摘要
肺癌是肿瘤相关死亡的首要死因,在所有的恶性肿瘤中其预后最差,其中超过80%的肺癌是非小细胞肺癌(non-small cell lung cancer,NSCLC)。虽然传统的肺癌治疗方式已取得了显著的进步,但其治疗价值仍较为有限,患者生存期较短,预后不甚理想,所以急需新的更为有效的治疗方法,而这就要求我们对于肿瘤发生发展过程中的分子机制有更为深入的了解。
包括肿瘤和炎症在内,机体许多病理过程都存在乏氧。实体肿瘤由于增殖过快并且血管发育不良经常出现缺氧区域,肿瘤组织中氧分压较之正常组织明显为低。大量证据表明缺氧是肿瘤发生发展过程的关键因素。缺氧诱导因子(hypoxia-inducible factors,HIFs)是一种转录因子,在缺氧调节过程中发挥核心作用。HIFs是一种氧依赖的转录激活因子,由一个调节表达的亚基(HIF-α)和持续表达的亚基(HIF-β)所组成,其中HIF-β也被称为芳香烃受体核转位蛋白(arylhydrocarbon receptor nuclear translocator,ARNT)。HIF-α蛋白表达受氧浓度即时调节,而HIF-β则为持续表达不受氧浓度的影响。HIFs的功能取决于HIF-α亚基,在常氧下,HIF-α仅有微弱表达,而在缺氧情况下,其在细胞内积聚,转运入核与HIF-β形成二聚体,并与靶基因的缺氧反应元件(hypoxia-responseelement,HRE)结合从而激活一群涉及肿瘤能量代谢、血管生成、细胞生长、凋亡、侵袭转移和化疗耐药等特性的基因表达。
目前,已有三种HIF-α亚基被克隆并命名为HIF-1α、HIF-2α和HIF-3α。其中HIF-1α和HIF-2α结构相似,都包含碱性环-螺旋-环(basic helix-loop-helix,bHLH)结构、PAS(Per-ARNT-Sim)结构、转录活性结构域(transactivation domain,TAD)和氧调节的死亡结构域(oxygen-dependentdeath domain,ODDD)。然而,许多分子、生化及生理研究结果都表明二者并不完全等同而是有着各自独特的生理功能。HIF-1α已为众多研究证明其直接或间接地参与调节许多肿瘤发生发展的过程,而另一方面,对于HIF-2α的作用尤其在NSCLC中的作用则所知较少。
实验课题对HIF-2α基因在NSCLC中的生物学作用作了较为系统的研究。实验从三个方面进行:①首先采用免疫组化和组织芯片技术对上百例NSCLC手术标本中HIF-2α及一些生物标记蛋白表达水平进行了检测并对HIF-2α蛋白表达和这些分子表达之间的相关性进行了分析,进一步研究了NSCLC组织中HIF-2α蛋白的表达与NSCLC的临床病理因素、复发转移及预后的关系,初步探讨了HIF-2α之于NSCLC在增殖、侵袭转移、血管生成及化疗耐药方面的影响;②建立了体外A549细胞缺氧培养模型,研究了缺氧态下HIF-2α及一些肿瘤相关基因的表达情况及其之间的关系;③成功构建了高效的靶向HIF-2α的siRNA表达载体,并将之转染A549细胞特异性沉默HIF-2α基因表达,探讨HIF-2α基因沉默对于细胞在增殖、凋亡、侵袭转移、血管生成及化疗耐药方面的影响。
实验结果表明HIF-2α的表达与NSCLC的发生发展密切相关。开发以HIFs为靶点的抗癌治疗药物已越来越为人们所重视。因此,本实验有助于更好的理解HIF-2α在NSCLC耐受乏氧及其发展过程中的作用,并为开展有关HIFs的靶向治疗研究提供了实验依据。
第一部分HIF-2α在人非小细胞肺癌组织中的表达及其临床意义
目的本部分实验通过检测HIF-2α蛋白和一些生物标记蛋白在NSCLC组织中的表达情况及其相互关系,并结合各临床病理因素及随访资料,初步探讨HIF-2α在NSCLC增殖、侵袭转移、血管生成及化疗耐受方面的作用及其对NSCLC手术患者预后的影响。
方法采用组织芯片技术及免疫组化Envision法检测140例NSCLC手术标本中HIF-2α和一些生物学指标PCNA、Mdr-1和肿瘤MVD的表达情况,探讨HIF-2α与这些生物学指标以及相关临床病理因素的关系。采用二分类Logistic回归分析法分析了HIF-2α蛋白表达以及各临床病理因素对NSCLC术后复发转移的影响。采用Kaplan-Meier法对按各因素分组的1至3年累计生存率进行了计算,相应绘制了生存曲线并应用log-rank检验进行比较。采用Cox比例风险模型对众多可能影响NSCLC患者术后生存的预后因素进行了单因素和多因素分析,筛选出影响NSCLC手术患者预后的独立危险因素。
