Research On The Roles Of Hypoxia-inducible Factor 2α In Human Non-small Cell Lung Cancer

Lung cancer is the leading cause of cancer-related mortality and its prognosis is the poorest in all cancer types,among which non-small cell lung cancer(NSCLC) accounts for more than 80%of all lung cancer cases. Although significant advances have been achieved in conventional therapies,the poor prognosis and short survival of patients,as well as the limited value of any sort of conventional therapy,are factors all demanding novel and more effective therapy which may requires our better understanding of the molecular mechanisms underlying carcinogenesis.Hypoxia is involved in many pathophysiological processes including neoplasm and inflammation.Solid tumors frequently harbor hypoxic areas due to rapid cellular proliferation and poor vascularization.The oxygen pressure in solid tumors is generally lower than that in the corresponding normal tissue.A growing body of evidence indicates that hypoxia is a key element involved in the development and progression of tumors.At the heart of the hypoxia regulatory system is hypoxia-inducible factors (HIFs),which are transcription factors.HIFs is an oxygen-dependent transcriptional activator which is composed of a regulated subunit (HIF-α) and a constitutive subunit(HIF-β),also known as ARNT(aryl hydrocarbon nuclear translocator).HIF-αprotein expression is immediately regulated by cellular oxygen whereas HIF-βprotein is constitutively expressed regardless of oxygen concentration.HIF-αis the unique subunit that determines the HIFs system activity.In normoxia, there is only trace expression of HIF-α,while under hypoxic condition, HIF-αprotein is accumulated in cells,which heterodimerizes with HIF-βand then enters the nucleus where it binds to hypoxia-responsible elements (HREs)and activates a network of genes transcription that control several aspects of tumor biology such as energy metabolism,angiogenesis, cellular growth,apoptosis,invasion and metastasis and chemotherapy resistance.To date,three HIF-αsubunits have been cloned and named HIF-1α, HIF-2α,and HIF-3α.Both HIF-1αand HIF-2αare structurally alike which contain a basic helix-loop-helix motif,a PAS domain, transcription-activation domains(TAD),and oxygen-dependent degradation domains(ODDD).However,several molecular,biochemical,and physiological studies have established that HIF-1αand HIF-2αare not redundant but have distinct functional roles.HIF-1αhas been directly or indirectly linked to the regulation of development and progression of many tumors.The corresponding role of HIF-2α,on the other hand,is less clear,especially in NSCLC.In this experiment subject,a systematic research was carried out on the biological function of HIF-2αgene in NSCLC.The experiment went on from three respects:①First,immunohistochemistry method and tissue chip technique were adopted to examine the expression of HIF-2αand some biological marker proteins in hundreds of NSCLC surgical specimens and correlations between HIF-2αand these proteins were also analyzed. Furthermore,the relationship of HIF-2αexpression in NSCLC tissue to clinicopathologic factors,recurrence and metastasis and prognosis of NSCLC was explored.All above made a preliminary probe into the influence of HIF-2αin NSCLC on the aspects of proliferation,invasion and metastasis,angiogenesis and chemotherapy resistance.②Secondly, hypoxic culture model for A549 cell line in vitro was conducted and the expression of HIF-2αand some tumor-associated genes were detected and the correlation between them was studied.③Finally,high efficient HIF-2α-targeted siRNA expression vector was successfully conducted, which was then transfected into A549 cells to specifically knock down the expression of HIF-2αgene.The impacts of HIF-2αgene silencing on the aspects of cell proliferation,apoptosis,invasion,angiogenesis and chemotherapy resistance was explored.Experimental results indicated that HIF-2αexpression was closely related with development and progression of NSCLC in many ways.There is a growing interest in the development of HIFs inhibitors as anticancer agents.Therefore,the data presented in this paper will provide an opportunity to better understand the roles of HIF-2αin adaptive mechanisms to hypoxia as well as in tumor development condition,which also produces experimental evidence for the research of targeting the HIFs system in cancer