Mir-29c-3p Targets Girdin And Induced Cell Apoptosis In Colorectal Cancer

Background:Colorectal cancer is one of the common malignancies.With the changes in economic development and eating habits,the incidence of colorectal cancer in China is increasing year by year,and the current surgical and chemotherapy methods can not achieve good prognosis results.Colorectal cancer has become a major problem in public health in China,and it is of great significance to explore new targets for the prevention and treatment of colorectal cancer.Girdin is a novel actin-binding protein that is abnormally expressed in various tumor tissues,and its expression level is related to clinicopathological features,tumor proliferation and invasion.MicroRNAs(miRNAs)are a unique group of small non-coding RNAs that play an important role in regulating gene expression by binding to the 3' untranslated region(UTR)of the target mRNA.Studies have shown that miRNAs can affect tumor proliferation,apoptosis and migration.This study focused on the relationship between Girdin expression levels and apoptosis in colon cancer cells,and the regulation of Girdin by miR-29c-3p.Method:First,we used qPCR,Western blot,and immunohistochemical techniques to observe the difference of Girdin expression between human colorectal cancer and corresponding paracancerous tissues,and to analyze the relationship between Girdin expression level and prognosis of colorectal cancer patients according to the GEO database.Then,we constructed a lentiviral vector that overexpresses and inhibits Girdin expression,and transfected colon cancer cell lines interfere with Girdin expression.CCK-8 and plate clone formation were used to observe the effect of Girdin expression level on cell proliferation,and apoptosis of colon cancer cells was detected by AnnexinV/7AAD flow cytometry,DCFDA fluorescent probe and JC-1 fluorescent probe.Apoptosis-related cell activity,Western blot analysis of Girdin and expression of apoptosis-related proteins such as Bax,Bcl-2 and caspase-3 in colon cancer cells.Finally,we analyzed the difference in expression of miR-29c-3p between colorectal cancer and normal tissues by TCGA data,and then used qPCR to detect the expression level of miR-29c-3p in human normal colon epithelial cells and colon cancer cells.The dual luciferase assay verified whether miR-29c-3p binds to the 3'-UTR of Girdin to regulate Girdin.qPCR and Western blot were used to further detect the regulation of Girdin mRNA and protein expression by miR-29c-3p.Result:1.The results of qPCR,Western blot and immunohistochemistry showed that the mRNA and protein content of Girdin in human colorectal cancer tissues were significantly higher than those in adjacent tissues.The GEO dataset GSE24551 showed that patients with low expression of Girdin had significantly higher expected survival rates than those with high expression.2.The results of CCK-8 and plate clone formation showed that overexpression of Girdin could significantly increase the proliferation of colon cancer cells in vitro,and inhibition of Girdin expression could inhibit the proliferation of colon cancer cells.The results of flow cytometry of AnnexinV/7AAD suggest that the inhibition of Girdin expression in colon cancer cells can induce apoptosis.The results of DCFDA fluorescent probe and JC-1 fluorescent probe suggest that inhibition of Girdin expression increases ROS levels and mitochondrial membrane potential in colon cancer cells,and the above pro-apoptotic behavior can be reduced by broad-spectrum caspase inhibitors.Western blot showed that the ratio of caspase-3,caspase-9 and Bax/Bcl-2 was significantly up-regulated in Girdin.3.TCGA database analysis and q-PCR assay showed that the expression of miR-29c-3p in colorectal cancer tissues was significantly lower than that in normal tissues.The dual luciferase reporter showed that miR-29c-3p binds to Girdin 3'-UTR.q-PCR and Western blot further confirmed that miR-29c-3p can regulate Girdin mRNA and protein expression in colorectal cancer cells.Conclusion:Inhibition of Girdin inhibits the proliferation of colon cancer cells and promotes apoptosis.Girdin may be a marker for the prevention and treatment of colorectal cancer and an effective new target.