements, BGC-823 cells were infected at different multiplicity of infection (MOI) of H. pylori.

Lentiviral Vector Construction and Transfection

PGRN knockdown lentivirus pLKO.1-PGRN shRNA-GFP vector and PGRN overexpression lentivirus Plenti6/V5-PGRN vector, and the corresponding negative control vector were successfully constructed and preserved in the laboratory. Lentiviruses CDK4-RNAi-13, CDK4-RNAi-14, CDK4-RNAi-15, and their control vector were purchased from Shanghai Genechem Co., Ltd. (China). BGC-823 cells were seeded in 6-well plates, and the virus was infected when the cell density reached ~70%. The PGRN knockdown group (represented by SI) and the control group (empty vector, represented by NS), the PGRN-overexpressing group (represented by PGRN) and the control group (empty vector, represented by GFP) were transfected with Lipofectamine 2000 (Invitrogen, USA). All experiments were carried out in triplicate according to the manufacturer's instructions.

RNA Extraction and Quantitative Real-Time PCR (qPCR)

Based on the manufacturer's instructions, total RNA was extracted with TRIzol (Invitrogen). cDNA was synthesized from 2 μg of extracted RNA using a ReverTra Ace qPCR RT Kit (Toyobo, Japan). The SYBR Green Pro Taq HS Premix qPCR Kit (AG, China) and the ABI 7900HT System (ABI, USA) were used for qPCR of cDNA expression. β-Actin was utilized as the normalization control. The relative expression fold changes of mRNA were calculated using the 2-ΔΔCt comparative threshold cycle method [37]. The primer sequences used were as follows: PGRN: forward-5’-GGACAGTACTGAAGACTCTG-3’, reverse-5’-GGATGGCAGCTTGTAATGTG-3’; CDK4: forward-5’-GGGCCGAGAGGACAGAATGG-3’, reverse-5’-GCTGTTCTAATCACCAGGGTAGGCC-3’; β-actin: forward-5’-AGTTGCGTTACACCCTTTCTTG-3’, reverse-5’-CACCTTCACCGTTCCAGTTTT-3’.

Western Blot Analysis

Gastric cancer cell proteins were collected using RIPA lysis buffer with PMSF protease inhibitor (Solarbio, China). The total protein was separated by SDS-PAGE and then transferred to a PVDF membrane. The PVDF membrane was blocked with 5% skim milk in TBST buffer for 1 h at room temperature and then incubated with primary antibody at 4°C overnight. Following that, it was incubated with HRP-linked anti-mouse IgG (1:2000, #7076, Cell Signaling Technology, USA) or anti-rabbit IgG (1:2000, #7074, Cell Signaling Technology) at room temperature for 1 h. The bands were visualized by a chemiluminescence ECL detection system (EMD Millipore, USA). The primary antibodies used were: anti-PGRN (1:200, sc-377036, Santa Cruz, USA), anti-CDK4 (1:1000, #12790, Cell Signaling Technology), anti-β-actin (1:1000, sc-47778, Santa Cruz), anti-Akt (1:1000, 4691S, Cell Signaling Technology), and anti-p-Akt (1:2000, #4060S, Cell Signaling Technology).

Immunohistochemical Analysis

Paraffin-embedded tissue sections were dewaxed and dehydrated using xylene and ethanol, respectively. After antigen retrieval, the samples were blocked with goat serum working solution (ZSGB Biotech, China). They were then incubated at 4°C overnight after adding mouse anti-PGRN antibody (1:100, sc-377036, Santa Cruz) or rabbit anti-CDK4 antibody (1:800, #12790, Cell Signaling Technology) according to the instructions. According to the difference in the primary antibody, the secondary antibody biotin-labeled goat anti-mouse IgG (1:700, ZSGB Biotech) or goat anti-rabbit IgG (1:700, ZSGB Biotech) was applied, followed by drop-wise HRP-conjugated Streptavidin working solution (ZSGB Biotech). Then, DAB chromogenic solution (ZSGB Biotech) was used for color development followed by hematoxylin staining for 1 min and 1% hydrochloric acid alcohol for color separation for 3–4 s. Then, 0.2% ammonia and neutral gum (Biosharp, China) were used for sealing after dehydration. Images were analyzed with Image Pro Plus 6.0 (Media Cybernetics, USA) software.

Flow Cytometry

The cells were cultured to a density of 90%, collected, washed 3 times with PBS, and fixed in 1 ml of 70% low-temperature ethanol at 4°C overnight. After washing with PBS, cells were stained with 0.5 ml propidium iodide (25 μl propidium iodide, 10 μl RNaseA, Beyotime, China). Following that, the cells were re-suspended and bathed in water at 37°C for 30 min. FACSVerse flow cytometry (BD, USA) was used to assess the cell cycle.

Colony Formation Assay

The cells were or were not infected with H. pylori for 3 h, then 300 cells were counted, seeded in a 6-well plate, and cultured at 37°C and 5% CO2 for 14 days. After fixation with methanol, they were stained with Giemsa dye (Solarbio), counted, and photographed.

Statistical Analysis

All data are indicated as the mean ± SD and were statistically analyzed using GraphPad Prism 8.0 (GraphPad Software Inc., USA). Comparison between groups was tested by paired t-test. One-way ANOVA was used to determine the differences in multiple comparison. Correlations of protein expression were done using the Spearman rank correlation test. p<0.05 represents statistical significance.

Results

PGRN and CDK4 Are Overexpressed in Gastric Cancer Tissues

The expression levels of PGRN and CDK4 in adjacent normal tissue and gastric cancer were assessed by immunohistochemical staining. Compared with adjacent normal tissue, PGRN (Fig. 1A) and CDK4 (Fig. 1B) expressed significantly higher in gastric cancer tissue. By analyzing the correlation between PGRN and CDK4, we found that PGRN was positively associated with CDK4 in gastric cancer, and the correlation coefficient r was 0.452 (Table 1).

Table 1 . Correlations between PGRN and CDK4 in gastric cancer and adjacent normal tissues analyzed by linear regression..

IOD(×103)rP
PGRN38.095 ± 1.600.4520.023
CDK418.342 ± 0.84


Figure 1. Differences of PGRN and CDK4 protein expression between gastric cancer tissue and adjacent normal tissue. (A, B) Expression levels of PGRN (A) and CDK4 (B) in both gastric cancer and adjacent normal tissues as measured by immunohistochemistry. The results represent the mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, paired t-test. All data are mean values of three biological replicates.

