HIF-1α Promotes Residual Hepatocellular Carcinoma Progression After Insufficient Thermalablation By Regulating Transketolase

Objective:Hepatocellular carcinoma(HCC)is one of the most common digestive tract tumors in the world,ranking sixth in the incidence of cancer in the world,and posing a great threat to people’s life,health and property safety.Radiofrequency ablation(RFA)is a safe and effective treatment,especially in HCC smaller than 3cm in diameter,with an overall curative effect comparable to surgical hepatectomy.However,due to the differences in energy distribution during ablation,irregular shape,inaccurate edges of tumors,and other factors,cancer cells often remain at the edges of tumors after ablation.Meanwhile,due to the destruction of blood supply,the residual tumor cells after insufficient ablation were in a hypoxic microenvironment.Local and distant recurrence,enhanced invasiveness and treatment resistance are common in residual tumors after insufficient ablation.In this study,in vitro insufficient ablation model was constructed to explore the changes in tumor characteristics of residual liver cancer cells after incomplete ablation and to preliminarily investigate the regulatory effect of Hypoxia-inducible factor-1α(HIF-1α)and Trasketolase(TKT)on residual HCC cells after insufficient ablation and its related mechanism.MethodsHuman hepatocellular carcinoma cell lines HCC-LM3(LM3)and MHCC-97H(97H)were selected for this study.The thermal effect of insufficient ablation and the local hypoxic microenvironment caused by insufficient ablation were simulated in vitro to establish the model of incomplete ablation of HCC.Short-hairpin RNA(sh RNA)-based lentiviral approach was used to knockdown HIF-1α and TKT.The changes of cell proliferation,migration,invasion,and chemotherapy resistance due to silence of HIF-1α and TKT were observed by CCK8 and Transwell assays.The protein and m RNA expression of target gene were detected by Western Blot and qRTPCR.ChIP-q PCR,Western-Blot and qRT-PCR were used to detect whether HIF-1αcould bind to the promoter sequence of TKT.Dual luciferase assay was used to detect whether HIF-1α enhanced the promoter activity of TKT.Results(1)Firstly,to evaluate the changes in the characteristics of residual HCC cells after incomplete ablation,in vitro insufficient ablation model was realized by simulating the thermal effect of insufficient ablation and the local hypoxic microenvironment.The results of CCK-8 assays showed that compared with that of untreated control group,insufficient ablation not only enhanced the proliferation ability,but also increased chemotherapy resistance of residual HCC cells.Transwell assays showed that the invasion and migration ability of residual liver cancer cells was also significantly enhanced after insufficient ablation.Western-Blot and qRT-PCR were used to detect the changes in protein and m RNA expression of EMT-related indicators.The results showed that compared with untreated control group,the expression of mesenchymal related markers(Snail,Slug,N-cadherin,MMP-9,Vimentin)was significantly up-regulated and the expression of E-cadherin and Claudin-1 was markedly down-regulated in residual HCC cells after insufficient ablation.Western Blot assay showed that HIF-1α expression was significantly up-regulated in residual HCC cells after insufficient ablation.(2)The cell lines with HIF-1α stable knockdown were constructed by short-hairpin RNA(sh RNA)-based lentiviral approach and the knock-down efficiency was detected by Western-Blot.CCK-8 assay showed that downregulation of HIF-1α expression reduced the proliferation ability and increased sensitivity to chemotherapy.The results of Transwell assays showed that downregulation of HIF-1α expression significantly reduced the invasion ability of residual HCC cells after insufficient ablation.Western Blot and qRT-PCR showed that the downregulation of HIF-1α expression significantly increased the expression of E-cadherin and Claudin-1 and decreased the expression of mesenchymal related markers(Snail,Slug,N-cadherin,MMP-9,Vimentin)in residual HCC cells after insufficient ablation.(3)Reactive oxygen species(ROS)detection showed that knockdown of HIF-1αsignificantly increased ROS levels in residual HCC cells after insufficient ablation.Meanwhile,the ratio of NADPH/NADP+ and GSH/GSSG decreased in residual HCC cells after insufficient ablation due to knockdown of HIF-1α.Results of qRT-PCR showed the expression of several antioxidant-related genes in residual HCC cells after insufficient ablation was significantly down-regulated after HIF-1α knockdown.Among them,TKT was the most significant.(4)The online bioinformatics website Jaspar predicted HIF-1α has four binding sites on the TKT promoter.The results of ChIP-q PCR and Dual luciferase assay showed that HIF-1α could bind to the binding site on the TKT promoter.(5)Western Blot and qRT-PCR were used to detect the efficiency of TKT knockdown in residual HCC cells after insufficient ablation.CCK-8 assays showed that TKT knockdown decreased the proliferation ability and increased sensitivity to chemotherapy.The results of Transwell assays showed that the invasion ability of residual HCC cells after insufficient ablation was significantly weakened by TKT knockdown.Western Blot assay also showed that the PI3K/AKT signaling pathway of residual HCC cells after insufficient ablation was significantly down-regulated after TKT knockdown.Conclusion:(1)Insufficient ablation promotes invasion,migration,proliferation,and resistance to chemotherapy of residual hepatocellular carcinoma cells(2)HIF-1α plays an important role in the progression of residual liver cancer after insufficient ablation and can regulate the expression of TKT.(3)TKT promotes the invasion,migration,proliferation,and chemotherapy resistance of residual HCC cells after insufficient ablation by regulating PI3K/AKT pathway.