Research On MiR-29c-3p Participates In Hippo Signaling Pathway Through DNMT3B To Regulate The Malignant Biological Behavior Of Hepatocellular Carcinoma

Objective:Hepatocellular carcinoma（HCC）is an important part of primary liver cancer（PLC）,and it is also one of the most common malignant tumors in clinic,causing 788000 deaths every year.At the same time,HCC is also the main cause of cancer death in China.Most of the patients are in the middle and late stage,accompanied by intrahepatic and extrahepatic metastasis,with poor prognosis.At present,there is a lack of effective prevention and treatment measures.The 5-year metastasis and recurrence rate after radical resection is as high as 61.5%.The recurrence and metastasis of HCC is still the main reason for the failure of treatment,which brings heavy burden to the family and society.Importantly,the development of HCC is a complex pathophysiological process involving multiple signaling pathways.Therefore,there is an urgent need to find the key molecular targets and signal pathways for the occurrence and development of HCC,to provide a precise direction for the prevention and control of HCC,and to improve the quality of life of HCC patients.MicroRNA（miRNA）is a kind of single stranded non coding microRNA with length of 20-24nt.miRNAs bind to the target gene’s mRNA transcripts in a sequence specific manner,leading to the degradation or translation inhibition of the corresponding mRNA and regulating the expression of the target gene.The latest research shows that abnormal expression of miRNAs can be found in many diseases.These abnormal expression of miRNAs are believed to be related to the occurrence and development of cancer.A large number of scientific studies have shown that miR-29c-3p,as an important member of miRNAs,plays an important role in the occurrence and development of tumors.However,the expression and role of miR-29c-3p in HCC cells are not clear,and which signaling pathway is involved in the malignant biological behavior of HCC is still unknown.In this study,we will analyze the expression of miR-29c-3p in HCC and the relationship between miR-29c-3p and the clinicopathological characteristics and prognosis of HCC patients,study the role of miR-29c-3p and its target gene in the malignant biological behavior of HCC,and further find the molecular mechanism of miR-29c-3p regulating the malignant biological behavior of HCC through signal pathway.This study will provide a scientific theoretical basis for the accurate diagnosis and treatment of HCCMethods:1.The expression of miR-29c-3p in HCC and its adjacent tissues was detected by real-time quantitative PCR and MiRNA in situ hybridization.Kaplan Meier survival analysis,univariate and multivariate logistic regression analysis were used to analyze the relationship between the expression of miR-29c-3p and the adverse prognosis of patients with HCC.2.The expression of miR-29c-3p in different hepatoma cell lines was detected by real-time quantitative PCR.Pre-miR-29c-3p and miR-29c-3p inhibitor were synthesized to interfere with the expression of miR-29c-3p.The proliferation of HepG2 and MHCC-97H cells was detected by EDU,cell clone and CCK8.The effect of interfering the expression of miR-29c-3p on the migration of HepG2 and MHCC-97H cells was detected by cell scratch test.The DNA methylation level of LATS1 in HepG2 and MHCC-97h cells was detected by methylation specific PCR and bisulfite sequencing.A subcutaneous xenograft model in nude mice was constructed,and the effect of overexpression of miR-29c-3p in HepG2 and MHCC-97H cells was evaluated.3.Using bioinformatics online website to find and verify the downstream target gene of miR-29c-3p.The binding of miR-29c-3p and DNMT3B was confirmed by luciferase labeling.The effect of overexpression of DNMT3B on the proliferation of HepG2-miR-29c-3p and MHCC-97H-miR-29c-3p cells was detected by EDU,cell clone and CCK8.The effect of overexpression of DNMT3B on the migration of HepG2-miR-29c-3p and MHCC-97H-miR-29c-3p cells was detected by cell scratch test.The effect of overexpression of DNMT3B on DNA methylation of LATS1 in HepG2-miR-29c-3p and MHCC-97H-miR-29c-3p cells was detected by methylation specific PCR.Western blot was used to detect the effect of overexpression of DNMT3B on the expression of Hippo signaling pathway.Results:1.The expression of miR-29c-3p was low in 77.6%of HCC tissues,while it was high in 68.9%of matched HCC tissues.Statistical analysis showed that the expression of miR-29c-3p in HCC tissues was significantly lower than that in matched HCC tissues（P<0.05）.Statistical analysis showed that the expression of miR-29c-3p was closely related to tumor size（P=0.018）,tumor number（P=0.011）and intrahepatic metastasis（P=0.035）.The overall survival time of HCC patients with low expression of miR-29c-3p was significantly