H. pylori Infection Regulated the Proliferation and Cycle Progression of Gastric Epithelial Cells

To evaluate whether H. pylori infection influenced the proliferation of gastric epithelial cells, we co-incubated BGC-823 cells with H. pylori at an MOI of 50:1. The results showed that, compared with the non-infected group, the proliferative capacity of BGC-823 cells was substantially increased after infection with H. pylori (Fig. 2A), indicating that H. pylori infection increases cell proliferation.

Figure 2. H. pylori infection promotes cell cycle progression and cell proliferation. (A) BGC-823 cells cocultured with H. pylori at a multiplicity of infection (MOI) 50:1 for 3 h, and their clonogenic potential were then assessed. (B) Flow cytometry results of BGC-823 cells infected with H. pylori 26695 at a MOI of 50:1 for 6, 12, and 24 h. (C) Flow cytometric results of H. pylori 26695 infected BGC-823 cells at different MOIs (10:1, 20:1, 50:1, 100:1, and 200:1). (D) Flow cytometry results of BGC-823 cells pre-treated with BAY11-7082 (5 μM), LY294002 (10 μM) and UO126 (10 μM) for 2 h before incubation with or without H. pylori at a MOI of 50:1 for 12 h. The results represent the mean ± SD of three independent experiments. ns, not significant, HP, Helicobacter pylori, *p < 0.05, **p < 0.01, ***p < 0.001.

To explore the mechanisms by which H. pylori promotes cell growth, we analyzed the effect of H. pylori infection on the cell cycle. BGC-823 cells were infected with H. pylori at an MOI of 50:1 for 6, 12, and 24 h. Flow cytometry analysis showed that, compared with uninfected cells at each time point, cells entering G2/M phase increased significantly after H. pylori infection, and with the prolongation of infection time, the proportion of cells entering G2/M phase gradually increased (Fig. 2B). Next, BGC-823 cells were infected with H. pylori at varying MOIs of 10:1, 20:1, 50:1, 100:1, and 200:1 for 12 h. Flow cytometry analysis demonstrated that with the increase of MOI, the proportion of cells entering G2/M phase gradually increased (Fig. 2C). These results suggested that H. pylori infection regulates gastric epithelial cell cycles in a time- and dose-dependent manner.

H. pylori was found to activate a set of main signaling molecules that include NF-κB, PI3K/Akt, and mitogen-activated protein kinases (MAPKs). To clarify the signaling pathways regulating the H. pylori-induced cell cycle, three signal molecule inhibitors were added to BGC-823 cells 2 h prior to H. pylori infection at an MOI of 50:1. Flow cytometry results indicated that only PI3K/Akt inhibitor LY294002 (10 μM) was capable of inhibiting a higher proportion of cells entering G2/M significantly stimulated by H. pylori and decreased the cell cycle to basal level, and there was no significant difference in the cell cycle using NF-κB inhibitor BAY11-7082 (5 μM) and MAPK inhibitors UO126 (10 μM) (Fig. 2D). Therefore, H. pylori infection may regulate the cell cycle via the PI3K/Akt signaling pathway.

PGRN Promotes H. pylori-Induced Gastric Epithelial Cell Cycle Progression and Cell Proliferation

Our previous studies have demonstrated that H. pylori increases PGRN expression via the p38MAPK and MEK1/2 pathways in gastric epithelial cells. Therefore, we sought to examine the role of PGRN in the gastric epithelial cell cycle progression and the cell proliferation induced by H. pylori infection. We knocked down and overexpressed PGRN in BGC-823 cells by lentivirus pLKO.1-PGRN shRNA-GFP (represented by SI), the control group (empty vector, represented by NS), the PGRN overexpressing lentivirus Plenti6/V5-PGRN (represented by PGRN), and the control group (empty vector, represented by GFP). The qPCR verified the effectiveness of lentivirus infection (Fig. 3A). Then, we co-incubated BGC-823 cells with H. pylori at an MOI of 50:1. Colony formation assay showed that repression of PGRN markedly reduced the foci numbers as well as sizes but overexpression of PGRN led to a significant increase. H. pylori infection could obviously increase the colony formation, but downregulation of PGRN nearly decreased the proliferative ability promoted by H. pylori infection, while overexpression of PGRN significantly enhanced the proliferation induced by H. pylori (Fig. 3B). Consistent with these results, the proportion of cells progressing to G2/M after PGRN knockdown was markedly less than that in the control group, but the proportion was markedly higher in PGRN overexpression. H. pylori infection could accelerate cell cycle progression to G2/M, but knockdown by PGRN almost reduced these activities induced by H. pylori, while overexpression of PGRN enhanced these activities (Fig. 3C). These results indicated that upregulating PGRN is associated with cell cycle progression and cell proliferation induced by H. pylori infection.

Figure 3. PGRN promotes cell cycle progression and cell proliferation in gastric cancer cells with or without H. pylori infection. (A) qPCR analysis of the expression of PGRN after transfection with pLKO.1-PGRN shRNA-GFP and Plenti6/V5-PGRN lentivirus. (B, C) Colony formation assays (B) and cell cycle assays (C) of BGC-823 cells transfected with pLKO.1-PGRN shRNA-GFP and Plenti6/V5-PGRN and their negative control lentivirus and cocultured with H. pylori at a MOI of 50: 1 for 3 h. The results represent the mean ± SD of three independent experiments. SI, PGRN knockdown group, NS, the control group of PGRN knockdown, GFP, the control group of PGRN overexpressing, HP, Helicobacter pylori, *p < 0.05, **p < 0.01, ***p < 0.001.

H. pylori Increases Expression of CDK4 to Promote the Cell Cycle through the Upregulation of PGRN

We have confirmed that the expression of PGRN and CDK4 was both increased and positively correlated in gastric cancer, but it was not clear whether the upregulated PGRN regulated the cell cycle via CDK4. BGC-823 cells were cocultured with H. pylori at an MOI of 100:1. Compared with the non-infected group, CDK4 mRNA and protein expression were both apparently upregulated after H. pylori infection. Furthermore, CDK4 expression was elevated in a time-dependent manner (Figs. 4A and 4B).

Figure 4. PGRN positively regulates CDK4 to promote cell cycle progression. (A, B) qPCR and western blot analysis of the expression of CDK4 in BGC-823 cells infected with H. pylori 26695 at a MOI of 100: 1. (C, D) qPCR and western blot analysis of the expression of CDK4 after transfection with pLKO.1-PGRN shRNA-GFP and Plenti6/V5-PGRN lentivirus. (E, F) CDK4 expression after transfection with CDK4-RNAi-13, CDK4-RNAi-14, CDK4-RNAi-15 of CDK4-knockdown lentivirus. (G) Flow cytometry analysis of the cell cycle changes of CDK4 knockdown and coculture with H. pylori in 50:1 MOI in BGC-823 cells. (H) Flow cytometry analysis of the cell cycle changes of CDK4 knockdown cell lines infected with PGRN-knockdown /overexpressed lentivirus and cocultured with H. pylori at a MOI of 50: 1. The results represent the mean ± SD of three independent experiments. SI, PGRN knockdown group, NS, the control group of PGRN knockdown, GFP, the control group of PGRN overexpressing, ns, not significant, HP, Helicobacter pylori, *p < 0.05, **p < 0.01, ***p < 0.001.