therapy.PartⅠThe expression of HIF-2αin human non-small cell lung cancer and its clinical significanceObjective:In this part of experiment,the expression status of HIF-2αand some biological marker proteins in human NSCLC tissue were examined and their mutual relationship was studied.Furthermore,combining clinicopathologic factors and follow-up data of cases,the role of HIF-2αin the proliferation,invasion and metastasis,angiogenesis and chemotherapeutic resistance of NSCLC and its impact on the prognosis of NSCLC surgical patients were explored.Methods:Immunohistologic expression of HIF-2αand some biology markers such as PCNA,Mdr-1 and tumor MVD were assessed in a tissue microarray of 140 NSCLC surgical specimens by immunohistochemical Envision method. The Relationship between the expression of HIF-2αprotein and clinicopathological factors or molecular markers was studied.The influence of HIF-2αexpression and clinicopathological factors on postoperative recurrence and metastasis of patients with NSCLC was analyzed by using logistic regression model.The cumulative survival rates of 1-3 year were calculated by Kaplan-Neier method in each factor group and survival curves were also plotted and compared using the log-rank test.Univariate and multivariate analyses with all possible prognostic fators for NSCLC patients were made by Cox proportional hazards model to screen out independent risk factors for the prognosis of NSCLC surgical patients.Results:HIF-2αprotein showed a mixed cytoplasmic/nuclear pattern of expression in cancer cells with different degrees,while normal lung tissue in control cores showed a negative or weak reactivity.Cases with high HIF-2αprotein expression were noted in 46%(64/140) of 140 NSCLC surgical specimens.A significant correlation was noticed between the expression level of HIF-2αprotein and tumor size(P＜0.05) or TNM stage (P＜0.01).The Spearman correlation analysis demonstrated that the expression level of HIF-2αprotein was significantly positively correlated with PCNA(r=0.268,P＜0.11) and Mdr-1(r=0.222,P＜0.01) protein expression and cases with high HIF-2αprotein expression were correlated with higher tumor MVD as compared to those with low HIF-2αprotein expression(P＜0.05).The 3-year postoperative recurrence and metastasis rate was 52.9%(74/140) in 140 NSCLC surgical patients.Univariate logistic analysis revealed that the protein expression of HIF-2α(OR=2.625,P＜0.01),tumor size(OR=3.500,P＜0.05),lymph node metastasis (OR=4.102,P＜0.01) and tumor TNM stage(OR=3.430,P＜0.01) were correlated with recurrence and metastasis after resection in NSCLC patients.Based on the univariate analysis,further binary logistic regression model showed that the protein expressin of HIF-2α(OR=2.327,P＜0.05) and lymph node metastasis(OR=3.784,P＜0.01) were independent risk factors for postoperative recurrence and metastasis of NSCLC.Kaplan-Meier survival analysis showed that the 1-,2- and 3-year cumulative survival rates of 140 NSCLC surgical patients were 80.7%,67.9%,57.8%,respectively. Therein,the 1-,2- and 3-year cumulative survival rates in stageⅠpatients were 89.6%,80.6%,73.0%,respectively,and the 1-,2- and 3-year cumulative survival rates in stageⅡ-Ⅲpatients were 72.6%, 56.2%,43.8%,respectively.The survival distribution in each group stratified by all possible factors that affected survical time was described and Log-rank test manifested that there was a significant difference in cumulative survival rates between each group stratified by the protein expression level of HIF-2α(P＜0.01),PCNA(P＜0.01),Mdr-1 (P＜0.01)or tumor MVD(P＜0.01),and some clinicopathologic factors such as age(P＜0.05),tumor size(P＜0.05),lymph node metastasis(P＜0.01)and TNM stage(P＜0.01).Cox regression model showed that HIF-2αhigh expression(RR=1.810,P＜0.05),Mdr-1 positive expression(RR=2.562, P＜0.01),advanced age(RR=1.027,P＜0.05),and lymph node metastasis (RR=2.411,P＜0.01) were independent prognostic factors for NSCLC surgical patients.Conclusion:Taken together,the results presented in this part of experiment provide evidence that the expression of HIF-2αis closely related to the clinical feature of NSCLC and some genes known to be implicated in tumorigenic processes.HIF-2αmay play an important role in tumor cell proliferation,angiogenesis,invasion,metastasis and chemotherapeutic resistance and appears to be of obvious value as a significant prognostic factor in NSCLC.PartⅡHypoxia-inducible