MicroRNA-346 Regulates Proliferation,Migration And Invasion Of Hepatocellular Carcinoma By Targeting BRMS1

BackgroundHepatocellular carcinoma(HCC)is one of the most common human malignancies.With the popularity of serum AFP detection,the progress of imaging examination technology and the improvement of people’s health awareness,the diagnosis rate of early-stage liver cancer in recent years has been significantly improved.Although surgical techniques and minimally invasive techniques are increasingly mature and widely promoted,the overall prognosis of liver cancer is not ideal,this is closely related to the invasive and metastatic ability of liver cancer.MicroRNA(miRNA)is an endogenous non-coding RNA with regulatory function of about 20-25 nucleotides.More and more studies have shown that miRNAs are closely related to the occurrence,metastasis,invasion and prognosis of hepatocellular carcinoma.miR-346 plays an oncogene function in many kinds of tumors,but miR-346 is rarely reported in the development of liver cancer.We first examined the expression level of miR-346 in hepatocellular carcinoma tissues and hepatocellular carcinoma cells,and analyzed the relationship between the clinicopathological characteristics and prognosis of hepatocellular carcinoma and the effect on the biological function of hepatoma cells.Then,we predicted and verified in vitro and in vivo that miR-346 promoted the proliferation,migration and invasion of hepatocellular carcinoma cells and reduced the apoptosis of hepatocellular carcinoma cells by inhibiting the expression of BRMS1.It provided a new target for the treatment of hepatocellular carcinoma.Part1 Expression and biological function of miR-346 inhepatocellular carcinomaPurposeTo study the expression of miR-346 in hepatocellular carcinoma and its correlation with clinicopathological features and prognosis,and to analyze the effect of miR-346 on the biological behavior of HCC cell lines.Methods1.The specimens were collected.Real-time PCR was used to detect the expression of miR-346 in hepatocellular carcinoma(HCC)and paired paracancer tissues.The correlation between the expression of miR-346 and clinicopathological features and prognosis was also analyzed.2.Real-time PCR was used to detect the expression of miR-346 in five HCC cell lines and human normal liver epithelial cells.3.Up-regulation or down-regulation of miR-346,analysis of abnormal expression of miR-346 on the biological behavior of HCC cell lines.Results1.The expression level of miR-346 in HCC was higher than that in matched paracancer tissues,and correlated with TNM stage(P = 0.038),tumor size(P = 0.028)and metastasis(P = 0.037).There was no significant correlation with age,sex and AFP(P>0.05).2.The expression level of miR-346 in TNM stage Ⅲ and Ⅳ hepatocellular carcinoma patients was higher than that in stage Ⅰ and Ⅱ.In patients with liver cancer more than 5cm was higher than that in no more than 5cm.In patients with metastasis was higher than that without metastasis,and survival time of patients with high expression of miR-346 was significantly shorter than that with low expression of miR-346.3.Overexpression of miR-346 promoted the proliferation,migration and invasion of HCC cells and reduced the apoptosis.4.Low expression of miR-346 inhibited the proliferation,migration and invasion of HCC cells and promoted cell apoptosis.5.miR-346 had no effect on the cell cycle.Part2 Research on the relationship between miR-346 and BRMS1PurposeBioinformatics software predicted target gene of miR-346,analyzed the relationship between target gene and clinicopathological features and prognosis of HCC patients,cytology experiments analyzed the effects of target gene on the biological behavior of HCC cell lines.Methods1.The online prediction of miR-346 target gene by bioinformatics software Targetscan and Pictar.2.Detected the expression of BRMS1 at mRNA and protein levels in HCC cell lines up-regulation or down-regulation miR-346.3.Dual luciferase reporter assay confirmed that miR-346 negatively regulates the expression of BRMS1.4.Real-time PCR was used to detect the expression of BRMS1 in hepatocellular carcinoma tissues and adjacent tissues.5.The expression of BRMS1 protein in hepatocellular carcinoma tissues and paracancerous tissues was analyzed by tissue microarray immunohistochemical staining.Then,we analyzed the relationship