The Expression Of Hypoxia-inducible Factor-1a Protein In Primary Hepatocellular Carcinoma And The Effects And Mechanisms Of It On Sorafenib Resistance

Objective. To investigate the expression of hypoxia-inducible factor-la (HIFIa) protein in hepatocellular carcinoma (HCC), and its effects on the sorafenib resistance in clinical study. In vitro study,we further investigate the role of HIF1a induced sorafenib resistance and possible mechanisms.Materials & Methods. A total of 67 patients with advanced hepatocellular carcinoma who received sorafenib and have tissue specimen at our hospital from January 2006 to January 2014 were enrolled in this study. Tissue HIF1? was analyzed by immunohistochemistry.All patients were divided into 2 groups according to HIF1? protein expression: the positive staining group and the negative staining group. Clinical-pathological characteristics, oncologic outcomes were collected and compared between the 2 groups.We analyze the the efficacy of sorafenib and the impact of HIF1a protein expression on the efficacy of sorafenib. In vitro study we culture hepatocarcinoma cells and construct hypoxia model. Silencing and overexpression of HIF1a was achieved by way of lentiviral transduction of the specific vectors. After transduction, stable cell lines expressing the LV-shHIF1a or LV-HIF1a were isolated by way of selection with puromycin. The effect and mechanism of HIF1a on sorafenib resistance were further analyzed by Western Blot,CCK8 cell proliferation test, apoptosis test and cell cycle test.Results. (1) Patients included 59 men and 8 women. 30 patients (44.8%) with the positive staining of HIF1a protein, and 37 patients (55.2%) with the negative staining of HIF1a protein. The positive staining of HIF1a protein was brown and granule-like and was mainly present in the cytoplasm. (2)HIF1? protein expression correlate with tumor size,AFP level and tumor grading. (3)7 patients (10.4%) were partial remission (PR), 44 patients (65.7%) were stable disease (SD) and 10 patients (14.9%) were disease progression (PD), 6 patients(9%) have not the efficacy evaluation information. The rate of HIFla protein positive expression was 80% in PD group, 36.4% in SD group and 14.3%in PR group(P=0.0126). (4) In a Cox proportional hazards model,positive staining of HIF1? protein, old age, family history of cancer, lower KPS, intrahepatic metastasis,lower tumor grading, tumor size more than 5cm and not receiving TACE treatment were associated with a lower OS. In addition, positive staining of HIF1? protein, family history of cancer, lower KPS, intrahepatic metastasis, lower tumor grading, and not receiving TACE treatment were associated with a lower PFS.(5)Cell proliferation test results show that under the hypoxia condition, or overexpression of HIF1a ,the effect of sorafenib inhibiting liver cancer cell proliferation was significantly reduced. Apoptosis test suggest that under hypoxia condition, or overexpression of HIF1a, the effect of sorafenib promoting tumor cell apoptosis was significantly reduced. (5) pVEGFR2, pPDGFR?,pRAF, pMEK1, pERK were detected by Western Blot after transduction of HIF1a overexpressing lentivirus, and we found that the target protein expression level was up-regulation.Conclusion. (1) HIF1? is an independent risk factor for predicting the efficacy of sorafenib and the prognosis of hepatocellular carcinoma. (2)HIF1? protein, family history of cancer, lower KPS, intrahepatic metastasis, lower tumor grading, and not receiving TACE treatment are poor prognostic factor affecting overall survival time and progression-free survival. (3)Hypoxia and HIF1? can induce sorafenib resistance by antagonizing sorafenib inhibition of hepatocellular carcinoma cell proliferation and promoting cell apoptosis.(4)The molecular mechanism of HIFla induced sorafenib resistance may be the up-regulation of the phosphorylation level of the sorafenib targets.