| Objective Malignant proliferation and invasion are one of the causes of recurrence and metastasis of hepatocellular carcinoma,but the specific mechanism is still unclear.miRNA is a small non-coding RNA,with a length of 19 to 22 nucleotides.Through binding with(UTRS)of mRNAs 3 'untranslated region to regulate the transcription or post-transcriptional expression of genes,it plays a role in regulating the expression of genes in many tumors.Previous studies have shown that miR-142 plays an important role in cancer,viral infection,inflammation and immune tolerance,especially low expression and regulating tumorigenesis and development in a variety of cancer,but the role in hepatocellular carcinoma is not clear,the reason for the imbalance of expression is unknown.The purpose of this study is to investigate the difference of expression of miR-142-3p in hepatocellular carcinoma(HCC)and its relationship with the poor prognosis of HCC patients,as well as to analyze the causes of the imbalance from the perspective of epigenetics,and find out the key target genes of miR-142-3p in regulating the occurrence and development of hepatocellular carcinoma,to provide evidence for revealing the mechanism of carcinogenesis and development of HCC and finding new markers for detection and potential therapeutic targets.Method1.The changes of TGF-?1 expression after overexpression of miR-142-3p by qR-TPCR and WB showed that there was a correlation between them.2.The proliferation activity of cells atfter overexpression of miR-142-3p was detected by flow cytometry,the ability of invasion and migration was detected by transwell test,and the epithelial mesenchymal transformation was verified by WB.The anterior angiogenesis was tested and the changes of TGF-?1 and VEGF cytokines in hepatocelluar carcinoma cells were detected by Elisa.3.miR-142-3p promoter methylation was detected by BSP methylation.After adding methylation inhibitor 5-Aza,the proliferation activity of hepatoma cells was detected by flow cytometry,the ability of cell invasion and migration is detected by flow cytometry and is detected by qRT-PCR,and the epithelial mesenchymal transformation was verified by WB.The changes of TGF-?1 and VEGF cytokines were detected by Elisa.Results1.Low expression of miR-142-3p is associated with poor prognosis of hepatocellular carcinoma50 pairs of HCC tissues and their corresponding paracancerous tissues were detected.qRT-PCR was used to detect the expression of miR-142-3p in HCC tissues and paracancerous tissues.The correlation between the expression of miR-142-3p and TGF-?1 and clinical data was analyzed statistically.The target gene of miR-142-3p was predicted by bioinformatics,and the binding relationship between miR-142-3p and target gene TGF-?1 was verified by double luciferase assay.2.TGF-?1 is a direct target of miR-142-3p in HCCThe results showed that TGF-?1 was significantly increased in hepatocellular carcinoma and cell line HepG2,SMMC7721,and negatively correlated with the level of miR-142-3p in clinical specimens.In addition,overexpression of miR-142-3p inhibited the expression of TGF-?1 in HepG 2 and SMMC7721 cells,while miR-142-3p inhibitor could restore the level of TGF-?1 mRNA and protein in the process.The results showed that miR-142-3p mimic could significantly decrease the luciferase activity of wild type TGF-?1-3'UTR,but no significant effect on mutant one.3.miR-142-3p inhibit the progression of HCC via TGF-?1The results showed that the viability of two kinds of hepatoma cells was significantly decreased after transfection of miR-142-3p mimics,inhibited the migration,invasion and secretion of VEGF,TGF-?.of HCC,up-regulated the expression of E-cadherin and decreased the expression of N-cadherin,inhibit tubular angiogenesis,but these results can be recovered by overexpression of TGF-?1.4.Hypermethylation of the Promoter region of miR-142-3p leads to its deletion expression.The results of BSP showed that the degree of miR-142-3p methylation in HCC tissues was significantly higher than that in normal tissues.This similar result was confirmed in HL-7702,HepG2 and SMMC7721 cells.In addition,5-Aza,a methylation inhibitor,inhibited the methylation of miR-142-3p to restore the expression of miR-142-3p in HepG 2 and SMMC 7721 cells,accompanied with decrease of the expression of TGF-?1.5.The methylation inhibitor 5-Aza can restore the expression of miR-142-3p and its anti-tumor effectThe results showed that 5-Aza decreased cell viability and proliferation,inhibited EMT transformation,migration and invasion by restoring the expression of miR-142-3p,weakened the angiogenic ability of miR-142-3p inhibitor,all of these results could reversed by miR-142-3p inhibitor.CONCLUSIONSThe low expression of miR-142-3p,as a tumor suppressor in HCC,may be related to the poor prognosis of HCC.TGF-?1 is the target gene of miR-142-3p and miR-142-3p targeting the TGF-?1 signal axis inhibits the occurrence and development of HCC,including proliferation,migration and invasion,EMT,tumor angiogenesis,secreting cytokines TGF-?1 and VEGF. |
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