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

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.


Introduction
. In the early G 1 -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 granulinepithelin 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][19][20][21][22][23][24][25][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][28][29][30][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 tumorassociated 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.

Tissue Samples
One hundred gastric cancer tissue and adjacent normal tissue samples were provided by Weifang People's 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.

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 o C under microaerophilic conditions containing 10% CO 2 , 5% O 2 and 85% N 2 . 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.

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 o 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% lowtemperature ethanol at 4 o 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 o 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 o C and 5% CO 2 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.

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).

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.
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 G 2 /M phase increased significantly after H. pylori infection, and with the prolongation of infection time, the proportion of cells entering G 2 /M phase gradually increased (Fig. 2B). Next, BGC-823 cells were infected with H. pylori at varying MOIs of   H. pylori was found to activate a set of main signaling molecules that include NF-κB, PI3K/Akt, and mitogenactivated 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 G 2 /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 G 2 /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 G 2 /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.

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).
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  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 G 2 /M was significantly reduced after CDK4 repression and the repression of CDK4 could attenuate the higher progression to G 2 /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 G 2 /M in PGRN and CDK4 both repression groups compared with repression of PGRN or CDK4 alone. The proportion of cells entering G 2 /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][42][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 ERpositive 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][53][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 p16 ink4c , p15 ink4c , P18 ink4c , and P19 ink4c ) and CIP/Kip families (p21 cip1 , p27 kip1 , and p57 kip2 ) [60]. In a p21 cip1 and p27 kip1 -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 G 1 phase [62,63]. However, Sherr and Shirin found that H. pylori inhibits the expression of p27 kip1 , which binds to cyclin E and CDK2 and inhibits the transition from G 1 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 G 1 phase and increases the proliferation rate [64,65]. Some studies have suggested that H. pylori infection caused cell cycle arrest [66][67][68][69]. In this study, we found that H. pylori accelerated the cell cycle process from G 1 to G 2 /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 G 2 /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 triplenegative 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 G 2 /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.