结果HIF-2α在对照孔的正常肺组织中无或仅有微量表达,而在癌细胞中则表现为程度不等的细胞质和细胞核的混合染色。140例NSCLC手术标本中HIF-2α蛋白高表达病例占46%(64/140),其表达水平与肿瘤大小(P＜0.05)及肿瘤的TNM分期(P＜0.01)密切相关。Spearman相关分析显示HIF-2α蛋白表达水平与PCNA(r=0.268,P＜0.01)、Mdr-1(r=0.222,P＜0.01)蛋白表达正相关。同时,与HIF-2α蛋白低表达组相比,HIF-2α蛋白高表达组其肿瘤MVD较高(P＜0.05)。在140个NSCLC手术患者中,术后三年复发转移率为52.9%(74/140)。采用logistic单因素分析显示HIF-2α蛋白高表达(OR=2.625,P＜0.01)、肿瘤大小(OR=3.500,P＜0.05)、淋巴结转移(OR=4.102,P＜0.01)以及肿瘤TNM分期(OR=3.430,P＜0.01)与NSCLC患者术后复发转移密切相关。基于单因素分析的结果,进一步进行二分类logistic回归多因素分析表明其中HIF-2α蛋白高表达(OR=2.327,P＜0.05)和淋巴结转移(OR=3.784,P＜0.01)是影响NSCLC术后复发转移的独立危险因素。Kaplan-Meier生存分析显示在140例NSCLC手术患者中,其1、2、3年的累计生存率分别为80.7%、67.9%、57.8%。其中,在Ⅰ期患者中其1、2、3年累计生存率分别为89.6%、80.6%、73.0%;在Ⅱ-Ⅲ期患者中其1、2、3年累计生存率分别为72.6%、56.2%、43.8%。按各种可能影响生存时间的因素进行分组并对各组的生存分布进行了描述,Log-rank检验显示其中HIF-2α(P＜0.01)、PCNA(P＜0.01)、Mdr-1蛋白表达(P＜0.01)、肿瘤MVD(P＜0.01)及患者临床病理因素如年龄(P＜0.05)、肿瘤大小(P＜0.05)、淋巴结转移(P＜0.01)、TNM分期(P＜0.01)各分组生存率均存在明显差异。Cox回归分析显示HIF-2α高表达(RR=1.810,P＜0.05)、Mdr-1阳性表达(RR=2.562,P＜0.01)、高龄(RR=1.027,P＜0.05)及淋巴结转移(RR=2.411,P＜0.01)是NSCLC手术患者的独立预后因素。
结论这部分实验表明HIF-2α表达与NSCLC的临床特征及一些在肿瘤发展过程中起明确作用的基因表达密切相关。HIF-2α可能在NSCLC增殖、侵袭转移、血管生成及化疗耐受方面发挥重要作用,同时作为判断NSCLC预后不良的生物标记也具有相当价值。
第二部分缺氧诱导人肺腺癌A549细胞株HIF-2α、VEGF、PCNA、Mdr-1的表达
目的细胞在多种生理或病理的情况下均会发生缺氧,尤其在组织快速生长超出血液供给致氧浓度低于5%时。这种缺氧微环境会激发复杂的由缺氧诱导因子(hypoxia-inducible factors,HIFs)介导的保护性反应。由缺氧环境所引起的反应过程与许多肿瘤的恶性表型密切相关,其中的分子运转机制以及如何对肿瘤的生物学特性产生影响值得关注。缺氧上调HIF-α表达,其与β亚基形成二聚体,通过作用于靶基因增强子序列中的乏氧反应元件从而激活基因表达。已知有超过100种的HIFs靶基因被发现,其中包括:促进血管生成的因子如血管内皮生长因子(vascular endothelial growth factor,VEGF)和血小板源性生长因子(platelet-derived growth factor,PDGF)等;糖酵解酶如aldolase A等;细胞周期调控因子如P21和P27等;另外还包括调节细胞基质重构以及细胞增殖、分化和凋亡方面的蛋白。这些基因的活化表达使肿瘤细胞能够适应不利的外界环境得以存活。为此,这部分实验测定了不同时长的缺氧培养A549细胞中HIF-2α、VEGF、PCNA、Mdr-1在mRNA和蛋白水平的表达,并探讨了HIF-2α和这些基因之间可能存在的内在关系。
方法将人肺腺癌细胞株A549缺氧培养不同时段(0～72h),采用qRT-PCR和Western Blot法以及ELISA法检测不同缺氧时段HIF-2α、VEGF、PCNA、Mdr-1的mRNA和蛋白表达情况,比较不同处理组的表达差异并研究了HIF-2α和其他基因之间表达的相关性。
结果常氧情况下A549细胞中HIF-2α在mRNA和蛋白水平上表达较低。与常氧组相比,在急性缺氧期,其mRNA水平在缺氧诱导12h后出现缓慢上调并在24h后增至2.69倍,随后在长期的乏氧环境下稳定在该峰值水平。同样地,HIF-2α蛋白在缺氧诱导下增加缓慢,在缺氧12h开始出现上调,至24h到达峰值,在随后的慢性缺氧期保持该水平稳定。其他基因VEGF、PCNA和Mdr-1的表达也表现出相同的趋势。进一步相关性分析显示无论在mRNA还是蛋白水平上HIF-2α的表达均与VEGF、PCNA和Mdr-1正相关。
结论这些结果表明在A549细胞中HIF-2α可能主要参与并调节细胞慢性乏氧过程,VEGF、PCNA和Mdr-1可能是HIF-2α的靶基因。HIF-2α通过活化这些基因的表达来调节肿瘤细胞的功能,从而促进了肿瘤的发展。
第三部分靶向抑制HIF-2α基因的siRNA干扰质粒的构建及筛选
目的构建siRNA_(HIF-2α)真核表达载体,从中筛选出最有效的siRNA序列,为进一步探讨HIF-2α的功能奠定基础。