between the expression of BRMS1 and clinicopathological features and prognosis of hepatocellular carcinoma patients.6.The expression of BRMS1 was up-regulated by the expression vector pcDNA3.1-BRMS1 in HCC cells,and the effect of BRMS1 overexpression on the biological behavior of HCC cells was analyzed.7.Co-transfected with miR-346 inhibitors and siBRMS1 to analyze whether the interference of BRMS1 expression could reverse the inhibitory effect of miR-346 inhibitors on the biological behavior of HCC cells.Results1.In HCC cell lines,miR-346 was able to target and inhibit the expression of BRMS1.2.Real-time PCR detection BRMS1 mRNA expression levels in 40 pairs liver cancer tissues and paired paracancerous tissues,the results found that BRMS1 expression levels were significantly lower in HCC tissues than that in adjacent tissues(P<0.05).3.Tissue chip immunohistochemical staining further validated the PCR results at protein level.According to the immunohistochemistry scores,patients were divided into BRMS1 low expression group and high expression group,and their relationship with clinicopathological features and prognosis were analyzed.From the statistical analysis,we found that BRMS1 expression level was correlated with TNM stage(P=0.009)and metastasis(P=0.005),but there was no significant difference between age(P=0.875),gender(P=0.628),AFP(P=0.369)and tumor size(P=0.093).In addition,the overall survival of HCC patients with low BRMS1 expression was shorter.4.Overexpression of BRMS1 inhibited the proliferation,migration and invasion of HCC cells and promoted apoptosis.This was consistent with miR-346 inhibitors results.5.Interference of BRMS1 expression could reverse the inhibitory effect of miR-346 inhibitors on the biological behavior of HCC cells.Part3 The mechanism research on miR-346 in nude micetumor formationPurposeThrough nude mouse tumorigenesis experiment,miR-346 was further verified in vivo to regulate the expression of BRMS1 to promote the proliferation of HCC cells and inhibit the apoptosis of HCC cells.Methods1.SMMC-7721 cells overexpression of miR-346 and negative control SMMC-7721 cells were injected subcutaneously into nude mice to observe the tumorigenesis of nude mice.2.Small animal live imaging system dynamic observation of tumor size changes.3.Vernier caliper measures the size of the tumors,and drawed a line map of the volume of the tumor.4.The expression of BRMS1 and caspase-3 in the tumor were detected by immunohistochemistry.The protein expression of BRMS1,PCNA,Bcl-xL and caspase-3 were detected by Western blot.5.The expression level of miR-346 and BRMS1 mRNA in the tumor were detected by RT-PCR,and the correlation was analyzed.Results1.Overexpression of miR-346 could promote the growth of xenografts in nude mice.2.Immunohistochemistry:The expression levels of BRMS1 and caspase-3 were decreased in the miR-346 overexpression group.Western blot:In the miR-346 overexpression group,the expression of BRMS1 protein decreased,the expression of PCNA protein increased,the expression of Bcl-xL protein increased,and the expression of caspase-3 protein decreased.3.The level of miR-346 in the tumor was negatively correlated with the expression level of BRMS1 mRNA.4.BRMS1 was a downstream target gene of miR-346.miR-346 promoted the proliferation of HCC cells and inhibited the apoptosis of HCC cells by targeting the expression of BRMS1.Full text conclusion1.The expression of miR-346 in hepatocellular carcinoma tissues was significantly increased,which was correlated with TNM staging,tumor size and metastasis in patients with hepatocellular carcinoma,and the high expression of miR-346 was associated with shorter overall survival.It can be seen that miR-346 played a role of oncogene in the development of liver cancer.2.BRMS1 was low expressed in HCC tissues,and the low expression level of BRMS1 was associated with poor prognosis in patients with liver cancer.It suggested that BRMS1 play a role of tumor suppressor gene in the development of liver cancer.3.In vitro and in vivo experiments confirmed that BRMS1 was a direct target of miR-346.MiR-346 participated in the progress of liver cancer through negative regulation of BRMS1.4.MiR-346/BRMS1 regulatory axis played an important role in the development of hepatocellular carcinoma.It was expected to become a new target for the treatment of liver cancer.