The Role And Mechanism Research Of MiRNA-345 Expression In Hepatocellular Carcinoma

BackgroundHepatocellular carcinoma ranks third in global cancer-related deaths(1),known as "the king of cancer".Although the treatment has improved in recent years,but the incidence of cancer and mortality rate is still increasing year by year and the prognosis is not ideal.The central cause of high mortality in hepatocellular carcinoma is the metastasis and recurrence of hepatocellular carcinoma,and due to its exact mechanism remains unclear,there is still a lack of effective means of prevention and treatment.Therefore,the study of the molecular mechanism of liver cancer metastasis and take effective prevention and treatment measures is to improve the efficacy of patients with liver cancer,liver cancer patients to improve the prognosis of the breakthrough.MircoRNAs(miRNAs)are endogenous non-coding single-stranded small molecule RNAs that are 20-23 bases long and widely present in eukaryotes.They are linked to the 3'-untranslated region of the target gene m RNA(3'UTR)interaction to regulate the level of its target gene after transcription,and thus participate in cell proliferation,differentiation,apoptosis,metabolism and other processes.Abnormal expression of miRNAs and a variety of diseases,including malignant tumors are closely related to the occurrence and development of the tumor play a role in canceror tumor suppressor gene.Recently,the relationship between tumor invasion and metastasis and miRNAs has been paid more and more attention.By regulating the expression of genes related to tumor cell adhesion,matrix degradation,epithelial mesenchymal transformation(EMT),angiogenesis and other related genes Play a role in many metastases associated with miRNAs molecules have been identified.Compared with normal samples,miRNA-345 was highly expressed in malignant mesothelioma(5).In addition,the increased expression of miRNA-345 may play a key role in oral malignant transformation(6).miRNA-345 was associated with cisplatin resistance of MCF-7 by targeting multidrug resistance-associated protein 1(MRP1)(7).In colorectal cancer,low expression of miRNA-345 conferred lymph node metastasis and worse histological type,and its recovery inhibits cancer cell proliferation and invasion(8,9).In addition,miRNA-345 expression was down-regulated in non-small cell lung cancer(NSCLC),and its low expression was associated with malignant clinical parameters and poor prognosis(10).It is reported that the absence of miRNA-345 confers resistance to pancreatic cancer(PC)cell apoptosis(11),miRNA-345 inhibits proliferation and migration of prostate cancer cells by inhibiting Smad1(12).Rare studies have reported the correlation between miRNA-345 and HCC.Shiu et al.Reported that hepatitis C virus(HCV)core protein up-regulates miRNA-345 expression,miRNA-345 inhibits curcumin-induced apoptosis by targeting p21 in Huh7 cells(Jiang et al),Jiang et al.The high survival rate of HCC patients with high miRNA-345 expression levels compared with those with poor survival rates(14).Therefore,it is worth studying the biological effects of miRNA-345 and its underlying mechanisms in HCC.But does miRNA-345 participate in the invasion and metastasis of liver cancer?The role of miRNA-345 in the invasion and metastasis of HCC is achieved by IRT1-mediated mTOR / STAT3 / AKT pathway inhibition of EMT? Is interferon targeting miRNA-345 effectively reversing the metastatic phenotype of hepatocellular carcinoma cells? Therefore,miRNA-345 in the role of liver cancer development mechanism with the need for in-depth study.AimTo investigate the inhibitory effect of miRNA-345 on hepatocellular carcinoma metastasis and its molecular mechanism,And to provide a theoretical basis for the application of miRNA in the treatment of liver cancer.Methods1.The expression of miRNA-345 in hepatocellular carcinoma tissues and adjacent tissues was detected by Real-time PCR.The expression of miRNA-345 was analyzed with the clinicopathological features and prognosis of patients with hepatocellular carcinoma(HCC)and corresponding tissues relationship.The hepatoma cell lines SMMC-7721,HepG2 and normal liver cell line L02 were cultured with different invasion and metastasis potentials.QRT-PCR The expression of miRNA-345 was analyzed and the relationship between the expression of miRNA-345 and the migration and invasion of hepatoma cells was analyzed.2.To observe the effect of miRNA-345 on the biological characteristics of hepatocarcinoma cells: To investigate the effect of miRNA-345 mimi(HmiR0210-MR04)and inhibitor(HmiR-AN0437-AM04)on liver cancer cells,respectively,and to overexpress and inhibit hepatocellular carcinoma(MTT method),migration(Transwell experiment),invasion(Matrigel test)and other biological behavior of the cells were observed in the level of miRNA-345 in the cells.3.The miRNA-345 overexpressing lentiviral vector was constructed and the miRNA-345 overexpressing vector was