Transglutaminase 2 Mediates Stemness And Promote Cell Migration And Invasiveness Of Hepatocellular Carcinoma

Objectives:Hepatocellular carcinoma(HCC)is the fifth most common cancer in the world,and currently ranks third in cancer mortality.The onset of HCC is hidden,the degree of malignancy is high,most patients are found with intrahepatic or extrahepatic metastasis,often miss the best time for surgery and other treatment,the overall treatment effect is still not good.The metastasis of cancer cells is usually related to the existence of tumor stem cell(CSCs).Transglutaminase 2(Tissue transglutaminase,TG2)is a member of the transglutaminase family,which exists widely in cells.Studies have shown that TG2 is related to stem cell characteristics,metastasis,diffusion and drug resistance of tumor.The expression of TG2 in HCC was determined by detecting the expression of TG2 in different differentiated HCC tissues and different metastatic potential HCC cell lines.After silencing TG2,the effects of TG2 on CSCs markers and metastatic ability of hepatoma cells were detected to explore the mechanism that TG2 regulates the downstream pathway to affect the metastasis of hepatocellular carcinoma cells by binding to integrinβ1(ITGB1).Materials and Methods:1.26 cases of HCC and its adjacent tissues with the same degree of differentiation were selected.according to the degree of differentiation of HCC,they were divided into low differentiation group(n=15),high differentiation group(n=11)and corresponding paracancerous tissues(n=5).The expression of TG2 in HCC and paracancerous tissues was detected by immunohistochemical method,and the results were compared among different groups and analyzed by one-way ANOVA.2.HCC-LM3(highly metastatic human hepatoma cell line),SMMC-7721(l human hepatoma cell line)and LO2(human hepatocyte cell line)with different migration potential were selected,and the expression level of TG2 in different migration potential HCC cell lines was detected by RT-PCR and Western blot.According to the relative expression level of TG2,the cell model of gene silencing and overexpression was selected.3.HCC-LM3 cells with high expression of TG2 were selected and silenced TG2(shTG2)was transfected with lentivirus.The unloaded virus was used as blank control group(NC).SMMC-7721 cells with low expression of TG2 were selected and overexpressed TG2(OE-TG2)was transfected with plasmid(short hairpin RNA,shRNA,and the empty plasmid was used as blank control group(OE-NC).The levels of CSCs markers CD133,CD44,SOX2 and OCT4、CD133~+of sh-TG2 and OE-TG2 in liver cancer were detected by q-PCR,and compared with those of NC and OE-NC.Scratch and transwell experiments were used to verify the effect of TG2 on migration and invasion.The regulation of downstream ITGB1-mediated pathway by shTG2 and OE-TG2 was detected by Western Blot.Results:1.The expression levels of TG2 were different in well-differentiated and poorly differentiated HCC tissues and corresponding paracancerous tissues.The expression level of TG2 in poorly differentiated HCC tissues was the highest,followed by well differentiated HCC tissues,and almost no expression in paracancerous tissues.The difference was statistically significant(P<0.05).2.The expression level of TG2 was different in different metastatic potential cell lines.HCC-LM3(highly metastatic human hepatoma cells)is higher than SMMC-7721(low metastatic human hepatoma cells),but almost no expression in LO2(normal hepatocytes).3.The levels of CD133,CD44,SOX2,OCT4 and the population of CD133~+cells in sh-TG2 group were significantly lower than those in NC group,while the levels of CD133,CD44,SOX2 and the population of CD133~+cells in OE-TG2 group were significantly higher than those in OE-NC group.4.The ability of migration and invasion in sh-TG2 group was weaker than that in NC group,and the expression of ITGB1 decreased with the decrease of TG2,but on the contrary in OE-TG2 group.Conclusion:1.The expression of TG2 is related to the differentiation degree of HCC,and is highly expressed in poorly differentiated HCC tissues and high migration potential HCC cell lines,relatively low in well differentiated HCC tissues and low migration potential cell lines,and not expressed in paracancerous tissues and human liver cell lines.2.TG2 can mediate HCC cancer stemness.3.TG2 may affect the invasion and metastasis of HCC by combining with ITGB1 to regulate the downstream pathway.