shortened.Low expression of miR-29c-3p was an independent risk factor for poor prognosis of HCC patients.2.The expression of miR-29c-3p in SMMC-7721,Huh-7,MHCC-97H and HepG2 cells was significantly lower than that of LO2,and the expression of MHCC-97H was the lowest.The results of CCK8 cell proliferation experiment,Edu uptake experiment and clone formation experiment suggested that the effectively increasing the expression of miR-29c-3p,the cell proliferation ability,Edu uptake ability and cell clone formation rate of HCC cells are significantly lower than that of the control group,statistical analysis showed that increasing the expression of miR-29c-3p can significantly inhibit the proliferation of HepG2 and MHCC-97H cells.The results of cell scratch experiments suggested that the effective increase of miR-29c-3p expression in HCC cell scratch width is significantly greater than the control group,statistical analysis shows that increasing the expression of miR-29c-3p can significantly inhibit HepG2and MHCC-97H cells migration ability.The results of animal models suggested that after effectively increasing the expression of miR-29c-3p,the formation and growth of HCC cells in nude mice subcutaneously transplanted tumors was significantly lower than that of the control group,statistical analysis showed that increasing the expression of miR-29c-3p could significantly inhibit the growth of HepG2 and MHCC-97h cells.WB results showed that the effectively increasing the expression of miR-29c-3p,the expression of LATS1,p-YAP^s127,and BAX were significantly higher than those of the control group,while the expression of YAP,BCL-2,and KI-67 were significantly lower than that of the control group,statistical analysis showed that increasing the expression of miR-29c-3p can significantly promote the expression of Hippo signaling pathway in HepG2and MHCC-97H cells.3.Bioinformatics online website and luciferase labeling experiments confirmed that DNMT3B was an effective target gene of miR-29c-3p.The results of methylation specific PCR indicated that the DNA methylation of LATS1 gene was significantly reduced after miR-29c-3p was effectively increased,and the DNA methylation of LATS1 gene was significantly restored and improved after DNMT3B was overexpressed,statistical analysis showed that overexpression of DNMT3B could partially reverse the inhibition of miR-29c-3p on DNA methylation of LATS1 gene in HepG2 and MHCC-97h cells.The results of CCK8 cell proliferation experiment,Edu uptake experiment and clone formation experiment suggested that the cell proliferation ability,Edu uptake ability and cell clone formation rate of HCC cells are significantly higher than the control group after effectively increasing the expression of DNMT3B in overexpression of miR-29c-3p,statistical analysis showed that overexpression of DNMT3B can partially reverse the inhibition of proliferation of HepG2 and MHCC-97H cells by overexpression of miR-29c-3p.The results of cell scratch test indicated that the scratch width of HCC cells was significantly lower than that of the control group after effectively increasing the expression of DNMT3B in overexpression of miR-29c-3p,statistical analysis showed that overexpression of DNMT3B could partially reverse the inhibition of migration of HepG2 and MHCC-97h cells by overexpression of miR-29c-3p.WB results showed that the expression of YAP,Bcl-2,Ki-67 was significantly higher than that of the control group,and the expression of LATS1,p-YAP^s127,Bax was significantly lower than that of the control group after effectively increasing the expression of DNMT3B in overexpression of miR-29c-3p,statistical analysis showed that overexpression of DNMT3B could partially reverse the effect of overexpression of miR-29c-3p on the expression of Hippo signaling pathway in HepG2 and MHCC-97h cells.Immunohistochemical results indicated that the expression of DNMT3B in HCC tumor tissue was significantly higher than that in matched adjacent tissues.The statistical analysis of prognosis showed that the prognosis of patients with high expression of DNMT3B was poor（P<0.05）.Immunohistochemical results indicated that the expression of LATS1 in HCC tumor tissue was significantly lower than that in matched adjacent tissues.The statistical analysis of prognosis showed that the prognosis of patients with low expression of LATS1 was poor（P<0.05）.Conclusions:1.The low expression of miR-29c-3p in HCC patients was significantly correlated with the prognosis of HCC patients,which was an independent risk factor for the prognosis of HCC patients.2.MiR-29c-3p regulates the DNA methylation and protein expression of LATS1 through DNMT3B.3.MiR-29c-3p affects the expression of LATS1 through DNMT3B and participates in the regulation of malignant biological behavior of HCC by Hippo signaling pathway.