transfected into hepatocarcinoma cells.The miRNA-345 overexpressing miRNA-345 was injected into the nude mice through the tail vein,The effect of miRNA-345 on the invasion and metastasis of HCC was observed in vivo.Construction of miRNA-345 overexpressing lentiviral vector and miRNA-345 +IFR1 overexpression lentiviral vector were transfected into hepatocellular carcinoma cells to regulate the expression of miRNA-345 and IFR1 respectively.The biological behavior of miRNA-345 and IFR1 was different To investigate the effect ofmiRNA-345 on IFT1-mediated mTOR / STAT3 / AKT pathway on EMT and metastasis of liver cancer.Results1 In hepatocellular carcinoma tissue and liver cancer cells,the positive rate of miRNA-345 expression was significantly lower than that in adjacent normal tissues and normal liver cells(P<0.05),with statistical significance.The expression of miR-345 was negatively correlated with multiple tumor lymph nodes,venous invasion and advanced stage of tumor lymph node metastasis(TNM)in patients with hepatocellular carcinoma(P<0.05).2 ChIP detection of histone H3 and H4 acetylation levels in the miRNA-345 promoter region in hepatocellular carcinoma and hepatocellular carcinoma cells using ChIP technique revealed acetylation of histone H3 in the miRNA-345 promoter region in healthy liver and normal liver cells The level was significantly higher than that in hepatocellular carcinoma and hepatocellular carcinoma cells,P<0.05,with statistical significance;while there was no obvious change in the acetylation of H4.The level of acetylation of histone H3 in the miRNA-345 promoter region is positively correlated with the expression of miRNA-345.3 The expression of IFR1 mRNA in healthy liver tissues was significantly lower than that in HCC tissues(P<0.05),which was statistically significant.The data from liver cancer cells showed that miR-345 overexpression significantly down-regulated IRF1expression(P<0.05),with statistical significance.By Spearman rank correlation analysis,miR-345 expression level was negatively correlated with IRF1 mRNA level in hepatoma cells(r =-0.415,P<0.05).4 Compared with normal liver tissue,miRNA-345 expression in SMMC-7721 cells transfected with HmiR0210-MR04 was significantly increased,P<0.05,with statistical significance.The expression of mTOR,STAT3 and AKT protein in mTOR /STAT3 / AKT pathway was significantly lower than that in normal liver tissue by Western blot,P<0.05,with statistical difference.The expression of E-cadherin,Claudin-1,N-cadherin and snail in EMT-related protein in normal liver tissue wassignificantly higher than that in normal liver tissue,the expression of Claudin-1,N-cadherin and snail protein were decreased Statistical differences.Overexpression of miR-345 has an inhibitory effect on mTOR / STAT3 / AKT signaling pathway and EMT.5 The expression of miRNA-345 in HepG2 cells transfected with HmiR-AN0437-AM04 was significantly lower than that in normal liver tissue(P<0.05).The protein expression of mTOR,STAT3 and AKT in mTOR / STAT3 /AKT pathway was significantly higher than that in normal liver tissue by Western blot(P<0.05).The expression of E-cadherin,Claudin-1,N-cadherin and snail in EMT-related protein were significantly lower than those in normal liver tissue,the expression of Claudin-1,N-cadherin and snail protein were increased Statistical differences.Interfering miR-345 expression can inhibit the mTOR / STAT3 / AKT signaling pathway and EMT.6 Rapamycin treatment of SMMC-7721 inhibition of mTOR / STAT3 / AKT pathway,the expression of mTOR,STAT3,AKT was significantly reduced,P<0.05,with statistical differences.Western blot showed that the expression of E-cadherin,Claudin-1,N-cadherin and snail protein were significantly decreased,while the expression of Claudin-1,N-cadherin and snail proteins were increased Statistical differences.The mTOR / STAT3 / AKT signaling pathway has a positive influence on the EMT process of hepatoma cells and promotes the invasion and metastasis of hepatocellular carcinoma cells.7 IFR1 can relieve the inhibitory effect of miRNA-345 on the expression of mTOR/ STAT3 / AKT pathway protein.On the promotion of hepatocellular tumor invasion.8 Transfected with empty vector SMMC-7721 cells after the mice were infected,the tumor volume daily growth was significantly greater than infected transfected miR0210-MR04 cell line SMMC-7721,the tumor volume was significantly different,p <0.05,with statistical significance.Lung metastasis was significantly higher in nude mice infected with SMMC-7721 cells transfected with miR0210-MR04,with a significant difference of P<0.05.The results show that the expression of miR-345 has inhibitory effect on tumor growth and lung metastasis in mice.conclusion miRNA-345 inhibits the invasion and metastasis of hepatocellular carcinoma cells by inhibiting IRT1-mediated mTOR / STAT3 / AKT pathway-induced EMT.The results of this study have a high clinical value for the prevention and metastasis of liver cancer.