| Background and purposeThe global cancer statistics of 2018 indicated that colorectal cancer(CRC)is the third most common malignant tumors in the world with the incidence of 10.2%(colon 6.1%and rectum 3.9%)and the second leading cause of cancer associated death with the mortality of 9.2%(colon 5.8%and rectum 3.2%),claiming 881,000 lives in 2018[1].In China,the incidence of colorectal cancer ranks among the top five malignant tumors[2].Most of the patients are diagnosed at a later period as the effect of surgical treatment is poor,and chemotherapy resistance is easy to occur.Therefore,exploring the pathogenesis of colorectal cancer,finding new molecular targets and promoting individualized treatment are important research directions for researchers.MicroRNA is a kind of small non-coding RNA with the length of 18-25 bp.Its most common function is to bind with the 3'-UTR of the target genes,inhibit the expression of the target genes.MicroRNA has been proved to play an important role in a set of malignant tumors[3].It can regulate proliferation,invasion,migration and apoptosis of cancer cells in colorectal cancer[4],and is closely related to colorectal cancer stem cells[5].It is one of the key areas of basic medical research.In this study,we found that miR-532-3p is a low-expressed microRNA in colorectal cancer tissues.It can inhibit the malignant biological behaviors of colorectal cancer cells such as chemotherapeutic resistance,proliferation and migration.We also explored the possible molecular mechanism.Methods1.Verification of the expression level of miR-532-3p in colorectal tissues and cell lines1)The expression of miR-532-3p in colon adenomas and tumors was analyzed within GSE41655.2)RT-PCR assay was used to detect the expression of miR-532-3p in colorectal tumor tissues and paired normal tissues,colorectal cancer cell lines and normal intestinal epithelial cell lines.2.MiR-532-3p inhibits malignant biological behaviors of colorectal cancer cells1)Gene microarray of miR-532-3p-over-expressed cells was analysed,and signal pathways related to cell functions with different changes were screened;2)Cell apoptosis and chemotherapeutic resistance:The effect of miR-532-3p on cell apoptosis and chemotherapeutic resistance of colorectal cancer cells was verified by apoptosis assay.3)Proliferation in vitro:miR-532-3p mimics and inhibitors were transfected into colon cancer cells.Cell cycle experiments and CCK8 assay were carried out after verifying the efficiency of transfection.4)Proliferation in vivo:Subcutaneous tumorigenesis assay of cells with overexpression or knock-down of miR-532-3p were performed in nude mice and the tumor tissues were stained with IHC of Ki-67.5)In vitro migration:Cells transfected instantaneously with miR-532-3p mimics and inhibitors were performed scratch healing assay and transwell chamber migration assay.3.Validation of the mechanism of the wnt/?-catenin signaling pathway regulated by miR-532-3p targeting ETS1/TGM2 axis1)The down-regulated genes in cells with over-expression of miR-532-3p and predicted target genes were intersected to identify the proposed target genes,which were verified by RT-PCR,Western Blot and IHC.The direct binding of miR-532-3p with target genes was verified by dual-luciferase reporter assay.2)The effect of miR-532-3p on Wnt/?-catenin signaling pathway was confirmed by western blot of downstream genes.3)The functions of ETS1 and TGM2 in colorectal cancer were verified by CCK8,scratch and apoptosis assays,and the transcriptional regulation of ETS1 on TGM2 was verified by ChIP(Chromatin immuoprecipitation)assay.4.ETS1/TGM2 axis participates in the regulation of colorectal cancer by miR-532-3pRecovery experiments(CCK8,cell cycle,apoptosis,scratch healing and transwell assays)were used to verify that ETS1/TGM2 axis is involved in the regulation of colorectal cancer by miR-532-3p.Results1.Compared with normal tissues,the expression of miR-532-3p in colorectal adenomas and tumors was significantly lower,and the expression of miR-532-3p was lower in LOVO and HT29 cells with higher malignancy,and the highest in FHC cells.2.The microarray of over-expression of microRNA-532-3p suggested that the p53-related apoptosis signaling pathway was significantly activated.Western blot also confirmed this conclusion.The apoptosis rate of cells with over-expression of miR-532-3p increased after treated with 5-fluorouracil and cisplatin,suggesting that miR-532-3p can promote cell apoptosis and inhibit the chemotherapeutic resistance of colorectal cancer cells to 5-fluorouracil and cisplatin.3.Overexpression of miR-532-3p slowed down the proliferation of colorectal cancer cells,blocked G1/M phase transformation,and cell cycle-related proteins could also support this conclusion;migration behavior was inhibited,and the detection of EMT marker proteins accorded with the above conclusion.4.MiR-532-3p directly targeted ETS1 and TGM2 and participated in Wnt/?-catenin signaling pathway.ETS1 can directly regulate the expression level of TGM2 as a transcription factor of TGM2.Both of them have the ability to promote the progress of colorectal cancer.5.Recovery experiments showed that over-expression of ETS1 and TGM2 could counteract the inhibitory effect of miR-532-3p on malignant biological behaviors of colorectal cancer.ConclusionThis study confirmed for the first time that the expression of miR-532-3p was low in colorectal adenomas and colorectal tumors,and that the overexpression of miR-532-3p could inhibit the abnormal activation of Wnt/?-catenin signaling pathway by targeting the ETS1/TGM2 axis,thus inhibiting the chemotherapeutic resistance,proliferation and migration of colorectal cancer. |
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