The Mechanism Of IL-6/STAT3/SNAIL Pathway Mediating Epithelial-mesenchymal Transitionin In Recurrence Of Liver Cancer After Radiofrequency Ablation

Background and Objective:Hepatocellular carcinoma(HCC)is the fifth most common cancer in the world wild,and its incidence is increasing in recent years,which is a serious threat to human health.It is reported that there is not significantly difference of 5-year survival rate between raidofrequency ablation(RFA)and liver resection when the tumor's diameter is less than 5cm..However,in recent years,more and more articles have reported the recurrence and progression of liver cancer after radiofrequency ablation.And epithelial-mesenchymal transition(EMT)is thought playing a key role in these phenomenons..Based on these backgrounds mentioned above,we aim to investigate the mechanism of the recurrence of liver cancer after radiofrequency ablation by establishing models in vitro and in vivo..Methods:1.Submerged H22 cells under water bath at the temperature 45? for 5min.The expression of E-cadherin,Vimentin,Snail and p-STAT3 are monitored by Western Blot and RT-PCR and the expression of IL-6 is assessed by Elisa.2.H22 cells were treated with IL-6 for 4h and the expression of EMT markers and p-STAT3 were detected by Western Blot.H22 cells were both treated with IL-6 and heat treatment and detected the expression of EMT markers and p-STAT3 by Western Blot.3.H22 cells were incubated with specific inhibitor of JAK/STAT3 signaling AG490 for 4h and detected these markers mentioned previously.H22 cells were both treated with AG490 and heat treatment and detected the expression of EMT markers and p-STAT3 by Western Blot.4.We established ten orthotopic transplantated mouse model.After Subcutaneous injected 1×x106H22 cells in the Right hind limb for 1 week,we collected the tumor and chopped it with a diameter of 1mm and transplantated it under the liver capsule.One week later,we selected five mouse randomly as experiment group to receive insufficient radiofrequency ablation at 45? for 5 min and the others named control group.We sacrificed all these ten mouse and recorded the condition of both mouse and tumors.We collected the tumor of transplanted and recurrent respectively to compare the variation of EMT bio-markers E-cadherin?Vimentin?snail and p-STAT3 by Western Blot and Immunohistochemistry.Results:1.We found the expression of E-cadherin decreased obviously.Vimentin and Snail increased clearly by exposed to 45? for 5min.2.The expression of IL-6 detected by Elisa showed that the heat treatment can promote the secretion of IL-6.And treated cells with IL-6 induced the the phosphorylation of STAT3 and down-regulated E-cadherin and up-regulated Vimentin and snail meanwhile.What's more,IL-6 treated combined with heat treatment could enhance EMT.3.After treated cells with AG490 for 4h,the change of EMT markers did not variate obviously as well as combined with heat treatment.And AG490 treated combined with heat treatment could suppress the occurrence of EMT.4.We established ten mouse model of orthotopic transplantation and the success rate is 100%.After received insufficient RFA for 1 week,we observed the abdomen was protuberant in all the mouse of experiment gorup.There was a new lesion at the edge of RFA area.The results of Western Blot and IHC showed us we the expression of E-cadherin decreased obviously,the expression of Vimentin?Snail and p-STAT3 increased clearly.Conclusions:The insufficient ablation performed by low temperature can successfully induce EMT in vitro and in our mouse model by regulating the expression of IL-6,p-STAT3 and snail and low temperature may cooperate with IL-6 in promoting the recurrence of liver cancer.And we also verified blocked this signaling pathway by AG490 could reverse EMT-like change in vitro,which may be a potential treatment combined with RFA in liver cancer.