HIF-2α Regulates Proliferation,Invasion,and Metastasis Of Hepatocellular Carcinoma Cells Via VEGF/Notch1 Signaling Axis After Insufficient Radiofrequency Ablation

Background:Hepatocellular carcinoma(HCC)has a high degree of malignancy,rapid development,and difficult early diagnosis.It is the third leading cause of cancer death in the world,among which China’s new cases and death cases rank first in the world.In recent years,in addition to hepatectomy and liver transplantation,local treatment has become one of the essential treatment methods for HCC,especially radiofrequency ablation(RFA)treatment,which has become an indispensable means for HCC treatment due to its advantages of accurate curative effect,convenient operation,minor trauma to patients and short hospital stay.However,it has been reported that due to the tumor’s size,location,and proximity,in addition to certain surgical complications,insufficient ablation will lead to local tumor recurrence,metastasis,and even rapid disease deterioration in a short time.Insufficient ablation leads to accelerated tumor recurrence and metastasis,which is the biggest bottleneck affecting the efficacy of radiofrequency ablation of liver cancer.Based on previous research and literature review,we speculate that HIF-2α may play an essential role in the recurrence of HCC after insufficient radiofrequency ablation.This study aims to explore the relevant factors causing insufficient ablation of HCC and the possible causes and mechanisms of inducing tumor recurrence by reviewing and analyzing the clinical data of radiofrequency ablation of hepatocellular carcinoma patients and the pathological samples of insufficient ablation in our center.Constructing an in vitro cell model provides a new idea for clinical prevention and treatment of HCC recurrence after insufficient ablation and a theoretical basis for developing specific targeted drugs.Methods:Firstly,we reviewed the data of patients admitted to the Department of Hepatobiliary and Pancreatic Surgery of the Affiliated Hospital of Guangdong Medical University for radiofrequency ablation of HCC between 2018.6.1 and 2021.6.30 to assess the possible factors associated with insufficient ablation;the expression of HIF-2α,VEGF,and Notchl in resected specimens of patients with insufficient ablation were detected by tissue immunohistochemical staining.An in vitro cell model of insufficient ablation was constructed using MHCC97H human hepatocellular carcinoma cell line.The HIF-2a inhibitor PT2385 was used to interfere with the HIF-2a pathway.The effects of HIF-2α inhibition on the viability,migration,and invasion ability of insuficient RFA cells were observed using the MTT method scratch assay and Transwell assay.HIF-2α,VEGF,and Notch1 mRNA expression and protein were detected using RT-PCR and Western blotting techniques.Results:Compared with the ordinary site group for hepatocellular carcinoma,there was no statistically significant difference in the first complete ablation rate and the local control rate of the lesions at one year in the extraordinary site group patients(P=0.635).Regarding complications,36.2%(25/95)of the extraordinary site group had higher pain in the operative area than 16.3%(29/178)of the ordinary site group.The difference between the two groups was statistically significant(P=0.048).There was one case each of colonic injury and biliary fistula in the extraordinary site group,suggesting that there is still some risk of RFA in the extraordinary site.Immunohistochemistry showed that HIF-2α expression was more pronounced in resected incompletely ablated hepatocellular carcinoma tissues than in paracancerous tissues,and Notch1 and VEGF levels were also upregulated.In vitro,cellular assays revealed that hepatocellular carcinoma treated with heat shock stimulation had enhanced proliferation and invasion ability(P<0.001)and migration ability(P=0.046)compared with unstimulated hepatocellular carcinoma cells.The HIF-2α-specific inhibitor PT2385 downregulated the migration and invasion ability of heat shock stimulation-treated hepatocellular carcinoma cells(P=0.009).Furthermore,insufficient ablation upregulated the expression of HIF-2α,VEGF,and Notch1 mRNA and protein expression,and the use of PT2385 downregulated HIF-2α,VEGF,and Notch1 mRNA and protein expression.Conclusion:The extraordinary site is an important factor affecting the efficacy and safety of radiofrequency ablation of clinical HCC;precise preoperative planning,optimization of ablation techniques,and development of individualized treatment strategies can improve the ablation rate of lesions.Insufficient ablation can induce hepatocellular carcinoma cell proliferation,invasion,and migration through HIF-2α/VEGF,notch1 signaling axis.Targeting HIF-2α is a new strategy to treat the recurrence of HCC insufficient ablation.