To define the role of PGRN on CDK4 expression, we transfected BGC-823 cells with lentivirus pLKO.1-PGRN shRNA-GFP and lentivirus Plenti6/V5-PGRN. Western blot confirmed that the repression of PGRN markedly decreased the expression of CDK4, and overexpression of PGRN apparently promoted the expression of CDK4 (Figs. 4C and 4D). Furthermore, H. pylori infection increased CDK4 expression in BGC-823 cells; however, this increased expression was decreased after knockdown of PGRN and upregulated after overexpression of PGRN (Figs. 5C and 5D). These results suggested that H. pylori regulated CDK4 expression via PGRN.

Figure 5. PGRN regulates CDK4 through the PI3K/Akt signaling pathway and promotes progression of the gastric mucosal epithelial cell cycle. (A) qPCR analysis of the expression of PGRN after transfection with pLKO.1-PGRN shRNA-GFP and Plenti6/V5-PGRN lentivirus and cocultured with H. pylori at a MOI of 100: 1. (B) Western blot analysis of CDK4 protein expression in cells pretreated with a PI3K signal pathway inhibitor (LY294002) for 2 h before transfection with pLKO.1-PGRN shRNA-GFP and Plenti6/V5-PGRN lentivirus. (C) qPCR analysis of the expression of CDK4 after transfection with pLKO.1-PGRN shRNA-GFP and Plenti6/V5-PGRN lentivirus and cocultured with H. pylori at a MOI of 100: 1. (D) Western blot analysis of CDK4, Akt, and p-Akt protein expression in cells transfected with pLKO.1-PGRN shRNA-GFP and Plenti6/V5-PGRN lentivirus and cocultured with H. pylori at a MOI of 100: 1. The results represent the mean ± SD of three independent experiments. SI, PGRN knockdown group, NS, the control group of PGRN knockdown, GFP, the control group of PGRN overexpressing, HP, Helicobacter pylori, *p < 0.05, **p < 0.01, ***p < 0.001.

To verify the effect of CDK4 on the cell cycle, we transfected BGC-823 cells with lentiviruses CDK4-RNAi-13, CDK4-RNAi-14, and CDK4-RNAi-15. qPCR and western blot showed that CDK4-RNAi-13 successfully inhibited the expression of CDK4, and the inhibition efficiency of CDK4-RNAi-14 and CDK4-RNAi-15 was relatively lower (Figs. 4E and 4F). We transfected BGC-823 cells with CDK4-RNAi-13 and assessed their cycle distribution by flow cytometry. The results indicated that the progression to G2/M was significantly reduced after CDK4 repression and the repression of CDK4 could attenuate the higher progression to G2/M induced by H. pylori (Fig. 4G).

To further verify that PGRN promoted the cell cycle of the gastric epithelial cells via CDK4, BGC-823 cells were co-transfected with CDK4-RNAi-13 and lentivirus pLKO.1-PGRN shRNA-GFP or lentivirus Plenti6/V5-PGRN. Fewer cells progressed to G2/M in PGRN and CDK4 both repression groups compared with repression of PGRN or CDK4 alone. The proportion of cells entering G2/M after overexpression of PGRN was markedly higher than that in the control group, while co-transfection with CDK4-RNAi-13 reduced this proportion nearly to the baseline (Fig. 4H). These results further indicated that H. pylori upregulated CDK4 expression, thereby promoting cell cycle progression via the upregulation of PGRN.

PGRN Regulates CDK4 via PI3K/Akt Signaling Pathway

We have already demonstrated that H. pylori infection may regulate the cell cycle via the PI3K/Akt signaling pathway. To analyze whether PGRN regulates CDK4 expression via the same pathway, we next applied the PI3K signal pathway inhibitor LY294002 to lentivirus pLKO.1-PGRN shRNA-GFP or lentivirus Plenti6/V5-PGRN infected BGC-823 cells. qPCR verified the effectiveness of PGRN knockdown and overexpression (Fig. 5A). LY294002 can markedly repress CDK4 expression stimulated by PGRN (Fig. 5B). To investigate whether PI3K signal pathway participates in the signal transduction process, western blot was used to evaluate that phosphorylation of Akt in response to PGRN knockdown or overexpression and interaction with H. pylori. We found that Akt phosphorylation and CDK4 expression decreased in PGRN knockdown cells, while their expression increased in overexpressing PGRN. Infected with H. pylori, Akt phosphorylation and CDK4 expression were higher than that without H. pylori, while the repression of PGRN could reduce the higher phosphorylation and CDK4 expression, and the overexpression of PGRN could augment them. (Figs. 5C and 5D). This indicated that the increased PGRN induced by H. pylori infection regulated CDK4 expression via PI3K/Akt signaling pathway. Consequently, the increased CDK4 promoted gastric epithelial cell cycle progression.

Discussion

As a multifunctional growth factor, PGRN is involved in cell growth, inflammation regulation, tumorigenesis, and many other important aspects. In a mouse model of PGRN-deficient arthritis, PGRN inhibits the binding of TNF to its receptor and blocks intracellular signaling pathways [38]. PGRN overexpression promotes the secretion of multiple inflammatory factors that contribute to the development of tumors and related diseases [39, 40]. Many studies have indicated that PGRN is overexpressed in various human cancers, for instance, ovarian cancer, colorectal cancer, and gastrointestinal tumors [41-43]. We previously showed that the upregulation of PGRN induced by H. pylori accelerates the cell proliferation and migration of gastric epithelial cells [36]. In this report, we showed that PGRN was upregulated by H. pylori infection in gastric epithelial cells, thereby stimulating the cell cycle and promoting cell proliferation by increasing the expression of CDK4. Immunohistochemical analysis demonstrated that the expression of PGRN and CDK4 in gastric cancer tissue was higher than that in adjacent normal tissue, and PGRN was positively associated with CDK4 in gastric cancer. As a highly tumorigenic growth factor, overexpression of PGRN in weakly tumorigenic cells significantly promotes tumor growth [44]. Inhibition of PGRN expression in highly tumorigenic mouse cells can reduce tumor formation [45]. This is consistent with our findings. In breast cancer, the tissue level of PGRN predicts the risk of recurrence of ER-positive invasive ductal carcinoma [46]. Monoclonal antibody against PGRN inhibits the growth of hepatocellular carcinoma in nude mice [47]. This shows that PGRN is a feasible target for developing new drugs against certain cancers.

