MiR-429 Inhibits The Proliferation Of Hepatocellular Carcinoma Cells By Targeting TRAF6 Expression

Objective:MiRNA abnormal expression in a variety of malignant tumors,and the occurrence and development of the tumor is closely related.This study was to detect the expression of miR-429 in hepatocellular carcinoma and its effect on the proliferation and migration of HCC.The expression of TRAF6 and the effect of TRAF6 on the proliferation and migration of HCC were analyzed.The occurrence of liver cancer,the development of the relationship.Methods:1.The expression of miR-429 was detected by Real time PCR in clinical specimens and cell lines.2.The effect of miR-429 on the proliferation of hepatocarcinoma cell line was detected by MTT assay.The effect of miR-429 on the proliferation of hepatocarcinoma cell line was detected by MTT assay.Healing and Transwell test were used to examine the effect of miR-429 on the migration ability of HCC cells.3.The target gene of miR-429 was detected by bioinformatics and luciferase reporter assay.4.The expression of miR-429 was detected by immunofluorescence and Western blot.The core signal pathway of miR-429 was detected by immunofluorescence and Western blot.The expression of miR-429 was detected by immunofluorescence and Western blot.5.The effect of miR-429 on the proliferation of HCC cells was detected by nude mice.Results:1.The expression level of miR-429 in hepatocellular carcinoma was significantly decreased by Real time PCR.The expression of miR-429 in paracancerous tissue was 1.47 times higher than that in normal hepatocellular carcinoma.The expression of miR-429 in hepatocarcinoma cells was down-regulated compared with normal hepatocyte HL7702 by Real time PCR.The expression of SK-HEP1 was the most obvious.2.Compared with miR-CON,miR-429 inhibited the proliferation and growth of hepatocyte line(MTT method).Suggesting that miR-429 can affect the proliferation of liver cancer.MiR-429 inhibits the migration of hepatocarcinoma cell lines.To investigate whether miR-429 affects the ability of hepatocarcinoma cells to migrate,we performed wound healing and Transwell experiments.Compared with the control group,the healing rate of SK-HEP1 transfected with miR-429 decreased to 65.52%.Transwell results were similar: the migration ability of SK-HEP1 transfected with miR-429 decreased to 45.67% compared with the control group.The above results showed that miR-429 had a significant inhibitory effect on the proliferation and migration of tumor cells in hepatocarcinoma cell lines.3.Detection of liver cancer TRAF6 is miR-429 target gene by luciferase reporter gene.Compared with the mutant vector TRAF6-3’UTR-MUT,the fluorescence signal of TRAF6-3’UTR-WT transfected cells was only 0.52 of the control.The value of the fluorescent signal in the transfected TRAF6-3’UTR-WT wild-type cells did not change compared to the blank vector.The above results show that miR-429,in combination with the binding site in the wild-type vector,induces the degradation of the vector and reduces the release of the fluorescent signal.We further examined the effect of miR-429 on TRAF6 protein expression by Western blot.Compared with hepatocarcinoma cells transfected with blank vector,the expression of TRAF6 protein was significantly decreased in miR-429 cells transfected with miR-429 cells.Therefore,the expression of increased miR-429 in hepatocarcinoma cells can effectively reduce the expression of TRAF6 protein.Conclusion: The expression level of TRAF6 in HCC is significantly higher than that in adjacent tissues.The expression of miR-429 in hepatocellular carcinoma was significantly down-regulated,suggesting that miR-429 may be present as a tumor suppressor in hepatocellular carcinoma.The inhibitory effect of miR-429 on the proliferation and invasion of hepatocellular carcinoma cells can be reversed by the high expression of TRAF6.TRAF6 is the target gene for miR-429.4.The expression of TRAF6 protein in hepatocellular carcinoma was higher than that in HCC tissues,but no expression of TRAF6 protein was found in hepatocellular carcinoma(P <0.01).TRAF6 is predominantly cytoplasmic predominant,with only scattered nuclei stained,with strong numbers of strongly expressed cases of 80%(20/25).In order to detect whether the expression of TRAF6 protein can be reversed by miR-429 tumor inhibition.We first transfected the HCCLM3 cell line using siTRAF6.Compared with the control group,the cell proliferation ability and migration ability of the siTRAF6 group were significantly inhibited.However,when the cells were transfected with anti-miR-429,the proliferation and migration ability of the hepatoma cells were significantly inhibited,Compared with the control group,no significant difference.In vitro cell lines,we infected HCC cells with LV-miR-429,and then immunofluorescence detection of P65 expression,the results show that P65 was significantly distributed in the cytoplasm,and by pcDNA-TRAF6 transfection,P65 again gathered in the nucleus Inside.This result suggests that miR-429 plays an important role in the regulation of NF-kB signaling pathways.MiR-429 transfection of HCC cells resulted in decreased expression of pTAK1,cMyc and TRAF6.After transfection of pcDNA-TRAF6,the expression of pTAK1,cMyc and TRAF6 was found to be consistent with that of the control group.The results indicated that TRAF6 expression Can offset the effect of miR-429 on NF-kB signaling pathways in HCC cell lines.The above results show that miR-429 exerts its biological effect through regulation of TRAF6 expression via NF-kB signaling pathway.5.We also used the nude mouse model to detect the effect of miR-429 on the growth of hepatocellular carcinoma in vivo.The results showed that the growth of miR-429 transfected subcutaneously implanted tumors was significantly inhibited compared to the subcutaneous implantation model of control miR-CON transfected cells.After 24 days of tumor cell injection,the mean volume of miR-429 transfected tumor was 53.39% of the tumor volume of the control group.The expression of TRAF6 and P65 was significantly decreased by immunohistochemistry.Therefore,miR-429 transfected hepatoma cells in vivo and in vitro experiments can significantly reduce the growth and proliferation of tumor cells.Conclusions:TRAF6 is a new target of miR-429,miR-429 can negatively regulate multiple factors of TRAF6 and NF-kB pathway,and can inhibit the growth,proliferation and metastasis of hepatoma cells.Therefore,in-depth study of miR-429 in the tumor function and its mechanism,activation of miR-429 or inhibition of TRAF6 expression may become another effective treatment of cancer treatment strategy.