LINC00174 Facilitates Proliferation and Migration of Colorectal Cancer Cells via MiR-3127-5p/ E2F7 Axis

The literature indicates that LINC00174 promotes the growth of colorectal cancer (CRC) cells, but its research needs to be enriched. We tried to explore the function and mechanism of LINC00174 in CRC cell proliferation and migration. Bioinformatics analysis predicted the binding relationship and expressions of lncRNA, miRNA and mRNA. Clinical study analyzes the relationship between LINC00174 and clinical data characteristics of CRC patients. The expressions of LINC00174, miR-3127-5p and E2F7 were verified by RT-qPCR, and the combination of the two was verified by dual luciferase analysis and RNA immunoprecipitation as needed. Western blot was used to detect the expression of EMT-related protein and E2F7 protein. Functional experiments were used to evaluate the function of the target gene on CRC cells. LINC00174 was up-regulated in CRC clinical samples and cells and was related to the clinical characteristics of CRC patients. High-expression of LINC00174, contrary to the effect of siLINC00174, promoted cell viability, proliferation, migration and invasion, up-regulated the expressions of N-Cadherin, Vimentin, E2F7, and inhibited the expression of E-Cadherin. MiR-3127-5p was one of the targeted miRNAs of LINC00174 and was down-regulated in CRC samples. In addition, miR-3127-5p mimic partially reversed the malignant phenotype of CRC cells induced by LINC00174. Besides, E2F7 was a target gene of miR-3127-5p, and LINC00174 repressed miR-3127-5p to regulate E2F7. Our research reveals that LINC00174 affected the biological characteristics of CRC cells through regulated miR-3127-5p/ E2F7 axis.

In addition, we obtained 7 miRNAs that bind to LINC00174 and are lowly expressed in rectal adenocarcinoma through bioinformatics analysis. Among them, miR-3127-5p has been rarely studied in diseases, and so far there have been only two reports. MiR-3127-5p was found to play a role in suppressing cancer in non-small-cell lung cancer (NSCLC) [22]. Moreover, as the target miRNA of lncRNA GACAT3, miR-3127-5p affects the progression of glioma by regulating the expression of ELAVL1 [23]. Although the latest research shows that miR-3127-5p is differentially expressed in CRC, its function and mechanism are still unclear [24]. Thus, we constructed clinical and cell assays accompanying bioinformatics analysis to investigate whether LINC00174 affects the biological process of CRC cells by regulating miR-3127-5p.

Ethics Statement and Tissue
Prior to this study, all patients were informed and signed informed consent. The clinical specimens were obtained from 80 CRC patients who came to our hospital from June 2019 to April 2020, after approval of the ethics committee of People's Hospital of Ningxia Hui Autonomous Region (CR201905020). The relationship between LINC00174 and clinical data characteristics of 80 CRC patients were shown in Table 1.

Real Time Quantitative Polymerase Chain Reaction
RNAiso Plus (9108Q, Takara, China) or RNAiso for Small RNA (9753Q) were used to isolate total RNA or miRNA, as required. Reverse transcription step was refered to the instructions of PrimeScrip RT reagent Kit (RR037Q) produced by Takara, China. The RT-qPCR detection was constructed in the Mx3000P Real-Time QPCR System (Agilent, USA) with Takara TB Green Premix Ex Taq II (RR820Q, China), followed by the conditions set as below: pre-denaturation (95°C, 3 min), denaturation (95°C, 15 s) and annealing (60°C, 1 min) for a total of 40 cycles, and finally extended (68°C, 7 min). GAPDH or U6 were used as control for mRNA or miRNA, and the results were quantified in the form of 2 -ΔΔCt method [16]. Sequences of the primers were listed as follows

Cell Counting Kit (CCK)-8
After transfected for 24 h, SW480 or LOVO cells were replaced with fresh medium to continue culturing. Cells in good growth condition was taken to make a single cell suspension (1 × 10 4 cells/ml) and seeded in 96-well plates. When SW480 or LOVO cells were cultured for 24, 48, or 72 h, 10 μl of CCK-8 reagent (C0037, Beyotime, China) was added to each well for routine incubation for 1 h. Finally, iMark microplate reader from Bio-Rad (USA) was applied to detect the optical density value (OD450) wavelength of each well to indicate cell activity.

Cell Colony Formation Assay
Consistent with the above experiment, first, each group of cells was adjusted to a cell suspension (1 × 10 4 cells/ml) and routinely cultivated in a 6-well plate. At about 14 days of culture, macroscopic cell colonies grew in the culture plate. Next, each well was fixed with 4% paraformaldehyde for 15 min, and Giemsa staining reagent (32884, Sigma, USA) was added for staining for 15-20 min. Finally, the optical microscope (DMi8, Leica, Germany) took pictures to record the cell proliferation.