expression of HIF-2α,VEGF,PCNA and Mdr-1 in human lung adenocarcinoma A549 cell lineObjective:Hypoxia occurs in a number of physiological and pathophysiological settings,particularly when rapid tissue growth exceeds blood supply and available oxygen concentration falls below 5%,triggering a complex protective adaptation mediated primarily through transcription by hypoxia-inducible factors(HIFs).Associations between cell response to microenvironmental hypoxia and aggressively malignant phenotypes are observed in a range of cancers,focusing attention on molecular operative mechanism of the process and how they contribute to tumor biology.Hypoxia up-regulates the expression of HIF-α,which in turn dimerizes with HIF-βand binds to the hypoxia response element in the enhancer region of target genes,thus activating them.More than 100 HIFs-targeted genes have been identified.These include genes that encode angiogenesis-promoting factors such as vascular endothelial growth factor(VEGF)and platelet-derived growth factor(PDGF),glycolytic enzymes such as aldolase A,and cell cycle regulators such as p21 and p27,in addition to other proteins involved in extracellular matrix remodeling, differentiation and apoptosis.Induction of these genes is part of the cellular response to an adverse environment and may give cells a survival advantage.Therefore,in this study,to reveal the internal relation between the expression of HIF-2αand some genes such as VEGF,PCNA and Mdr-1 during hypoxia,we investigated the expression of these genes both at mRNA and protein levels in A549 cells exposed to different length of time in hypoxic culture.Methods:A549 cells were cultured and exposed to hypoxia for various hours (0～72h).The expressions of HIF-2α,VEGF,PCNA and Mdr-1 at mRNA and protein levels were determined by qRT-PCR and Western blot analysis as well as ELISA method,and the discrepancies in the gene expression among these groups were compared and gene expression correlation between HIF-2αand other genes was studied.Results:The expression of HIF-2αmRNA and protein were low under normoxic condition in A549 cells,while when cultured under hypoxia,the amount of HIF-2αmRNA was gradually raised since 12h during acute hypoxia period compared with that in normoxia,then increased over time and reached 2.69-fold at about 24h,which then stabilized at that peak level under long-term hypoxia exposure.Likewise,the protein level of HIF-2αrose slowly after hypoxic culture,which was initially induced at 12h and up-regulated to the peak level at 24h,and then the protein level kept stable during chronic hypoxia.The expression of other genes such as VEGF, PCNA and Mdr-1 exhibited the same trend with exposure to hypoxia condition in A549 cells.Furthermore,the results of correlation analysis showed that the mRNA and protein levels of HIF-2αpositively correlated to those of VEGF,PCNA and Mdr-1 gene respectively in A549 cells.Conclusion:These results have opened up the possibility that HIF-2αmay induce a longer response and control the response to chronic hypoxia in A549 cells,suggesting that VEGF,PCNA and Mdr-1 gene might be HIF-2αtarget genes.HIF-2αcould regulate tumor cell function through activation of these tumor-associated genes,which in turn contributes to its tumor promoting activity. PartⅢConstruction and selection of the siRNA recombination plasmid targeting HIF-2αmRNAObjective:To contruct small interfering RNA(siRNA) eukaryotic expression vector targeting HIF-2αgene by which to screen out the most effective siRNA and to observe the effects of gene silencing of HIF-2αby RNA interference on A549 cells,which lay a foundation for further research of HIF-2αfunction.Methods:The siRNA eukaryotic expression vectors targeting HIF-2αgene were designed and synthesized.The recombined plasmids were transformed into E.coli JM109,which was then selected and enlarged cultured.The plasmid of DNA was extracted and identified by enzyme digestion analysis and DNA sequencing.Further,the siRNA expression vectors were transfected into A549 cells via reagent of Lipofectamin 2000 followed by incubation for 24h or 48h under hypoxic condition.Real-time quantitative PCR and Western blot analysis of HIF-2αmRNA and protein levels respectively validated the inhibitory effects of siRNA on HIF-2αgene expression to select the most effective siRNA expression vector.Results:The successful construction of HIF-2α-targeted