H. pylori, as the main pathogenic factor of gastric cancer, has a significantly increased infection rate in premalignant lesions and gastric cancer. Eradication of H. pylori reduces the incidence of gastric cancer [48, 49]. Current research has shown that a variety of virulence factors produced by H. pylori, for instance, CagA, VacA, HtrA, Baba, Saba, and oipa, can help it attach to gastric epithelial cells, cause the host immune system to release various pro-inflammatory cytokines and chemokines and activate multiple signal pathways, such as the NF-κB, Wnt/β-catenin, and PI3K/Akt/mTOR pathways, which affect cell proliferation and differentiation, and promote the transformation of normal gastric epithelial cells into cancer cells [50, 51]. In this study, we demonstrated that H. pylori increased the proliferative activity of gastric epithelial cells in a certain range, and the increased activity was positively correlated with the number of bacteria loaded. In addition, we found that the more proliferative activity induced by H. pylori was caused by accelerated cell cycle progression. The cell cycle is an important event associated with development, apoptosis, DNA repair, and tissue regeneration [52-54]. This also confirms that H. pylori plays a crucial role in tumorigenesis and metastasis [55]. Normal proliferation of cells is regulated by cell cycle checkpoints, and once the cell cycle checkpoint is defective, cells may proliferate uncontrollably [56, 57]. When cancer occurs, the control of checkpoints often becomes dysfunctional, including abnormal expression of the RB gene and mis-regulation of CDKs, resulting in dysregulated cell cycle activity, causing hyper-proliferation leading to cancer or enabling cell loss [16, 58]. The CDK-cyclin complex regulates the cell cycle process by phosphorylating its substrates, and the cycle process is negatively regulated by cell cycle-dependent kinase inhibitors (CDKIs), which can halt the process by binding inhibition before or after DNA replication in response to DNA damage [50, 59]. CDKIs are divided into INK4 families (including p16ink4c, p15ink4c, P18ink4c, and P19ink4c) and CIP/Kip families (p21cip1, p27kip1, and p57kip2) [60]. In a p21cip1 and p27kip1-deficient mouse model, the tumor growth rate was accelerated [61]. The action of H. pylori on the cell cycle may be connected with its regulated CDKs. Ahmed and Li detected that H. pylori infection caused DNA damage, increased p53 expression, which induced p21 expression, and bound to the CDK2-cyclin E complex to block the cell cycle in G1 phase [62, 63]. However, Sherr and Shirin found that H. pylori inhibits the expression of p27kip1, which binds to cyclin E and CDK2 and inhibits the transition from G1 to S. Similarly, other studies have shown that H. pylori promotes the expression of cyclin D1, which causes activation of CDK4 and CDK6, initiates the inactivation of the phosphorylation-dependent RB tumor suppressor protein and the release of transcription factor E2F, and shortens G1 phase and increases the proliferation rate [64, 65]. Some studies have suggested that H. pylori infection caused cell cycle arrest [66-69]. In this study, we found that H. pylori accelerated the cell cycle process from G1 to G2/M in a time- and dose-dependent manner, clarifying the promoting action of H. pylori on the cell cycle. This effect may be related to the time in coculture and the dose of H. pylori. Therefore, H. pylori may affect the proliferation of cancer cells by disrupting the balance of each stage of the cell cycle by affecting the expression of proteins in each phase of the cell cycle.

Recent studies reported that loss-of-function of PGRN caused the accumulation of TDP-43 protein to inhibit CDK6 expression, and then abnormally activated the Wnt5a signal and showed cell cycle disorder [70, 71]. To evaluate the function of PGRN in the cell cycle induced by H. pylori infection, cell cycle distribution was analyzed in gastric epithelial cells. We found that H. pylori infection promoted progression to G2/M, but knockdown of PGRN reduced these activities promoted by H. pylori infection, while PGRN overexpression enhanced these activities. This indicated that the cell cycle-promoting effects induced by H. pylori infection may be mediated through PGRN. To further understand the molecular mechanisms of PGRN regulating cell cycle progression, we turned our attention to CDK4, which is positively correlated with PGRN expression in gastric cancer. It has been reported that the synergy of PI3K and CDK4/6 inhibitors increases apoptosis and cell cycle arrest in triple-negative breast cancer cells, and that tumor immunogenicity is enhanced [72]. CDK4/6 inhibitor inhibits tumor growth in xenograft mouse model [73]. Here, we demonstrated that CDK4 is the downstream target of PGRN. Knockdown of PGRN significantly inhibited CDK4 expression, and overexpression of PGRN markedly promoted CDK4 expression. Moreover, we found that CDK4 expression was apparently upregulated in gastric epithelial cells after H. pylori infection. Repression of PGRN inhibited the higher expression of CDK4 promoted by H. pylori, while overexpression of PGRN further promoted the expression of CDK4, indicating that H. pylori increased CDK4 expression through PGRN. Repression of CDK4 could also decrease the cell cycle process induced by H. pylori infection. Meanwhile, knockdown of CDK4 expression inhibited the cell cycle progression promoted by PGRN overexpression. These findings demonstrated that H. pylori upregulated CDK4 expression to promote cell cycle progression via the upregulation of PGRN.

The PI3K/Akt, NF-κB, and MEK/ERK signaling pathways are important pathways that participate in the process by which PGRN regulates tumor growth [74, 75]. Here, we cocultured cells with various signal pathway inhibitors and found that H. pylori regulated the cell cycle via the PI3K/Akt signal pathway. Furthermore, we cocultured PI3K/Akt signaling pathway inhibitors with PGRN-overexpressing cells and found that this inhibitor reduced the expression of CDK4. To determine whether PI3K signal pathway participated in the signal transduction process, the phosphorylation of Akt was detected. The Akt was activated by H. pylori infection, and inhibition of PGRN reduced the higher activation, while overexpression of PGRN increased this level. These data showed that the enhanced expression of PGRN stimulated by H. pylori activated the PI3K signaling pathway, thereby increasing the expression of CDK4, accelerating the entry of cells into G2/M phase, which increased the proliferation of gastric epithelial cells and promoted tumorigenesis. Additionally, other studies have suggested that the growth of mucosal epithelial cells after H. pylori colonization may be mediated by a gastrin-dependent mechanism [76]. This also provides a novel approach for extensive exploration of its mechanisms in the future.