方法设计并合成靶向HIF-2α的siRNA真核表达载体,经大肠杆菌E.coliJM109转化、筛选克隆与扩增并对质粒进行酶切和DNA测序鉴定。通过脂质体Lipofectemin 2000将重组质粒转染A549细胞并在之后分别缺氧培养24h和48h,采用qRT-PCR和Western Blot方法检测HIF-2α表达的抑制情况,从中筛选出最为有效抑制靶基因表达的siRNA干扰表达载体。
结果重组siRNA_(HIF-2α)表达载体经酶切和测序鉴定证实siRNA真核表达载体构建成功。在将siRNA_(HIF-2α)表达载体转染A549细胞后,相应检测到细胞HIF-2α在mRNA和蛋白水平的表达均有明显的下调。qRT-PCR检测显示实验构建的第4号siRNA表达载体对于HIF-2αmRNA表达抑制效应最强,相较于空白组,其转染后24h和48h的对HIF-2α基因mRNA表达水平的特异性抑制率分别为(60.63±5.10)%和(80.00±3.55)%。对转染后细胞进行Western Blot检测证实同样是第4号siRNA表达载体对于HIF-2α蛋白表达抑制效应最强,其转染后24h和48h的抑制率分别为(31.69±11.56)%和(82.87±4.09)%。
结论所构建的siRNA表达载体可以有效的抑制靶基因HIF-2α在mRNA和蛋白水平的表达,并筛选出一条高效的4号siRNA表达载体,为进一步的研究HIF-2α在NSCLC的作用奠定基础。
第四部分HIF-2α基因沉默对人肺腺癌细胞株A549生物学行为的影响
目的肿瘤细胞处于缺氧时常表现为大量基因的表达改变,其中很多基因启动子或增强子包含的缺氧反应元件(HRE)能与缺氧诱导因子(HIFs)相互作用从而直接地受到HIFs的调控。HIF-2α是HIFs家族的重要成员,前面的实验已初步表明:在NSCLC中HIF-2α的表达与其增殖、侵袭转移、血管生成及化疗耐受方面关系密切。在这部分实验中,通过运用RNA干扰技术,抑制HIF-2α表达,观察其对人肺腺癌细胞株A549增殖凋亡、血管生成、侵袭转移及化疗耐药方面的影响。为进一步关于以HIF-2α为靶点治疗NSCLC提供实验依据。
方法实验细胞分为三组:正常对照组、阴性对照组以及siRNA_(HIF-2α)转染组。首先A549细胞转染siRNA_(HIF-2α)表达载体或经对照处理后置于缺氧环境下培养96h,每24h采用MTT法测定其细胞的生长水平。其次,各组细胞转染处理后置于缺氧环境下分别培养24h和48h,Annexin V/PI双染后用流式细胞仪检测以测定A549细胞的凋亡率。再次,采用Transwell法测定各组细胞的侵袭力。最后,采用ELISA法检测各组细胞VEGF蛋白的表达水平;Western Blot法测定PCNA和Mdr-1的蛋白表达水平。实验比较了实验组和对照组细胞在这些方面的表达差异。
结果MTT实验结果表明siRNA_(HIF-2α)转染组A549细胞生长速度较正常对照组明显减缓,差异具有统计学意义(P＜0.05)。与正常对照组相比,转染后缺氧培养24h和48h,实验组细胞的凋亡率明显上升(P＜0.05)。Transwell法细胞迁移试验中,通过计数穿过滤膜的细胞数来评价该组细胞的侵袭能力,实验发现在siRNA_(HIF-2α)组,其穿膜细胞数明显减少(P＜0.05),表明实验组细胞侵袭力下降。通过ELISA和Western Blot法测定了A549细胞相关蛋白的表达,发现在siRNA_(HIF-2α)转染组,VEGF、PCNA和Mdr-1蛋白表达水平明显下调(P＜0.01)。
结论这部分实验进一步揭示了HIF-2α与VEGF、PCNA、Mdr-1表达的密切关系,并且探讨了HIF-2α表达在A549细胞增殖、凋亡、侵袭转移方面的影响,从而更为深入地研究了HIF-2α在NSCLC的发生发展中的作用。研究发现采用小干扰RNA技术可有效抑制靶基因HIF-2α的表达从而转变人肺腺癌细胞株A549的表型,有可能成为NSCLC的一种有效的治疗手段。
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 significance
Objective: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 line
Objective: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-2αmRNA
Objective: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 A549
Objective: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.
引文
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