Wound Healing Assay
The pre-digested CRC cells (1 × 10 5 cells/ml) were added into ibidi cell plugin (ibidi, 81176, Germany) routinely incubated for 24 h. Next, the ibidi wound healing insert was raised with tweezers. Microscopic examination (CKX53, Olympus, Japan) was performed on the cells grown the day before (Magnification × 100).

Transwell
Matrix gel (354230, BD Biosciences, USA) was applied to the bottom membrane of the Transwell chamber (8 μm, BD Biosciences). Also, the Transwell chamber was inserted into the 24-well plate. 50 μl cell suspensions were seeded in the upper chamber, whilst RPMI-1640 medium with 10% FBS was added in lower chamber. After cells were cultured for 24 h, the chamber was removed. Invaded cells were fixed and then stained with 0.5% crystal violet for 30 min. Finally, five fields of the cells were randomly observed under the microscope to calculate the number of cell invasion (Magnification × 250).

Target Gene Verification
The wild-type or mutant sequences of LINC00174 or E2F7 were integrated into the pmirGLO vector (E1330, Promega, USA). Different recombinant plasmids together with miR-3127-5p (M) or mimic control (Blank) were transfected into SW480 or LOVO cells, respectively. Luciferase activities were measured in dual luciferase system (D0010-100T, Solarbio, China) by GloMax 20/20 detector (Promega). Imprint RNA Immunoprecipitation Kit (RIP, Sigma-Aldrich) was constructed to RNA immunoprecipitation (RIP) assays. Briefly, CRC cells were lysed using RIR lysisi buffer, and protein A/G beads conjugated with anti-Argonaute2 (Ago 2) was added to cell extract for 6 h at 4°C. After that, samples were incubated with roteinase K to isolate RNA-protein complexes. IgG was served as control. Finally, immunoprecipitated RNA was subjected to RT-qPCR detection.

Statistical Analysis
Data were described by mean ± SD; comparison between count data groups was analyzed using χ 2 test. The data of Fig. 1B used paired sample t test; one-way ANOVA for comparison between multiple groups; Tukey test for pairwise comparison between groups. All statistical analysis were implemented using Graphpad 8.0 software, with p < 0.05 considered as significant. Fig. 1A showed LINC00174 was highly expressed in colon adenocarcinoma (COAD) (P = 3.9E-9). Based on this, we collected 80 clinical samples of CRC patients, and found that the level of LINC00174 was elevated in tumor tissue (p < 0.001, Fig. 1B). In addition, we assessed the relationship between LINC00174 and the clinical data characteristics of CRC patients, and found that the level of LINC00174 was memorably associated with tumor size, lymphatic vessel invasion, tumor invasion depth, lymph node metastasis, distant metastasis and tumor stage (p < 0.05, Table 1). We further examined the situation of LINC00174 in the CRC cell line and found that the expression of LINC00174 in LS174T, HCT116, HCT-15, SW480, LOVO cells was notably higher than that of CCD-18Co cells (p < 0.01, Fig. 2A). Among all CRC cell lines, the expression of LINC00174 was highest in SW480 cells and lowest in LOVO cells (p < 0.01, Fig. 2A), so these two cell lines were selected as the next experimental cells.

Overexpression of LINC00174 Promoted the Biological Function of CRC Cells, while Silencing LINC00174 Was the Opposite
To understand the impact of LINC00174 on the biological characteristics of CRC, we transfected LINC00174 overexpression plasmids and shLINC00174 into SW480 cells or LOVO cells. As we wished, LINC00174 was successfully overexpressed or reduced (p < 0.05, Figs. 2B and 2C). Subsequently, we used functional experiments to explore the role of LINC00174. The cell viability of the LINC00174 group was dramatically increased, while shLINC00174 had the effect of inhibiting cell viability (p < 0.05, Figs. 2D and 2E). Similarly, overexpression of LINC00174 increased the number of cell clones, while knocking out LINC00174 decreased the number of cell