In conclusion, our study demonstrated that infection of gastric epithelial cells by H. pylori led to increased PGRN expression, which regulated the expression of CDK4 by activating the PI3K/Akt signal pathway. The increased CDK4 then regulated the cell cycle and promoted cell proliferation. This process not only provides a new direction for exploring the carcinogenic pathway of H. pylori, but also provides a new potential target for the early detection of and therapy for gastric cancer.

Acknowledgments

This study was supported by the National Natural Science Foundation of China (No. 81201262). We thank Prof. IC Bruce for critical reading of the manuscript.

Conflict of Interest

The authors have no financial conflicts of interest to declare.

Fig 1.

Figure 1.Differences of PGRN and CDK4 protein expression between gastric cancer tissue and adjacent normal tissue. (A, B) Expression levels of PGRN (A) and CDK4 (B) in both gastric cancer and adjacent normal tissues as measured by immunohistochemistry. The results represent the mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, paired t-test. All data are mean values of three biological replicates.
Journal of Microbiology and Biotechnology 2022; 32: 844-854https://doi.org/10.4014/jmb.2203.03053

Fig 2.

Figure 2.H. pylori infection promotes cell cycle progression and cell proliferation. (A) BGC-823 cells cocultured with H. pylori at a multiplicity of infection (MOI) 50:1 for 3 h, and their clonogenic potential were then assessed. (B) Flow cytometry results of BGC-823 cells infected with H. pylori 26695 at a MOI of 50:1 for 6, 12, and 24 h. (C) Flow cytometric results of H. pylori 26695 infected BGC-823 cells at different MOIs (10:1, 20:1, 50:1, 100:1, and 200:1). (D) Flow cytometry results of BGC-823 cells pre-treated with BAY11-7082 (5 μM), LY294002 (10 μM) and UO126 (10 μM) for 2 h before incubation with or without H. pylori at a MOI of 50:1 for 12 h. The results represent the mean ± SD of three independent experiments. ns, not significant, HP, Helicobacter pylori, *p < 0.05, **p < 0.01, ***p < 0.001.
Journal of Microbiology and Biotechnology 2022; 32: 844-854https://doi.org/10.4014/jmb.2203.03053

Fig 3.

Figure 3.PGRN promotes cell cycle progression and cell proliferation in gastric cancer cells with or without H. pylori infection. (A) qPCR analysis of the expression of PGRN after transfection with pLKO.1-PGRN shRNA-GFP and Plenti6/V5-PGRN lentivirus. (B, C) Colony formation assays (B) and cell cycle assays (C) of BGC-823 cells transfected with pLKO.1-PGRN shRNA-GFP and Plenti6/V5-PGRN and their negative control lentivirus and cocultured with H. pylori at a MOI of 50: 1 for 3 h. The results represent the mean ± SD of three independent experiments. SI, PGRN knockdown group, NS, the control group of PGRN knockdown, GFP, the control group of PGRN overexpressing, HP, Helicobacter pylori, *p < 0.05, **p < 0.01, ***p < 0.001.
Journal of Microbiology and Biotechnology 2022; 32: 844-854https://doi.org/10.4014/jmb.2203.03053

Fig 4.

Figure 4.PGRN positively regulates CDK4 to promote cell cycle progression. (A, B) qPCR and western blot analysis of the expression of CDK4 in BGC-823 cells infected with H. pylori 26695 at a MOI of 100: 1. (C, D) qPCR and western blot analysis of the expression of CDK4 after transfection with pLKO.1-PGRN shRNA-GFP and Plenti6/V5-PGRN lentivirus. (E, F) CDK4 expression after transfection with CDK4-RNAi-13, CDK4-RNAi-14, CDK4-RNAi-15 of CDK4-knockdown lentivirus. (G) Flow cytometry analysis of the cell cycle changes of CDK4 knockdown and coculture with H. pylori in 50:1 MOI in BGC-823 cells. (H) Flow cytometry analysis of the cell cycle changes of CDK4 knockdown cell lines infected with PGRN-knockdown /overexpressed lentivirus and cocultured with H. pylori at a MOI of 50: 1. The results represent the mean ± SD of three independent experiments. SI, PGRN knockdown group, NS, the control group of PGRN knockdown, GFP, the control group of PGRN overexpressing, ns, not significant, HP, Helicobacter pylori, *p < 0.05, **p < 0.01, ***p < 0.001.
Journal of Microbiology and Biotechnology 2022; 32: 844-854https://doi.org/10.4014/jmb.2203.03053

Fig 5.

Figure 5.PGRN regulates CDK4 through the PI3K/Akt signaling pathway and promotes progression of the gastric mucosal epithelial cell cycle. (A) qPCR analysis of the expression of PGRN after transfection with pLKO.1-PGRN shRNA-GFP and Plenti6/V5-PGRN lentivirus and cocultured with H. pylori at a MOI of 100: 1. (B) Western blot analysis of CDK4 protein expression in cells pretreated with a PI3K signal pathway inhibitor (LY294002) for 2 h before transfection with pLKO.1-PGRN shRNA-GFP and Plenti6/V5-PGRN lentivirus. (C) qPCR analysis of the expression of CDK4 after transfection with pLKO.1-PGRN shRNA-GFP and Plenti6/V5-PGRN lentivirus and cocultured with H. pylori at a MOI of 100: 1. (D) Western blot analysis of CDK4, Akt, and p-Akt protein expression in cells transfected with pLKO.1-PGRN shRNA-GFP and Plenti6/V5-PGRN lentivirus and cocultured with H. pylori at a MOI of 100: 1. The results represent the mean ± SD of three independent experiments. SI, PGRN knockdown group, NS, the control group of PGRN knockdown, GFP, the control group of PGRN overexpressing, HP, Helicobacter pylori, *p < 0.05, **p < 0.01, ***p < 0.001.
Journal of Microbiology and Biotechnology 2022; 32: 844-854https://doi.org/10.4014/jmb.2203.03053

Table 1 . Correlations between PGRN and CDK4 in gastric cancer and adjacent normal tissues analyzed by linear regression..

