| Objectives:Among the cases of primary liver cancer,the incidence of hepatocellular carcinoma（HCC）is high.The progression of HCC is a multi-step process involving multi-factor etiology and multi-gene alternation.Transmembrane protein 206（TMEM206）,also known as proton-activated chloride channel（PAC）,is a transmembrane protein.Many transmembrane proteins affect the migration and invasion of various malignant tumors and other malignant behaviors.However,whether TMEM206 plays a role in hepatocellular carcinoma,and what role it plays have not yet been studied.Therefore,this experiment aims to explore the expression of TMEM206 in liver cancer tissues and cells,its influence on the proliferation,migration and invasion of liver cancer cells and its potential molecular mechanisms.Methods: 1.The hepatocellular carcinoma patient data was download through the TCGA（The Cancer Genome Atlas,TCGA）database and the human protein atlas（THPA）online tool,evaluate the expression of TMEM206 in hepatocellular carcinoma tissues,and analyze clinicopathological characteristics.The univariate and multivariate Cox regression analysis was carried out to evaluate whether TMEM206 can be used as a potential biomarker,draw a survival curve to evaluate the effect of TMEM206 on the prognosis of patients with hepatocellular carcinoma,and perform GSEA（Gene Set Enrichment Analysis）enrichment analysis to find out the effect of TMEM206 on the prognosis of patients with hepatocellular carcinoma.2.The expression of TMEM206 in normal liver cells and liver cancer cells was detected by RT-q PCR and Western blot.Construct the TMEM206 lentiviral knockdown model,and detect the expression of TMEM206 in the cells by fluorescence observation,RT-q PCR and Western blot to ensure the success of the TMEM206 lentiviral knockdown model.3.CCK-8（Cell Counting Kit-8）and plate clone formation experiment was used to detect the effect of TMEM206 on the proliferation of hepatocellular carcinoma cells from the cell phenotype.Using the scratch test,Transwell migration and invasion test,the effect of TMEM206 on the migration and invasion ability of hepatocellular carcinoma was detected from the cell phenotype.RT-q PCR and Western blot were used to detect the expression levels of MMP2（matrix-metalloproteinase 2）,MMP9（matrixmetalloproteinase 9）,E-CAD（E-cadherin）and N-CAD（N-cadherin）,and the expression levels were in transcription and translation.Phase verification of the effect of TMEM206 on the migration and invasion ability of hepatocellular carcinoma.5.The effect of TMEM206 on the expression of PI3 K,AKT,p-PI3 K and p-AKT in liver cancer cells was detected by immunofluorescence,and RT-q PCR and Western blot was used to detect the expression of PI3 K,AKT,p-PI3 K and p-AKT,to verify the effect of TMEM206 on PI3K/AKT pathway in liver cancer cells.Results: 1.Download 374 cases of hepatocellular carcinoma tissues and 50 cases of normal tissues adjacent to hepatocellular carcinoma from the TCGA-STAD database,a total of 424 samples for analysis,and download immunohistochemical staining maps of hepatocellular carcinoma tissues and adjacent tissues from THPA.The results showed that TMEM206 was significantly overexpressed in hepatocellular carcinoma tissues.Analysis of the clinical and pathological characteristics of the samples found that the expression of TMEM206 has nothing to do with the age and gender of HCC patients,but is significantly related to the grade and staging of the tumor.Univariate and multivariate Cox regression analysis showed that TMEM206 can be used as a potential biomarker for liver cancer patients.2.At the cellular level,the expression of TMEM206 m RNA and protein in normal liver cell line HL-7702 and three liver cancer cell lines Hep G2,SMMC-7721,and Bel-7402 was detected.The results showed that the expression of TMEM206 in the three liver cancer cell lines was higher than that of normal liver cells.Among them,the expression of TMEM206 was the highest in the Hep G2 cell line.3.After lentiviral transfection and selection of stable cell lines,the green fluorescence was observed through an inverted microscope and it was found that the lentiviral transfection was successful.The results of RT-q PCR and Western blot showed that the expression of TMEM206 in the Hep G2 cell line of the experimental group was significantly knocked down,and the TMEM206 knockdown cell model was successfully constructed.4.The results of CCK-8 proliferation experiment and plate clone formation experiment showed that knockdown of TMEM206 can inhibit the proliferation and clone formation of Hep G2.Scratch experiments,Transwell migration and invasion experiments showed that knockdown of TMEM206 can inhibit the migration and invasion of liver cancer cells Hep G2.At the same time,the expression level of MMP2 and N-CAD decreased,and the expression level of E-CAD increased.5.The results of immunofluorescence showed that knockdown of TMEM206 significantly inhibited the expression of p-PI3 K and p-AKT,and RT-q PCR and Western blot also showed the same results.Conclusion: TMEM206 is highly expressed in hepatocellular carcinoma tissues and cells,and is related to the poor prognosis of HCC patients,and may be a biomarker of HCC.TMEM206 can act on the PI3K/AKT pathway to promote the proliferation,migration and invasion of HCC cells. |
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