| Background:Hepatocellular carcinoma(HCC)is a common malignant tumor of digestive system,accounting for 80%～90%of primary hepatic cancer.In China,primary liver cancer remains the fifth most prevalent tumor and the second most lethal tumor,which seriously threatens people’s life and health.Because liver cancer is mostly diagnosed at the late stage,the prognosis of liver cancer is extremely poor.In the past decade,liver resection and liver transplantation have been the main treatment methods for HCC.Additionally,immunotherapy has also begun to put into clinical trials and treatment for HCC,but the 5-year survival rate for advanced hepatic cancer is still less than 10%.Therefore,the study of the development mechanism of HCC can provide a new effective target and theoretical basis for the clinical treatment of HCC.Sohlh2(Spermatogenesis and oogenesis basic helix-loop-helix transcription factor 2)is one of the important members of bhlh transcription factor 2.It could bind to the conserved Ebox sequence of target gene promoter region,regulate target gene expression,and participate in the regulation of cell apoptosis,differentiation,proliferation and other biological behaviors.Our previous experimental results had confirmed that fatty liver and liver fibrosis could occur spontaneously in Sohlh2 knock-in mice.And Sohlh2 knock-in mice had more serious acute liver injury after injection of hepatocellular carcinoma inducer DEN.Bioinformatics analysis and preliminary experimental results showed that the expression of Sohlh2 in HCC cells was significantly up-regulated.These results suggest that Sohlh2 may play an important role in the occurrence and development of liver cancer.Autophagy is a conservative cellular pathway involving in the degradation of cytoplasmic macromolecules,aggregated proteins,damaged organelles,or pathogens.Autophagy plays an important role in the occurrence and development of a variety of tumors,including liver cancer.When autophagy is activated,Beclinl catalyzes the formation of the autophagic membrane,and then LC3b-Ⅰ catalyzes the formation of LC3b-Ⅱ on the autophagic membrane.LC3b-Ⅱ could bind to p62,then move into the autolysosome stage to complete autophagy.Autophagy promotes the metabolism,stress resistance,invasion,drug resistance and stemness of tumor cells,thereby promoting the development of tumors.TGM2(Transglutaminase type 2)increases in various inflammatory,fibrotic and neoplastic tissues.TGM2 could interact with p62 through its UBA domain to promote autophagy.Preliminary experimental results showed that the Sohlh2 could raise TGM2 expression and promote the autophagyin HCC cells.Therefore,we presumed that Sohlh2 may promote the progression of HCC by regulating TGM2-mediated autophagy.Methods:1.To analyze the expression of Sohlh2 in liver cancer tissues of patientsBioinformatics analysis using TCGA database was performed to analyze the difference of Sohlh2 expression in liver cancer tissues and adjacent tissues,as well as the correlation between Sohlh2 expression and prognosis of liver cancer.Immunohistochemical staining was performed on liver cancer clinical tissue chips to analyze the difference of Sohlh2 expression between cancer and adjacent tissues.2.To investigate the effects of Sohlh2 on the proliferation,EMT,migration and invasion of HCC cellsHuman HCC cell lines HepG2,Huh7 and snu387 were cultured in DMEM or 1640 medium with 10%FBS.Sohlh2 overexpression or Sohlh2 knockdown stable cell lines were prepared by lentivirus transfection.CCK-8 proliferation assay,colony formation assay,Transwell migration and invasion assays were perfomed to detect the effect of Sohlh2 on the proliferation,migration and invasion of HCC cells.q-PCR and Western-blot were used to detect the effect of Sohlh2 on the expression of EMT markers E-cadherin,ZO-1,N-cadherin and Vimentin in HCC cells.3.To investigate the effect of Sohlh2 on autophagy in HCC cellsAutophagy of HCC cells was induced by EBSS.Subsequently,q-PCR,Western-blot and immunofluorescence staining were used to detect the effect of Sohlh2 on the expression of autophagy markers p62,Beclinl and LC3b.Transmission electron microscopy and LC3 doublelabeled lentivirus system were used to analyze the effect of Sohlh2 on autophagy in HCC cells.HCC cells were treated with autophagy inhibitor 3-MA,and CCK-8 proliferation assay,Transwell migration and invasion assays were performed to detect whether Sohlh2 regulated the proliferation,migration and invasion of HCC cells via an autophagy-dependent way.4.To investigate the effect of Sohlh2 on subcutaneous tumor formation and metastasis of HCC cells in nude miceTen male nude mice were randomly divided into two groups,and each mouse was subcutaneously injected with 3×106 the control or Sohlh2 overexpression HepG2 cells.The volume of subcutaneous tumor was measured every two days,after four days of the injection,and growth curve was drawn ultimately.Four weeks after injection,the subcutaneous tumors of nude mice were collected.Then,q-PCR,Western-blot and immunohistochemical staining were performed to study the effects of Sohlh2 on autophagy and proliferation of HCC cells in subcutaneous tumors.Ten male nude mice were randomly divided into two groups,and 5×105 the control or Sohlh2 overexpression HepG2 were injected respectively through tail vein of mice.HE staining and statistical analysis were implemented to measure the effect of Sohlh2 on tumor metastasis 5.To explore whether Sohlh2 promotes autophagy by regulating the expression of TGM2q-PCR,Western-blot,immunohistochemistry and immunofluorescence staining of Sohlh2 overexpression HCC cells and subcutaneous tumors were utilized to dectect the effect of Sohlh2 on TGM2 expression.ChIP and dual double luciferase reporter assay were used to analyze whether Sohlh2 directly regulated the expresssion of TGM2.TGM2 RNAi was transiently transfected into Sohlh2 overexpression HepG2 cells,then q-PCR,Western-blot,CCK-8 proliferation assay