IOD(×103)rP
PGRN38.095 ± 1.600.4520.023
CDK418.342 ± 0.84

References

  1. Ford AC, Yuan Y, Moayyedi P. 2020. Helicobacter pylori eradication therapy to prevent gastric cancer: systematic review and metaanalysis. Gut 69: 2113-2121.
    Pubmed CrossRef
  2. Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, et al. 2021. Global Cancer Statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J. Clin. 71: 209-249.
    Pubmed CrossRef
  3. Karimi P, Islami F, Anandasabapathy S, Freedman ND, Kamangar F. 2014. Gastric cancer: descriptive epidemiology, risk factors, screening, and prevention. Cancer Epidemiol. Biomarkers Prev. 23: 700-713.
    Pubmed KoreaMed CrossRef
  4. Song Z, Wu Y, Yang J, Yang D, Fang X. 2017. Progress in the treatment of advanced gastric cancer. Tumour Biol. 39: 1010428317714626.
    Pubmed CrossRef
  5. Schistosomes, liver flukes and Helicobacter pylori. IARC Working Group on the Evaluation of Carcinogenic Risks to Humans. Lyon, 7-14 June 1994. IARC Monogr. Eval. Carcinog. Risks Hum. 61: 1-241.
    Pubmed KoreaMed
  6. Gonzalez CA, Megraud F, Buissonniere A, Lujan Barroso L, Agudo A, Duell EJ, et al. 2012. Helicobacter pylori infection assessed by ELISA and by immunoblot and noncardia gastric cancer risk in a prospective study: the Eurgast-EPIC project. Ann. Oncol. 23: 1320-1324.
    Pubmed CrossRef
  7. Hooi JKY, Lai WY, Ng WK, Suen MMY, Underwood FE, Tanyingoh D, et al. 2017. Global prevalence of Helicobacter pylori infection: Systematic review and meta-analysis. Gastroenterology 153: 420-429.
    Pubmed CrossRef
  8. Cho J, Prashar A, Jones NL, Moss SF. 2021. Helicobacter pylori infection. Gastroenterol. Clin. North Am. 50: 261-282.
    Pubmed KoreaMed CrossRef
  9. Wang F, Meng W, Wang B, Qiao L. 2014. Helicobacter pylori-induced gastric inflammation and gastric cancer. Cancer Lett. 345: 196-202.
    Pubmed CrossRef
  10. Hochegger H, Takeda S, Hunt T. 2008. Cyclin-dependent kinases and cell-cycle transitions: does one fit all? Nat. Rev. Mol. Cell Biol. 9: 910-916.
    Pubmed CrossRef
  11. Enders GH. 2012. Mammalian interphase cdks: dispensable master regulators of the cell cycle. Genes Cancer 3: 614-618.
    Pubmed KoreaMed CrossRef
  12. Gao X, Leone GW, Wang H. 2020. Cyclin D-CDK4/6 functions in cancer. Adv. Cancer Res. 148: 147-169.
    Pubmed CrossRef
  13. Tadesse S, Caldon EC, Tilley W, Wang S. 2019. Cyclin-eependent kinase 2 inhibitors in cancer therapy: An update. J. Med. Chem. 62: 4233-4251.
    Pubmed CrossRef
  14. Kastan MB, Bartek J. 2004. Cell-cycle checkpoints and cancer. Nature 432: 316-323.
    Pubmed CrossRef
  15. Ford HL, Pardee AB. 1999. Cancer and the cell cycle. J. Cell Biochem. Suppl 32-33: 166-172.
    Pubmed CrossRef
  16. Sherr CJ. 1996. Cancer cell cycles. Science 274: 1672-1677.
    Pubmed CrossRef
  17. Fong LY, Nguyen VT, Farber JL, Huebner K, Magee PN. 2000. Early deregulation of the the p16ink4a-cyclin D1/cyclin-dependent kinase 4-retinoblastoma pathway in cell proliferation-driven esophageal tumorigenesis in zinc-deficient rats. Cancer Res. 60: 4589-4595.
    Pubmed
  18. Guha R, Yue B, Dong J, Banerjee A, Serrero G. 2021. Anti-progranulin/GP88 antibody AG01 inhibits triple negative breast cancer cell proliferation and migration. Breast Cancer Res. Treat. 186: 637-653.
    Pubmed KoreaMed CrossRef
  19. Zhao J, Li X, Liu J, Jiang W, Wen D, Xue H. 2018. Effect of progranulin on migration and invasion of human colon cancer cells. J. Coll. Physicians Surg. Pak. 28: 607-611.
    Pubmed CrossRef
  20. Walsh CE, Hitchcock PF. 2017. Progranulin regulates neurogenesis in the developing vertebrate retina. Dev. Neurobiol. 77: 1114-1129.
    Pubmed KoreaMed CrossRef
  21. He Z, Ong CH, Halper J, Bateman A. 2003. Progranulin is a mediator of the wound response. Nat. Med. 9: 225-229.
    Pubmed CrossRef
  22. Wei J, Zhang L, Ding Y, Liu R, Guo Y, Hettinghouse A, et al. 2020. Progranulin promotes diabetic fracture healing in mice with type 1 diabetes. Ann. NY Acad. Sci. 1460: 43-56.
    Pubmed KoreaMed CrossRef
  23. Xu B, Chen X, Ding Y, Chen C, Liu T, Zhang H. 2020. Abnormal angiogenesis of placenta in progranulindeficient mice. Mol. Med. Rep. 22: 3482-3492.
    CrossRef
  24. Lu J, Huang J, Shan M, Hu X, Guo W, Xie W, et al. 2021. Progranulin ameliorates lung inflammation in an LPS-induced acute lung injury mouse model by modulating macrophage polarization and the MAPK pathway. Ann. Clin. Lab. Sci. 51: 220-230.
    Pubmed
  25. Li H, Zhang Z, Feng D, Xu L, Li F, Liu J, et al. 2020. PGRN exerts inflammatory effects via SIRT1-NF-kappaB in adipose insulin resistance. J. Mol. Endocrinol. 64: 181-193.
    Pubmed CrossRef
  26. Feng JQ, Guo FJ, Jiang BC, Zhang Y, Frenkel S, Wang DW, et al. 2010. Granulin epithelin precursor: a bone morphogenic protein 2-inducible growth factor that activates Erk1/2 signaling and JunB transcription factor in chondrogenesis. FASEB J. 24: 1879-1892.
    Pubmed KoreaMed CrossRef
  27. Abdulrahman A, Eckstein M, Jung R, Guzman J, Weigelt K, Serrero G, et al. 2019. Expression of GP88 (Progranulin) protein is an independent prognostic factor in prostate cancer patients. Cancers (Basel). 11: 2029.
    Pubmed KoreaMed CrossRef
