Chronic Alcohol Exposure Regulates CTCs Through MiR-22-3p/ TET2 To Promote Metastasis Of HCC

[Backgrounds] Hepatocellular carcinoma((HCC))is one of the most common cancers in the world,and it is also the main cause of cancer death.About 841000 patients worldwide are diagnosed with hepatocellular carcinoma(HCC)every year.Hepatocellular carcinoma(HCC)is the third leading cause of cancer-related death in China due to underlying liver disease,advanced diagnosis and limited treatment.Liver cancer has a high degree of malignancy and rapid progress.Recurrence and metastasis of liver cancer is one of the main reasons for the failure of treatment.Epidemiological statistics show that alcohol is one of the important risk factors in the occurrence and development of liver cancer.A large number of previous studies mainly focused on the effect of oxidative stress of acetaldehyde,the main product of alcohol metabolism on the disease.However,more and more studies have shown that alcohol metabolism affects the methylation and demethylation of DNA.DNA methylation refers to the catalysis of cytosine to 5-methylcytosine(5m C)in the presence of methyltransferase.Aberrant methylation of DNA,an important epigenetic mechanism of transcriptional regulation,is closely related to the occurrence and development of tumors.The TET(ten-eleven translocation)protein family is a group of DNA demethylases that can regulate the epigenetic response,and convert 5-methylcytosine(5m C)to 5-hydroxymethylcytosine(5hm C),which is a necessary stage of DNA demethylation.The previous study found that chronic alcohol exposure can cause a variety of mi RNAs expression differences.Micro RNAs(mi RNAs)is a kind of endogenous non-coding small RNAs,which plays an important role in the regulation of gene expression.Under certain conditions,mi RNA can be used as both oncogene and tumor suppressor.Studies have shown that abnormal expression of mi RNA can affect various characteristics of cancer,such as maintaining proliferation signals,escaping growth inhibitory factors,resisting cell death,activating invasion and metastasis,and inducing angiogenesis.The orthotopic transplantation model of liver cancer will be used to explore the role of alcohol in the progression of liver cancer.The possible mechanisms may be discussed through western blot and q-PCR.And then reveal the possible mechanism of alcohol-induced liver cancer metastasis.[Methods](1)Through lentivirus transfection and puromycin screening,cell lines stably expressing green fluorescent protein were obtained.(2)In order to examine the effect of chronic alcohol stress on liver cancer progression,we constructed an orthotopic liver cancer transplantation model in mice.(3)By magnetic bead sorting and fluorescence microscopy counting,the number of circulating tumor cells in the peripheral blood of nude mice at five and six weeks of drinking was detected.(4)H&E staining was used to observe the number and size of metastases on lung tissue in orthotopic transplantation model of nude mice.(5)The expression of TET2 protein in tumor tissue was detected by IHC.(6)At the cellular level,the protein expression of TET2 was detected by western blot,and the expression of mi R-22-3p was detected by q-PCR.(7)The expression level of TET2 in tumor tissue was detected by western blot,and the expression level of mi R-22-3p in tumor tissue was detected by q-PCR[Results](1)Successfully constructed stable transfected cell line of HCCLM3/GFP.(2)Chronic alcohol exposure promotes the proliferation of liver tumor,increasing the tumor volume and the number of metastases on the surface of the lung in alcohol group.(3)Chronic alcohol exposure increased the number of circulating tumor cells in peripheral blood.(4)H&E test found that in the alcohol group the number of metastases was more,large,and infiltrated with more tumor cells,and alveolar tissue areas formed large cancer nests.(5)Immunohistochemical results showed that chronic alcohol exposure decreased the expression of TET2 in hepatocellular carcinoma.(6)At the cellular level,the expression level of TET2 decreased and the expression level of mi RNA-22-3p increased after chronic alcohol exposure at different periods,related to the exposure time.(7)After chronic alcohol,low expression of TET2 and high expression of mi RNA-22-3p in liver tumor tissues.[Conclusion] Chronic alcohol exposure promotes the metastasis of liver cancer via mi R-22-3p/TET2 regulation of circulating tumor cells.