and Transwell migration and invasion assays were used to investigate whether TGM2 mediated the regulation of Sohlh2 on autophagy,proliferation,migration and invasion of HCC cells.6.To demonstrate the correlation between Sohlh2 and TGM2 in liver cancer clinical samplesUsing liver cancer clinical tissue microarray,immunohistochemical staining was performed to analyze the correlation between the expression of Sohlh2 and TGM2.Results:1.Sohlh2 is highly expressed in liver cancer tissuesBioinformatics analysis of TCGA database showed that the expression of Sohlh2 in liver cancer tissues was higher than that in adjacent tissues,and the overall survival of liver cancer patients with high Sohlh2 expression was shorter than that of liver cancer patients with low Sohlh2 expression,suggesting that Sohlh2 can be used as an independent prognostic indicator for liver cancer.Immunohistochemistry staining of liver cancer clinical sample chip showed that Sohlh2 was highly expressed in liver cancer tissues compared with adjacent tissues.2.Sohlh2 promotes proliferation,EMT,migration and invasion of HCC cellsThe results of CCK-8 proliferation assay,colony formation assay and Transwell migration and invasion assays showed that Sohlh2 overexpression could significantly promote the proliferation of HCC cells,and increase the number of colony sphere formation.Besides,migrating and invading cells of Sohlh2 overexpression group were notably higher than those of the control group.On the contrary,Sohlh2 knockdown could significantly reduce the proliferation ability of HCC cells,and decrease the number of colony sphere formation,migration and invasion of HCC cells.The results of q-PCR and Western-blot showed that Sohlh2 overexpression in HCC cells down-regulated the expression of epithelial markers Ecadherin and ZO-1,and up-regulated the expression of mesenchymal markers N-cadherin and Vimentin.And Sohlh2 knockdown resulted in the opposite.3.Sohlh2 promotes autophagy in HCC cellsEBSS was used to induce autophagy in HCC cells.The results of q-PCR,Western-blot,and immunofluorescence staining showed that Sohlh2 overexpression increased the mRNA and protein levels of Beclinl,decreased the mRNA and protein levels of p62,and promoted the transformation of LC3b-Ⅰ to LC3b-Ⅱ in HCC cells.And Sohlh2 knockdown reduced the mRNA and protein content of Beclinl,increased the mRNA and protein level of p62,and inhibited the conversion of LC3b-Ⅰ to LC3b-Ⅱ in HCC cells.Under the transmission electron microscopy,it was observed that the autophagosome was increased in Sohlh2 overexpression HCC cells,and decreased in Sohlh2 knockdown HCC cells.LC3 double-labeled lentivirus system results showed that autophagosome increased and autophagy was activated after Sohlh2 overexpression in HCC cells.3-MA was used to inhibit the autophagy in Sohlh2 overexpression HCC cells.The results of CCK-8 proliferation assay and Transwell migration and invasion assays showed that the proliferation rate of 3-MA+Sohlh2 group was slowed down,and the number of migrating and invading cells of 3-MA+Sohlh2 group was reduced compared with the Sohlh2 group.These results suggested that autophagy mediated the regulation of Sohlh2 on the proliferation,migration and invasion of HCC cells.4.Sohlh2 enhances subcutaneous tumor growth and metastasis of HCC cells in nude miceThe results of subcutaneous tumor formation experiment in nude mice showed that Sohlh2 overexpression promoted the subcutaneous tumor growth of HCC cells.The growth speed and weight of tumor in Sohlh2 overexpression group were much more than the control group.The results of q-PCR and Western-blot showed that S-ohlh2 overexpression up-regulated the mRNA and protein expression of Beclinl,down-regulated the mRNA and protein expression of p62,and elevated the transformation of LC3b-I to LC3b-II.Immunohistochemical staining results showed that the positive rate of Ki67 in Sohlh2 overexpression group were significantly higher than those in the control group.HE staining results showed that the number of tumor metastases in the lung and liver of the Sohlh2 overexpression group was higher compared with the control group.5.Sohlh2 regulates autophagy and promotes the development of liver cancer through upregulation of TGM2 expressionThe results of q-PCR,Western-blot,immunohistochemical and immunofluorescence staining showed that Sohlh2 overexpression increased TGM2 expression in HCC cells,while Sohlh2 knockdown decreased TGM2 expression.ChIP and double luciferase reporter assay showed that Sohlh2 could bind to the promoter region of TGM2 to enhance its transcriptional activity and promote TGM2 expression.After TGM2 RNAi was transiently transfected into Sohlh2 overexpression HCC cells to slient TGM2,the results of q-PCR and Western-blot showed that TGM2 knockdown increased the expression of p62 by Sohlh2 overexpression.LC3 double-labeled lentivirus system results showed that autophagosome decreased and autophagy was inhivited after silencing TGM2 in Sohlh2 overexpression HCC cells.Simultaneously,the results of CCK-8 proliferation experiment and Transwell migration and invasion assays showed the cell proliferation,and the number of migrating and invading cells decreased in the Sohlh2+siTGM2 group compared with the Sohlh2 group.These results indicated that TGM2 could mediate Sohlh2 regulation on autophagy,proliferation,migration and invasion of HCC cells.6.Sohlh2 is positively correlated with TGM2 expression in the liver cancer clinical samples,The results of immunohistochemistry staining and Spermann correlation analysis showed that the expression of Sohlh2 was positively correlated with the expression of TGM2 in liver cancer tissues(r=0.442,P=0.004).Concusion:1.Sohlh2 enhances the proliferation,migration and invasion of HCC cells and promotes the development of HCC.2.Sohlh2 promotes autophagy by up-regulating the expression of TGM2,thereby promoting HCC. |
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