  28. Feng T, Zheng L, Liu F, Xu X, Mao S, Wang X, et al. 2016. Growth factor progranulin promotes tumorigenesis of cervical cancer via PI3K/Akt/mTOR signaling pathway. Oncotarget 7: 58381-58395.
    Pubmed KoreaMed CrossRef
  29. Kimura A, Takemura M, Serrero G, Yoshikura N, Hayashi Y, Saito K, et al. 2018. Higher levels of progranulin in cerebrospinal fluid of patients with lymphoma and carcinoma with CNS metastasis. J. Neurooncol. 137: 455-462.
    Pubmed CrossRef
  30. Yang D, Wang LL, Dong TT, Shen YH, Guo XS, Liu CY, et al. 2015. Progranulin promotes colorectal cancer proliferation and angiogenesis through TNFR2/Akt and ERK signaling pathways. Am. J. Cancer Res. 5: 3085-3097.
    Pubmed KoreaMed
  31. Buraschi S, Neill T, Xu SQ, Palladino C, Belfiore A, Iozzo RV, et al. 2020. Progranulin/EphA2 axis: A novel oncogenic mechanism in bladder cancer. Matrix Biol. 93: 10-24.
    Pubmed KoreaMed CrossRef
  32. Yabe K, Yamamoto Y, Takemura M, Hara T, Tsurumi H, Serrero G, et al. 2021. Progranulin depletion inhibits proliferation via the transforming growth factor beta/SMAD family member 2 signaling axis in Kasumi-1 cells. Heliyon 7: e05849.
    Pubmed KoreaMed CrossRef
  33. Fang W, Zhou T, Shi H, Yao M, Zhang D, Qian H, et al. 2021. Progranulin induces immune escape in breast cancer via up-regulating PD-L1 expression on tumor-associated macrophages (TAMs) and promoting CD8(+) T cell exclusion. J. Exp. Clin. Cancer Res. 40: 4.
    Pubmed KoreaMed CrossRef
  34. Zhou C, Huang Y, Wu J, Wei Y, Chen X, Lin Z, et al. 2021. A narrative review of multiple mechanisms of progranulin in cancer: a potential target for anti-cancer therapy. Transl. Cancer Res. 10: 4207-4216.
    Pubmed KoreaMed CrossRef
  35. Liu B, Li X, Sun F, Tong X, Bai Y, Jin K, et al. 2019. HP-CagA+ regulates the expression of CDK4/CyclinD1 via reg3 to change cell cycle and promote cell proliferation. Int. J. Mol. Sci. 21: 224.
    Pubmed KoreaMed CrossRef
  36. Wang H, Sun Y, Liu S, Yu H, Li W, Zeng J, et al. 2011. Upregulation of progranulin by Helicobacter pylori in human gastric epithelial cells via p38MAPK and MEK1/2 signaling pathway: role in epithelial cell proliferation and migration. FEMS Immunol. Med. Microbiol. 63: 82-92.
    Pubmed CrossRef
  37. Livak KJ, Schmittgen TD. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 25: 402-408.
    Pubmed CrossRef
  38. Tang W, Lu Y, Tian QY, Zhang Y, Guo FJ, Liu GY, et al. 2011. The growth factor progranulin binds to TNF receptors and is therapeutic against inflammatory arthritis in mice. Science 332: 478-484.
    Pubmed KoreaMed CrossRef
  39. Arechavaleta-Velasco F, Perez-Juarez CE, Gerton GL, Diaz-Cueto L. 2017. Progranulin and its biological effects in cancer. Med. Oncol. 34: 194.
    Pubmed KoreaMed CrossRef
  40. Jian J, Li G, Hettinghouse A, Liu C. 2018. Progranulin: A key player in autoimmune diseases. Cytokine 101: 48-55.
    Pubmed KoreaMed CrossRef
  41. Demorrow S. 2013. Progranulin: a novel regulator of gastrointestinal cancer progression. Transl. Gastrointest. Cancer 2: 145-151.
    Pubmed KoreaMed CrossRef
  42. Dong T, Yang D, Li R, Zhang L, Zhao H, Shen Y, et al. 2016. PGRN promotes migration and invasion of epithelial ovarian cancer cells through an epithelial mesenchymal transition program and the activation of cancer associated fibroblasts. Exp. Mol. Pathol. 100: 17-25.
    Pubmed CrossRef
  43. Klupp F, Kahlert C, Franz C, Halama N, Schleussner N, Wirsik NM, et al. 2021. Granulin: an invasive and survival-determining marker in colorectal cancer patients. Int. J. Mol. Sci. 22: 6436.
    Pubmed KoreaMed CrossRef
  44. Wang W, Hayashi J, Kim WE, Serrero G. 2003. PC cell-derived growth factor (granulin precursor) expression and action in human multiple myeloma. Clin. Cancer Res. 9: 2221-2228.
    Pubmed
  45. Lu R, Serrero G. 2000. Inhibition of PC cell-derived growth factor (PCDGF, epithelin/granulin precursor) expression by antisense PCDGF cDNA transfection inhibits tumorigenicity of the human breast carcinoma cell line MDA-MB-468. Proc. Natl. Acad. Sci. USA 97: 3993-3998.
    Pubmed KoreaMed CrossRef
  46. Serrero G, Hawkins DM, Yue B, Ioffe O, Bejarano P, Phillips JT, et al. 2012. Progranulin (GP88) tumor tissue expression is associated with increased risk of recurrence in breast cancer patients diagnosed with estrogen receptor positive invasive ductal carcinoma. Breast Cancer Res. 14: R26.
    Pubmed KoreaMed CrossRef
  47. Ho JC, Ip YC, Cheung ST, Lee YT, Chan KF, Wong SY, et al. 2008. Granulin-epithelin precursor as a therapeutic target for hepatocellular carcinoma. Hepatology 47: 1524-1532.
    Pubmed CrossRef
  48. Wu JY, Lee YC, Graham DY. 2019. The eradication of Helicobacter pylori to prevent gastric cancer: a critical appraisal. Expert Rev. Gastroenterol. Hepatol. 13: 17-24.
    Pubmed KoreaMed CrossRef
  49. Zhou Z, Ye G, Peng J, He B, Xu S, Fan W, et al. 2021. Expression of Wnt3, beta-catenin and MMP-7 in gastric cancer and precancerous lesions and their correlations with Helicobacter pylori infection. Zhong Nan Da Xue Xue Bao Yi Xue Ban 46: 575-582.
  50. Molaei F, Forghanifard MM, Fahim Y, Abbaszadegan MR. 2018. Molecular signaling in tumorigenesis of gastric cancer. Iran Biomed. J. 22: 217-230.
