MiR-130a-3p Regulates Of Smad4 To Mediate Epithelial-mesenchymal Transition In Gemcitabine-resistant Hepatocellular Carcinoma Cells

Background and objective:Hepatocellular carcinoma is one of the common malignant tumors in China,and its mortality rate is the second of the cancer death rate in our country.In the newly issued HCC each year,China occupies 53.5% of the proportion,and the death rate is as high as 0.93.Obviously,China is a big country and hit the country in HCC.HCC onset occult,it is difficult to detect early,about 80% of patients have lost the chance of radical resection of the diagnosis,even after surgical resection of hepatocellular carcinoma is still prone to recurrence or metastasis.Therefore,the treatment of locally advanced or metastatic HCC that cannot be cured by radical resection has been a difficult problem that needs to be overcome and solved in clinical work.New cytotoxic drugs such as oxaliplatin,(OXA)and gemcitabine(GEM)in the treatment of HCC shows a certain prospect,but the primary or acquired resistance is still the main cause of chemotherapy failure.In order to reveal the mechanism of drug resistance in HCC and to explore the mechanism of drug resistance drugs,our research team in the pre constructed GEM multidrug-resistant hepatocellular carcinoma cell model(HepG2 GEM/10.0 and SMMC-7721 GEM/10.0.The following referred to as HepG2 GR and SMMC-7721 GR ",collectively referred to as" HCC GR ").It was found that HCC GR cell occurred between epithelial like transformation(Epithelial-to-mesenchymal transition,EMT).Further study showed that small RNAs,microRNAs(miRNAs)are involved in the occurrence of HCC cell GR EMT.It was found that HCC GR cell occurred between epithelial like transformation(Epithelial-to-mesenchymal transition,EMT).Further study showed that small RNAs,microRNAs(miRNAs)are involved in the occurrence of HCC cell GR EMT.In the past we use miRNA microarray to screen for differentially expressed miRNAs in HepG2 parents and HepG2 GR cell(hereinafter referred to as "HCC GR"),by bioinformatics analysis,we find that the and EMT molecular phenotype related transcription factors may be mir-130a-3p target genes.In view of the previous research results,we put forward "MiR-130a-3p".Regulation and expression of EMT related transcription factors involved in the academic hypothesis of GEM resistant hepatocellular carcinoma cells EMT.This study proposed by QRT PCR,Western blot and reporter gene,siRNA interference and cell biological analysis method,the effect of mir-130a-3p of target genes and gem resistant cell EMT phenotype,the analysis of the relationship between target gene and gem resistant cell EMT phenotype.In this study,we have revealed the mechanism of drug resistance of GEM in hepatocellular carcinoma cells,and it also provides a strategy to overcome the GEM drug resistance of HCC.Methods: 1.Bioinformatics analysis,in the past miRNA microarray technology to identify the differences in miRNAs identified in GEM resistant hepatocellular carcinoma cells may participate in the key miRNA EMTMiRNA prediction software(DIANAmT;miRanda;miRWalk;PICTAR4;Targetscan),determine the possible involvement of GEM resistant hepatocellular carcinoma cells miR-130a-3p EMT,and predict the possible downstream target gene Smand4.2.The correlation between miR-130a-3p and target gene Smad4 expression was investigated in the parental and drug resistant cells of liver cancer.Between hepatocellular carcinoma of the parental and resistant cells,expression of QRT PCR was used to determination of mir-130a-3p revealed mir-130a-3p in GEM resistant cells are extensively downregulated,respectively by QRT PCR and Western blot methods.Determination of the expression of target genes,Smad4.To analyze the correlation between the expression of mir-130a-3p and target genes.3.The effects of miR-130a-3p on the expression of target genes and the biological characteristics of EMT were investigated in the parental and resistant cells of liver cancer.1)The effect of transient silencing of miR-130a-3p on the expression of target gene was investigated in the parental cells of hepatocellular carcinoma.2)The effect of transient overexpression of miR-130a-3p on the expression of target gene and the biological characteristics of EMT cells was investigated.3)To construct a target gene transcription of the human full-length 3 'UTR(wild type)luciferase reporting system,to investigate whether the direct identification of miR-130a-3p target gene transcription 3' UTR to regulate its expression.4)In multidrug-resistant hepatocellular carcinoma cell,transfection mir-130a-3p simulation and negative control by morphological observation,migration / invasion analysis,scratch test,cell adhesion and detachment experiments and investigation of mir-130a-3p overexpression effects on the biological characteristics of resistant cell EMT.5)Effect of MTT assay on GEM drug sensitivity of hepatocellular carcinoma cells transfected with miR-130a-3p and negative control.4.Effects of miR-130a-3p target gene on phenotype and biological characteristics of hepatocellular carcinoma cell line EMT1)In multidrug-resistant hepatocellular carcinoma cell,transfection of target gene Smad4 siRNA transient silencing Smad4 expression in QRT PCR and Western blot were determined by target gene transcription regulation of EMT related target gene expression(E-caherin,vimentin and MMP2).2)In multidrug-resistant hepatocellular carcinoma cell,transfection of target gene Smad4 siRNA to transient silencing Smad4 expression by morphological observation,migration / invasion analysis,scratch test,cell adhesion and detachment experiments.The influence of target gene Smad4 transient silencing on the biological characteristics of resistant cell EMT.Results: 1.MiR-130a-3p in HCC GR cells in a wide cut,in HCC GR Smad4 cells are up,and the expression of miR-130a-3p reverse regulation Smad4Relative to HCC parental cells,miR-130a-3p was widely down regulated in GR HCC cells,while Smad4 was highly expressed in GR HCC.After the HCC parental cells were transfected with miR-130a-3p,the Smad4 expression was up-regulated;GR HCC cells were transfected with miR-130a-3p analog,and the expression of Smad4 was down regulated.2.Expression of miR-130a-3p targeting regulated Smad4 affected the biological characteristics of GEM resistant hepatocellular carcinoma cell line EMTLuciferase reporter assay demonstrated that miR-130a-3p can directly identify Smad4 transcripts in the 3 'UTR region.miR-130a-3p cells transfected with HCC GR simulation,cell motility and adhesion separation ability decreased,migration and invasion ability decreased.3.Downregulation of Smad4 expression was able to reverse the EMT molecular phenotype of GEM resistant hepatocellular carcinoma cellsHCC GR cells transfected with target gene siRNA Smad4,Smad4 expression was significantly down regulated,accompanied by a reversal of the EMT molecular phenotype: epithelial marker E-caherin expression was up-regulated,and the expression of Vimentin and MMP-2 was down regulated.4.Downregulation of Smad4 expression and the biological characteristics of GEM resistant hepatocellular carcinoma cell line EMTHCC GR cells transfected with target gene Smad4 siRNA,cell adhesion separation ability is weakened,cell migration and invasion ability decreased.Conclusion: 1.miR-130a-3p target Smad4 gene influence the biological characteristics of EMT in gemcitabine resistance of hepatoma cells.2.Down-regulation of Smad4 reverses EMT to MET in HCC GR cells.Smad4 mediated-EMT on the phenotype and molecular biological characteristics through the downstream gene of EMT in gemcitabine resistance of hepatoma cells.