Study On The Molecular Mechanism Of Signal Transducer And Activator Of Transcription 3 Aggravating Epithelial-to- Mesenchymal Transition Of Hepatoma Carcinoma Cell And Promoting Progression Of Hepatoma Carcinoma

Background and objectives:Occurrence,development,invasion and metastasis of tumor are closely related to epithelial-to-mesenchymal transition(EMT).Aberrant activation of Interleukin-6(IL-6)/Janus protein tyrosinekinase(JAK)/signal transducer and activator of transcription 3(STAT3)signaling pathway promoted the development of tumor by inducing epithelialto-mesenchymal transition(EMT)of tumor cells,but this phenomenon is rarely studied on the incidence of Hepatocellular carcinoma(HCC).The aim of this experiment is to investigate the underlying molecular mechanisms of EMT in hepatoma carcinoma cells and interaction of IL-6/JAK/STAT3 and Sekelsky mothers against d PP 3(Smad3)/TGF-?1 signaling pathways during EMT process.Methods:We cultivated human hepatic cancer cell lines(Hep G2,Bel7402,MHCC97 H,HCCLM3)in vitro.We chose the hepatic cancer cell lines(Hep G2,HCCLM3)for our study based on the protein expression level of phosphorylated Signal transducer and activator of transcription 3(p-STAT3).The hepatic cancer cell lines(Hep G2,HCCLM3)were treated with TGF-?1,AG490,IL-6 or combination of TGF-?1 and AG490,IL-6,respectively.The messenger Ribonucleic Acid(m RNA)expression level of Zinc finger transcriptional factor(Snail)was detected by quantitative real time polymerase chain reaction(q RTPCR).The protein expression levels of p-STAT3/STAT3,phosphorylated Sekelsky mothers against d PP 3(p-Smad3)/Smad3 and EMT related markers(Snail,E-Cadherin,Vimentin)were observed by Western Blotting.Results:The hepatic cancer cell line Hep G2 was incubated with TGF-?1(0,2,5,10ng/ml)for 24 hours,firstly,we observed Hep G2 cells gradually lost the original polarity,transformed from circular or oval(epithelial phenotype)into fusiform or typical fibroblast phenotype(mesecchymal phenotype).With the incubation concentration of TGF-?1 increasing,the change of fibroblast phenotype is also more obvious,the effect was most significant with TGF-?1 10ng/ml.Secondly,we extracted the protein of Hep G2 cells,we found that the protein level of E-Cadherin was decreased,while the protein level of Vimentin,Snail,p-STAT3 was increased,which were positively correlated with the doses of TGF-?1.Then we incubated hepatic cancer cell line Hep G2 cells with TGF-?1 10ng/ml for different time(0,0.5,1,2,4,8h)and extracted the protein of Hep G2 cells,we found that the protein levels of p-STAT3,Snail,p-Smad2,p-Smad3 were elevated,which were positively correlated with stimulation time.It indicated that TGF-?1 could induce occurrence of EMT in Hep G2,the effect was most significant with TGF-?1 10ng/ml for 8h.The hepatic cancer cell lines Hep G2,HCCLM3 were incubated with IL-6(0,50,100ng/ml)for 1 hour,we extracted the protein of Hep G2,HCCLM3 cells,we found that the protein level of p-STAT3 was increased,which was positively correlated with the doses of IL-6.Then we incubated the hepatic cancer cell lines(Hep G2,HCCLM3)with TGF-?1 5ng/ml,IL-6 50ng/ml or combination of TGF-?1 5ng/ml and IL-6 50ng/ml,respectively,firstly,we extracted the m RNA of Hep G2,HCCLM3 cells,we found that compared with group TGF-?1,the m RNA expression level of Snail in group IL-6+TGF-?1 was significantly elevated(TGF-?1+IL-6 versus TGF-?1: Hep G2 p=0.003;HCCLM3 p=0.008).Secondly,we extracted the protein of Hep G2,HCCLM3 cells,we found that compared with group IL-6 or TGF-?1,the expression levels of protein p-Smad3 and Snail in group IL-6+TGF-?1 were significantly upregulated.Compared to group IL-6 or TGF-?1,the expression level of protein E-Cadherin in group IL-6+TGF-?1 was lower,but Vimentin was higher,which indicated that the effection of IL-6+TGF-?1 on EMT progression in Hep G2 was more obvious than group IL-6 or TGF-?1.AG490 is a JAK2-specific inhibitor.The hepatic cancer cell lines Hep G2,HCCLM3 were incubated with AG490(0,50,100 u M)for 4 hour,we extracted the protein of Hep G2,HCCLM3 cells,we found that the protein level of p-STAT3 was discreased, which was positively correlated with the doses of AG490.Then we incubated the hepatic cancer cell lines(Hep G2,HCCLM3)with TGF-?1 5ng/ml,AG490 50 u M or combination of TGF-?1 5ng/ml and AG490 50 u M,respectively,firstly,we extracted the m RNA of Hep G2,HCCLM3 cells,we found that compared with group TGF-?1,the m RNA expression level of Snail in group AG490+TGF-?1 was significantly reduced(AG490+TGF-?1 vs TGF-?1: Hep G2 p=0.013;HCCLM3 p=0.022).Secondly,we extracted the protein of Hep G2,HCCLM3 cells,we found that compared with group TGF-?1,the expression levels of protein p-Smad3 and Snail in group AG490+TGF-?1 were significantly downregulated.Compared to group IL-6 or TGF-?1,the expression level of protein E-Cadherin in group IL-6+TGF-?1 was higher,but Vimentin was lower,which implied that AG490 inhibited the occurrence of EMT induced by TGF-?1 in hepatoma carcinoma cells.Conclusion:STAT3 as a positive regulator played a vital role in aggravating EMT induced by TGF-?1.STAT3 signaling transduction can aggravate EMT induced by TGF-?1 in hepatoma carcinoma cells,sequentially promote the progression of hepatocellular carcinoma.