    Pubmed KoreaMed CrossRef
  51. Padda J, Khalid K, Cooper AC, Jean-Charles G. 2021. Association between Helicobacter pylori and gastric carcinoma. Cureus 13: e15165.
    Pubmed KoreaMed CrossRef
  52. Kato MV, Sato H, Anzai H, Nagayoshi M, Ikawa Y. 1997. Up-regulation of cell cycle-associated genes by p53 in apoptosis of an erythroleukemic cell line. Leukemia 11 Suppl 3: 389-392.
    Pubmed
  53. Woodward TA, Klingler PD, Genko PV, Wolfe JT. 2000. Barrett's esophagus, apoptosis and cell cycle regulation: correlation of p53 with Bax, Bcl-2 and p21 protein expression. Anticancer Res. 20: 2427-2432.
    Pubmed
  54. Chinzei N, Hayashi S, Ueha T, Fujishiro T, Kanzaki N, Hashimoto S, et al. 2015. P21 deficiency delays regeneration of skeletal muscular tissue. PLoS One 10: e0125765.
    Pubmed KoreaMed CrossRef
  55. Megraud F, Bessede E, Varon C. 2015. Helicobacter pylori infection and gastric carcinoma. Clin. Microbiol. Infect. 21: 984-990.
    Pubmed CrossRef
  56. Evan GI, Vousden KH. 2001. Proliferation, cell cycle and apoptosis in cancer. Nature 411: 342-348.
    Pubmed CrossRef
  57. Kar S. 2016. Unraveling cell-cycle dynamics in cancer. Cell Syst. 2: 8-10.
    Pubmed CrossRef
  58. Pack LR, Daigh LH, Meyer T. 2019. Putting the brakes on the cell cycle: mechanisms of cellular growth arrest. Curr. Opin. Cell Biol. 60: 106-113.
    Pubmed KoreaMed CrossRef
  59. Campbell GJ, Hands EL, Van de Pette M. 2020. The role of CDKs and CDKIs in murine development. Int. J. Mol. Sci. 21: 5343.
    Pubmed KoreaMed CrossRef
  60. Canepa ET, Scassa ME, Ceruti JM, Marazita MC, Carcagno AL, Sirkin PF, et al. 2007. INK4 proteins, a family of mammalian CDK inhibitors with novel biological functions. IUBMB Life 59: 419-426.
    Pubmed CrossRef
  61. Jackson RJ, Adnane J, Coppola D, Cantor A, Sebti SM, Pledger WJ. 2002. Loss of the cell cycle inhibitors p21(Cip1) and p27(Kip1) enhances tumorigenesis in knockout mouse models. Oncogene 21: 8486-8497.
    Pubmed CrossRef
  62. Ahmed A, Smoot D, Littleton G, Tackey R, Walters CS, Kashanchi F, et al. 2000. Helicobacter pylori inhibits gastric cell cycle progression. Microbes Infect. 2: 1159-1169.
    Pubmed CrossRef
  63. Li N, Xie C, Lu NH. 2016. p53, a potential predictor of Helicobacter pylori infection-associated gastric carcinogenesis? Oncotarget 7: 66276-66286.
    Pubmed KoreaMed CrossRef
  64. Sherr CJ. 1995. D-type cyclins. Trends Biochem. Sci. 20: 187-190.
    Pubmed CrossRef
  65. Shirin H, Sordillo EM, Oh SH, Yamamoto H, Delohery T, Weinstein IB, et al. 1999. Helicobacter pylori inhibits the G1 to S transition in AGS gastric epithelial cells. Cancer Res. 59: 2277-2281.
    Pubmed
  66. Scotti C, Sommi P, Pasquetto MV, Cappelletti D, Stivala S, Mignosi P, et al. 2010. Cell-cycle inhibition by Helicobacter pylori Lasparaginase. PLoS One 5: e13892.
    Pubmed KoreaMed CrossRef
  67. Cover TL, Krishna US, Israel DA, Peek RM Jr. 2003. Induction of gastric epithelial cell apoptosis by Helicobacter pylori vacuolating cytotoxin. Cancer Res. 63: 951-957.
    Pubmed
  68. Kim KM, Lee SG, Kim JM, Kim DS, Song JY, Kang HL, et al. 2010. Helicobacter pylori gamma-glutamyltranspeptidase induces cell cycle arrest at the G1-S phase transition. J. Microbiol. 48: 372-377.
    Pubmed CrossRef
  69. Li H, Liang D, Hu N, Dai X, He J, Zhuang H, et al. 2019. Helicobacter pylori inhibited cell proliferation in human periodontal ligament fibroblasts through the Cdc25C/CDK1/cyclinB1 signaling cascade. J. Periodontal. Implant Sci. 49: 138-147.
    Pubmed KoreaMed CrossRef
  70. Alquezar C, Esteras N, Bartolome F, Merino JJ, Alzualde A, Lopez de Munain A, et al. 2012. Alteration in cell cycle-related proteins in lymphoblasts from carriers of the c.709-1G>A PGRN mutation associated with FTLD-TDP dementia. Neurobiol. Aging 33: 429.e427-420.
    Pubmed CrossRef
  71. Alquezar C, Esteras N, de la Encarnacion A, Alzualde A, Moreno F, Lopez de Munain A, et al. 2014. PGRN haploinsufficiency increased Wnt5a signaling in peripheral cells from frontotemporal lobar degeneration-progranulin mutation carriers. Neurobiol. Aging 35: 886-898.
    Pubmed CrossRef
  72. Teo ZL, Versaci S, Dushyanthen S, Caramia F, Savas P, Mintoff CP, et al. 2017. Combined CDK4/6 and PI3Kalpha inhibition is synergistic and immunogenic in triple-negative breast cancer. Cancer Res. 77: 6340-6352.
    Pubmed CrossRef
  73. Kollmann K, Briand C, Bellutti F, Schicher N, Blunder S, Zojer M, et al. 2019. The interplay of CDK4 and CDK6 in melanoma. Oncotarget 10: 1346-1359.
    Pubmed KoreaMed CrossRef
  74. Lu Y, Zheng L, Zhang W, Feng T, Liu J, Wang X, et al. 2014. Growth factor progranulin contributes to cervical cancer cell proliferation and transformation in vivo and in vitro. Gynecol. Oncol. 134: 364-371.
    Pubmed CrossRef
  75. He Z, Ismail A, Kriazhev L, Sadvakassova G, Bateman A. 2002. Progranulin (PC-cell-derived growth factor/acrogranin) regulates invasion and cell survival. Cancer Res. 62: 5590-5596.
    Pubmed
  76. Peek RM Jr, Wirth HP, Moss SF, Yang M, Abdalla AM, Tham KT, et al. 2000. Helicobacter pylori alters gastric epithelial cell cycle events and gastrin secretion in Mongolian gerbils. Gastroenterology 118: 48-59.
    Pubmed CrossRef