siRNA expression vectors was confirmed via enzyme digestion analysis and DNA sequences assay.After transfection with the HIF-2αsiRNA expression vectors into A549 cells,mRNA and protein expression of HIF-2αgene were markedly downregulated.The resuls of qRT-PCR showed that No.4 siRNA vector was most effective in suppressing HIF-2αexpression.In comparison with the blank control group,transfection of A549 with this HIF-2αRNAi vector resulted in sequence specific silencing with(60.63±5.10)%and(80.00±3.55)%decreases of HIF-2αmRNA transcription at 24h or 48h post-transfection respectively.The Western Blot test also domonstrated the best effectiveness of this expression vector with the inhibition rate of(31.69±11.56)%at 24h post-transfection and(82.9±4.09)%at 48h post-transfection.Conclusion:The constructed siRNA expression vectors could effectively suppress the mRNA and protein level expression of HIF-2α.A highly efficient NO.4 siRNA was selected out,which established the foundation for further study of HIF-2αfunction in NSCLC.PartⅣEffects of HIF-2αgene silencing on the biological behavior of human lung adenocarcinoma cell line A549Objective:The response of tumor cells to hypoxia is characterized by specific alterations in the expression of a large number of genes,many of which are regulated directly by hypoxia-inducible factors(HIFs) via binding to cis-acting hypoxia response elements(HREs) located in the enhancers and/or promoters of these genes.HIF-2αis an important member of hypoxia-inducible factor family.Part 1 and 2 presented preliminary proof for HIF-2αin close relation with proliferation,apoptosis, angiogenesis,invasion and metastasis and chemotherapy resistance of NSCLC.Here,the expression of HIF-2αwas specifically suppressed by adopting RNA interference technology.The impacts of HIF-2αexpression on the cell proliferation,apoptosis,migration and the protein expression of some genes such as VEGF,PCNA and Mdr-1 were explored.Methods:Cells were divided into three groups:cells treated with a mock transfection(blank control),negative control and the experiment group treated with siRNA for HIF-2α.First,A549 cells were transfected with the siRNA expression vector containing a HIF-2α-specific targeting sequence as well as control prior to the exposure of cells to oxygen deprivation for 96h.Cell growth level was measured by MTT colorimetric assay at each 24h.Moreover,annexin V and propidium iodide staining was also detected on flow cytometry for analysis of A549 cell apoptosis after subsequent incubation for 24h or 48h in hypoxia in each experimental group. Furthermore,the migration of A549 cells was assayed using transwell cell culture chambers.Finally,VEGF protein expression was determined by enzyme-linked immuno sorhent assay(ELISA),and the expression of PCNA and Mdr-1 protein was detected by means of Western blot analysis. Differences between experimental and control groups were compared.Results:MTT reduction assay showed a reduced cell growth in A549 cells transfected with siRNA targeting HIF-2αcompared to the control group, which is of statistical significance(P＜0.05).As compared with control, the apoptosis rate of A549 cells in the experimental group increased obviously after 24 or 48 hours of hypoxic incubation(P＜0.05).In migration analysis,the number of cells that had migrated through the filter was counted representing the migration ability of cells in each group.It was shown that hypoxically induced migration of A549 cells was much lesser in groups treated with siRNA for HIF-2αthan in control,which indicated a reduction in the invasive capacity of the experimental group. Western blot analysis and ELISA analysis of protein level from A549 cells after different treatment showed the protein expression of VEGF,PCNA and Mdr-1 was significantly down-regulated when A549 cells were treated with siRNA targeting HIF-2αtransfection.Conclusion:This part of study further revealed the close relationship between VEGF,PCNA,Mdr-1 and HIF-2αexpression,and the impacts of HIF-2αexpression on A549 cells in proliferation,apoptosis and invasiveness were also confirmed,resulting a more deep investigation on the roles of HIF-2αgene in NSCLC carcinogenesis and development.This research showed that siRNA technology could effectively inhibit the expression of HIF-2αand change A549 cell phenotype,which may provide a possible effective therapeutic means for patients with NSCLC.