Correlation Of Hypoxia-inducible Factor-2α Expression And Angiogenesis, Lymphangiogenesis And Epithelial-mesenchymal Transition In Non-small Cell Lung Cancer

BACKGROUND:Lung cancer is a malignant tumor with highest incidence and mortality in the world. About80%of lung cancer is non-small cell lung cancer (NSCLC). Although people fight NSCLC with a comprehensive treatment program, including surgical resection, chemotherapy, radiation therapy and targeted drug therapy according to different stages, from1975to2003, the improvement of overall five-year survival rate in NSCLC is not obvious, only increased from13%to16%, and5-year survival rate of early resectable NSCLC is only60%to70%,1/4to1/3of the patients still die of metastasis and recurrence finally. In order to further improve the effect of treatment and prognosis of patients with NSCLC, a research about the molecular mechanisms related to tumor invasion and metastasis is needed, and a new way of anti-invasion and metastasis may be found by studying its signaling pathways and key regulatory factors. A large number of studies have demonstrated that hypoxia can promote the occurrence and development of tumor, hypoxia inducible factor (hypoxia inducible factor, HIF) family plays a very important role in this process. HIF-1and HIF-2are the most important HIF factors involved in the regulation of tissue hypoxia. Although there is significant homology on gene sequences between HIF-1α and HIF-2α, they play important but not overlapping roles in tumor progression. In recent years, some studies of breast cancer, renal cell carcinoma, lung cancer, the neuroblastoma found that HIF-2α rather than HIF-1α is the major regulatory factor to promote tumor progression, HIF-2α correlates closely to tumor metastasis and prognosis, and HIF-2α may be a useful indicator of malignancy risk of NSCLC patients and a potential goal of tumor prevention. However, how does HIF-2a promote invasion and metastasis in NSCLC as well as the related molecular mechanism is still unclear. VEGF-A, VEGF-C is major regulator of angiogenesis and lymphangiogenesis respectively, tumor angiogenesis and lymphangiogenesis has an important role in metastasis of solid tumors. Epithelial-mesenchymal transition (EMT) has been one of the research foci of cancer in recent years, a large number of studies have shown that the invasiveness of tumor cells significantly enhanced after EMT, and metastasis is more likely to occur. Whether HEF-2α promotes tumor invasion and metastasis through the impact of tumor angiogenesis and lymphangiogenesis and EMT in NSCLC has no relevant reports.OBJECTIVE:To investigate the role of HEF-2α in the development progression of non-small cell lung cancer and its relationship with tumor angiogenesis and lymphangiogenesis and EMT.METHOD:1. The expression of HIF-2a, HIF-1α protein in52resected non-small cell lung cancer specimens was detected by immunohis-tochemistry (IHC). The relationship between their expression and clinicopathological features, prognosis was statistically analyzed.2. The expression of EMT-associated proteins was detected by IHC. The relationship between expression of EMT-associated proteins and expression of HIF-2α, HIF-1α protein was statistically analyzed.3. The expression of VEGF-A, VEGF-C protein was detected by IHC. CD31and D2-40was used to mark tumor microvessel and lymphatic microvessel by IHC respectively. The relationship between tumor angiogenesis and lymphangiogenesis and expression of HIF-2α, HIF-1α protein was statistically analyzed. 4. The specific HTF-2α small interference (siRNA) plasmid was constructed and transfected into lung adenocarcinoma A549cells using lipofectamine2000. Three groups of cells were used in the in vitro studies, including black control group (untransfected cells), negative control group (cells transfected with the non-silencing siRNA) and HIF-2a siRNA group (cells transfected with the HIF-2a siRNA).The changes of cell morphology, mRNA and protein level of these cell factors were detected by using light microscopy, immuno-fluorescence, Real time PCR and Western blotting to analyse the role of HIF-2a in the regulation of EMT and VEGF-A, VEGF-C expression in lung adenocarcinoma A549cell.RESULTS:1. Among52samples with non-small cell lung cancer, HIF-2a protein expression was not related with HIF-1α protein expression (P=0.131). HIF-2a protein expression was significantly correlated with tumor vascular invasion, lymphatic invasion and lymph node metastasis (all P<0.01). HIF-la protein expression was not correlated with all clinicopathological factors (P>0.05). In the COX univariate analysis, HIF-2a positive expression group had a remarkably lower5-year survival rate than the negative expression group (P=0.012). There was no statistical difference between HIF-1α positive expression group and negative expression group, regarding5-year survival rate (P>0.05). COX multivariate analysis showed that it was HIF-2a expression rather than HIF-1α expression that served as an independent prognostic factor for NSCLC patients.2. Among52samples with non-small cell lung cancer, the value of immature MVD and LMVD of HIF-2a positive expression group were greater than those of HIF-2a negative group (P=0.000,0.031, respectively). Differences between the value of MVD, immature MVD and LMVD of HIF-la positive/negative expression groups were not statistically significant (P>0.05). HCF-2α expression was positively related to VEGF-A and VEGF-C expression (r=0.428,0.316, P=0.002,0.022, respectively).3. Among52samples with non-small cell lung cancer, HIF-2a expression was negatively correlated with E-cadherin expression (r=-0451, P=0.001), while it was positively correlated with Vimentin expression (r=0.321, P=0.020).4. In hypoxic pulmonary adenocarcinoma A549cells, HIF-2a protein expression increased significantly, while E-cadherin protein expression was significantly reduced. Meanwhile, Vimentin, Twist, VEGF-A and VEGF-C protein expression were increased, and growth mode of cells changed from epithelial growth type to interstitial growth type.5. HIF-2a siRNA expression plasmid which can be efficiently transferred to A549cells was successfully constructed. Compared with the negative control group, among three pairs of siRNA-HIF-2α, HIF-2α-siRNA-1331has the most noticeable silencing effect toward HIF-2α protein (P<0.05). In transfected HIF-2α-siRNA-1331group, silencing efficiency of HIF-2a mRNA was70.27%. Compared with the negative control group and blank control group, hypoxic A549cells with HIF-2a-siRNA transfected had a significantly higher expression of E-cadherin mRNA (P<0.01) and lower expression of Twist, VEGF-A mRNA (all P<0.05) by Real time PCR, and Western blotting results showed that E-cadherin protein expression of A549.cells with HIF-2a-siRNA transfected was significantly higher (P<0.01), while vimentin, Twist, VEGF-A and VEGF-C protein expression were significantly lower (all P<0.01). CONCLUSION:1. Positive expression of HIF-2a protein is not only strongly related with the invasion and metastasis in NSCLC, but also used as one of the indicators that determine the prognosis of patients with NSCLC.2. HIF-2α plays a positive role in tumor angiogenesis and lymphangiogenesis. Increased LMVD and immature MVD possibly provide more ways for lymph node metastasis and blood metastasis, and consequently lead to a higher risk of diversion.3. HIF-2α is involved in epithelial-mesenchymal transition process of NSCLC, promoting tumor invasion and metastasis.4. HIF-2α siRNA led to the efficient and specific inhibition of HIF-2α expression in hypoxic pulmonary adenocarcinoma A549cells. By silencing the expression of HIF-2a, epithelial-mesenchymal transition and VEGF-A, VEGF-C expression can be inhibited in A549cells.