Synergistic Effect And Mechanism Of MicroRNA-100 Inactivation In HBx-induced Malignant Transformation Of Hepatocytes

[ Backgrounds]Hepatocellular tumor is the common malignant tumor disease,which seriously affects human health.About 90% of hepatocellular tumor is hepatocellular carcinoma(HCC).The occurrence of hepatocellular carcinoma is a complicated process,which is caused by environmental factors and genetic factors.Hepatitis B virus(HBV),hepatitis C virus(HCV),alcohol,aflatoxin B1 and other factors are involved in liver cell injury,which induces abnormal necrosis and proliferation of liver cells.Physical and chemical factors can also stimulate somatic cells resulting to inactivation of tumor suppressor genes and the activation of proto-oncogenes,synergistically inducing the malignant transformation of hepatocytes,which will eventually lead to chronic liver disease and cirrhosis.The end stage of the malignant transformation of hepatocytes is hepatocellular carcinoma(HCC).In China,the most cause of liver cancer is hepatitis B virus infection.The proportion of people infected with hepatitis B virus in China has exceeded 8%,the number is about112 million.Hepatitis B virus is a circular double-stranded DNA molecule.HBx is one of the four open reading frames in the hepatitis B virus genome.The encoded HBx protein is a transactivator.HBx protein is a key protein that HBV promotes the malignant transformation of hepatocytes.It can activate the Notch,PI3 K / m TOR,and Wnt / ?-catenin pathways,affect non-coding RNAs(lnc RNAs and miRNAs),regulate downstream signals,change Epigenetics(methylation and acetylation),affect DNA repair,oxidative stress and change immune system to cause malignant transformation of normal liver cells and promote the occurrence and development of hepatocellular carcinoma.Micro RNA(micro RNA,miRNA)is a type of endogenous non-coding single-stranded RNA that plays an important role.It encodes about 19-25 nucleotides in length and binds to the target gene by binding to the 3'-UTR of the target gene.Mi RNAs promote the malignant transformation of normal cells by regulating the transcription of intracellular proteins and other methods.Our previous research found that miR-100 inactivation can synergistically enhance the heavy metal pollutant arsenic promoting the occurrence of malignant transformation of normal lung epithelial cell lines through the activation of epithelial-mesenchymal transition of cells.Other studies have found that miR-34 a can kill non-small cell lung cancer tumor cells by cooperating with EGFR tyrosine kinase inhibitors.In our study,we also found that miR-100 inactivated in HL02 cells in vitro can promote malignant biological functions,leading to malignant transformation of normal cells.Based on the above background,our topic was devoted to to investigating whether miR-100 inactivation has a synergistic effect in HBx-induced malignant transformation of hepatocytes,which can lead to the occurrence of liver cancer,and to explore its possible mechanism.Therefore,we transfected HBx lentivirus into human normal liver cells(HL02)and HL02 with miR-100 inactivation to achieve stable cell lines.By observing the biological functions and tumor formation in nude mice of cells treated by different intervention factors to verify the synergistic effect of miR-100 inactivation on HBx-induced malignant transformation of liver cells,and further studies were conducted on the possible signaling pathways and key molecules.[Research methods]1.HL02 and miR-100 inactivated HL02 cells were transfected with HBx lentivirus to select HBx overexpression,miR-100 inactivated and HBx co-transfected cell lines.1.Through MTT,flat colony assay,migration and invasion assay and soft agar colony assay to explore the effects of biological behavior changes of normal hepatocytes HL02 treated with different intervention factors.3.Nude mouse tumor formation experiments were performed to explore the ability of tumorigenesis in nude mice of normal hepatocytes HL02 with different intervention factors.4.Western blot was used to detect changes in ERK / AKT / m TOR signaling pathway,and RT-PCR was used to detect the expression of cytokines.[Results](1)Successfully achieved the HL02 cells with HBx overexpression and HL02 cells with miR-100 inactivation and HBx overexpression co-transfected.(2)HBx promoted the proliferation,migration,invasion,colony forming ability of HL02 cells,and promoted the malignant transformation of HL02 cells.(3)The miR-100 inactivation synergistically promoted the proliferation,migration,invasion,colony-forming ability,and tumor-forming ability of nude mice of HBx over-expressed HL02 cells.(4)The miR-100 inactivation synergistically promoted ERK / AKT / m TOR activation and affected the expression of IL-6 of HL02 with HBx overexpression.[Conclusion]Mi R-100 inactivation plays a synergistic role in HBx-induced malignant transformation of hepatocytes.