Discussion
LncRNAs play various roles in the progression of CRC [18,25]. LncRNA MALAT1 may promote the biological characteristics of CRC through its target protein AKAP-9 [26]. Studies by Zhu et al. showed that high expression of MALAT1 can promote the development of CRC by regulating miR-145/SOX9 axis [27], suggesting that the same lncRNA has multiple regulatory mechanisms involved in tumor progression.
LINC00174 plays a pro-cancer role by regulating different functions of cancer cells. In glioma, LINC00174 accelerated carcinogenesis of glioma by promoting cell proliferation, migration, invasion and glycolysis of glioma cells [10,12]; moreover, LINC00174 down-regulation decreases chemoresistance to temozolomide in human glioma cells [11]. In laryngeal papilloma, LINC00174 enhanced the proliferation and apoptosis evasion of laryngeal papilloma cells by regulating miR-4500/BZW2 axis [15]. LINC00174 involved in cell migration and lipid metabolism, and is a novel prognostic factor in thymic epithelial tumors [14]. It has been reported that LINC00174 can be used as the ceRNA of miR-1910-3p, regulating the target gene TAZ to facilitate the progress of CRC [16], suggesting that LINC00174 can be used as a new drug therapy target for CRC. In this study, LINC00174 was up-regulated in CRC clinical samples and cells, and the abnormal expression of LINC00174 was significantly associated with poor prognosis in patients with CRC. In cell experiments, we found that LINC00174 promoted CRC cell viability, which is consistent with previous studies [16]. However, the difference is that heterologous overexpression of LINC00174 also accelerated the migration and invasion ability of CRC cells, and may promote EMT protein and E2F7 by competitively binding miR-3127-5p to play a cancer-promoting role in CRC. In was used to predict the miRNAs that LINC00174 may bind to, and the TCGA database (http://cancergenome.nih.gov/) was used to obtain miRNAs that were lowly expressed in rectal adenocarcinoma, using the Venny 2.1.0 website to obtain the intersection. (B-C) The RT-qPCR was used to determine the expression of target miRNAs in SW480 and LOVO cells. + p < 0.05, ++ p < 0.01, +++ p < 0.001 vs NC.
More and more studies have shown that lncRNA can affect the binding of miRNA and its target gene by binding to the miRNA site to regulate the expression of the target gene, which is the ceRNAs regulatory network [31]. For example, LncRNA UICLM as a ceRNA of miR-215 regulates the expression of ZEB2, thereby promoting the proliferation, invasion, EMT and stem cell characteristic functions of CRC, and promoting liver metastasis of CRC [19]. Similarly, we propose to assume that LINC00174 can be used as a "molecular sponge", blocking miR-3127-5p's post-transcriptional inhibition of downstream target genes, so that the function of target genes can be restored. Based on bioinformatics analysis, we examined the effects of LINC00174 and miR-3127-5p on the expression of E2F7, and found that LINC00174 can regulate the expression of E2F7 through miR-3127-5p in CRC cells. Interestingly, E2F7 has been shown to be highly expressed in CRC and has the promoting effect on the malignant biological function of CRC [32]. Of course, more downstream regulatory networks require further experimentation.
MiRNA plays a momentous role in the progression of CRC by up-regulating oncogenes or down-regulating oncogenes [33]. For example, the latest research found that miR-185-3p is down-regulated in CRC, and upregulating miR-185-3p can enhance the chemotherapy sensitivity of CRC cells by targeting AQP5 [34]. Ma et al. found that knocking down LINC02163 attenuated CRC cell proliferation and metastasis through the miR-511-3p/AKT3 axis [35]. MiR-3127-5p is the mature miR-3127 derived from miR-3127 during maturation. Yu et al. pointed out that miR-3127-5p was a target miRNA by SNHG1 to suppress the anti-cancer roles of baicalein in cervical cancer [36]. To our knowledge, though the latest research shows that miR-3127-5p is differentially control, LINC00174+miR-3127-5p mimic, miR-3127-5p mimic groups were determined by wound healing assay (Magnification × 100). (E-H) Transwell experiment was used to detect the invasion rate of CRC cells in each group (Magnification × 250). ** p < 0.01 vs Control; # p < 0.05, ## p < 0.01 vs LINC00174; ^^p < 0.01, ^^^p < 0.001 vs M. expressed in CRC, this is the first report of the effect of miR-3127-5p in CRC. It was found that miR-3127-5p may function as a tumor suppressor gene in CRC, revealing that miR-3127-5p may be a potential treatment target for CRC. In addition, whether LINC00174 will develop normal cells into cancer cells is need to further study.
In summary, the expression of LINC00174 is up-regulated and the expression of miR-3127-5p is downregulated in CRC cells. Intervention of LINC00174 expression can play a role in promoting the malignant phenotype and EMT of CRC cells through miR-3127-5p/E2F7 axis. This study provides an experimental basis for the application of LINC00174 and miR-3127-5p in the diagnosis and molecular targeting of CRC. The next step will continue to explore the impact of LINC00174/miR-3127-5p/ E2F7 axis on CRC in vivo. (http://mirdb.org/), starBase, Targetscan (http://www.targetscan.org/vert_72/docs/help.html) were used to predict miR-3127-5p target genes. The Cancer Genome Atlas (TCGA) database (http://cancergenome.nih.gov/) analyzed mRNAs that are abnormal expressed in rectal adenocarcinoma. Venn diagram was used to analyze the intersection of the data generated from the databases. (B-C) SHBG and E2F7 expressions were detected by RT-qPCR. (D-F) E2F7 bound to miR-3127-5p as predicted and verified by Targetscan and dual luciferase assay. ++ p < 0.01, +++ p < 0.001 vs MC.