摘要
研究背景:
目前对卵巢癌的治疗原则是以手术为主,辅以化疗等综合治疗。随着有效化疗方案广泛应用,卵巢恶性生殖细胞肿瘤的治疗效果有了明显提高,死亡率由90%降至10%,但卵巢上皮癌的治疗效果确一直未获根本改善,5年生存率徘徊于30%～40%。因此,卵巢癌的诊治仍面临许多困难和挑战,伴随临床标准化化疗而来的肿瘤细胞的原发性和/或继发性耐药已成为影响卵巢癌临床治疗效果的重要障碍。70%的卵巢癌患者在治疗后出现复发,关键原因之一是卵巢癌细胞对铂类化疗药物产生耐药。卵巢癌对铂类药物的耐药机制十分复杂,涉及药物蓄积、代谢、凋亡、DNA损伤修复等多方面,尚难以定论哪一个方面占主要地位,且许多机制仍不清楚。许多已知和未知的因素参与癌细胞产生耐药性的过程从而为耐药性的逆转带来困难。因此了解卵巢肿瘤细胞产生耐药的机制以及各种细胞生物学指标在耐药产生中所起的作用十分重要,可以指导我们更为合理的选择相应的化疗方案,并选择相应的特异的耐药修饰剂,可不同程度改善卵巢癌的预后。随着生命科学的飞速发展,分子生物学技术的不断改进,从基因水平去研究耐药机制已成为人类肿瘤研究的热点。积极寻找新的卵巢癌防治手段,进一步提高卵巢癌的总体治疗水平,在我国将具有积极而重要的现实意义。
凋亡抑制蛋白(inhibitor of apoptosis protein,IAPs)是一类在结构上具有同源性的细胞内源性凋亡抑制蛋白家族。人类IAPs的主要成员X连锁凋亡抑制蛋白(X-linked inhibitor of apoptosis protein,XIAP)是IAP家族中最有效力的caspase抑制物,也是分子结构研究得最清楚的IAP家族成员。研究发现XIAP的生物学功能多样,对肿瘤发生、发展及预后有多方面的影响,XIAP对细胞凋亡和细胞分裂周期有双重调控作用,具有选择性的组织细胞分布特征以及特殊的抑制细胞凋亡的作用机理,可能成为肿瘤诊断的标志物以及肿瘤基因治疗的新靶点。学者们对XIAP在肿瘤发病机制中的作用和靶向治疗中的应用等研究正逐步深入。本课题将利用腺病毒载体介导siRNA片段抑制XIAP基因的表达,进一步阐明卵巢癌多药耐药的可能机制,为卵巢癌的基因治疗提供一个新的可能的靶点及相关理论依据,为卵巢肿瘤的治疗开辟更广阔的路径。
目的:
利用RNA干扰(RNAi)技术,以XIAP基因为靶基因,以卵巢癌顺铂耐药细胞A2780/DDP为靶细胞,设计构建针对XIAP基因的siRNA真核表达质粒,进行测序鉴定及干涉效率的评价,利用腺病毒载体系统介导RNA干涉片段转染卵巢癌耐药细胞,获得XIAP基因表达抑制细胞,并检测该表达载体转染卵巢癌细胞系A2780/DDP后XIAP基因表达情况、卵巢癌细胞生物学行为以及多药耐药特性的影响,并结合基因芯片技术对其可能机制及作用通路进行初步研究。将有望发现逆转上皮性卵巢癌多药耐药的新途径,为卵巢癌的基因治疗提供新的技术方案和理论依据。
方法:
1.干扰质粒psiRNA—XIAP的构建、筛选及鉴定
①siRNA靶序列的选择及其模板DNA的设计:利用GeneBank得到XIAP的mRNA序列,根据siRNA靶序列遴选设计原则,并经BLAST序列同源性分析后,选择三条特异性siRNA靶序列,进而设计、合成其相应的模板DNA;
②干扰质粒psiRNA—XIAP的构建、筛选及鉴定:磷酸化各单链DNA片段之5'末端,退火产生相应的双链DNA,分别插入BglⅠ和HindⅢ双酶切空白质粒pSilencer,得到干扰质粒psiRNA—XIAP1、2、3,分别转化感受态大肠杆菌,挑选阳性菌落,裂解法小量抽提质粒DNA,BglⅠ和HindⅢ双酶切,1%琼酯糖凝胶电泳初步鉴定后进行测序;
③筛选XIAP mRNA敲除效率最佳的干扰质粒:中量提取各干扰质粒psiRNA—XIAP1、2、3,辅以脂质体Lipofectamine 2000分别转染人卵巢癌顺铂耐药细胞系A2780/DDP,对照组为空白对照组和空载体对照组。24 h后,RT-PCR、Western blot检测各组A2780/DDP细胞的XIAP表达水平,据此选择干扰效果最佳的的干扰质粒以构建重组腺病毒pAd-siRNA-XIAP;
2.重组腺病毒pAd-siRNA-XIAP的构建、鉴定、包装及滴度的测定:XbalⅠ和XholⅠ双酶切干扰质粒psiRNA-XIAP得到siRNA-XIAP表达片段,插入XbalⅠ和XholⅠ双酶切的腺病毒穿梭质粒pAdTrack,得到pAdTrack-siRNA-XIAP,PmeⅠ酶切线性化后,转化含有腺病毒骨架质粒pAdEasy-1的大肠杆菌BJ5183,二者同源重组产生重组腺病毒质粒pAd-siRNA-XIAP。卡那霉素抗性筛选,挑取阳性克隆,抽提质粒pAd-siRNA-XIAP,BamHⅠ酶切,0.5%琼酯糖凝胶电泳鉴定。阳性重组腺病毒质粒pAd-siRNA-XIAP电穿孔转化大量扩增后,PacⅠ酶切线性化,辅以脂质体Lipofectamine 2000转化293细胞包装产生重组腺病毒Ad-siRNA-XIAP。氯化铯滴度超离心纯化,倍比稀释法测定Ad-siRNA-XIAP滴度;
3.通过脂质体介导将Ad-siRNA-XIAP转染A2780/DDP细胞;综合运用MTT法、流式细胞仪分析、DNA凝胶电泳分析、Hoechs染色等方法,检测XIAP siRNA腺病毒载体作用于A2780/DDP细胞后,细胞增殖、细胞周期、DNA碎片、细胞凋亡及细胞耐药特性的改变。
4.应用Trizol一步法抽提卵巢癌及正常组织总RNA,分离纯化mRNA并逆转录合成荧光分子(Cy3/Cy5)标记cDNA探针,与含有8464种cDNA基因的表达谱芯片杂交,分析Ad-siRNA-XIAP转染前后A2780/DDP细胞差异表达的基因,对所获得的基因进行分子生物信息学分析。
结果:
1.干扰质粒psiRNA-XIAP1、psiRNA-XIAP2及psiRNA-XIAP3经限制性酶切鉴定和基因测序证实构建成功;分别转染A2780/DDP细胞24 h后,PCR检测XIAP mRNA水平显著下降,westernblot结果进一步证实psiRNA-XIAP1干扰效果最佳,因此选择干扰质粒psiRNA-XIAP1构建重组腺病毒Ad-siRNA-XIAP;
2.重组腺病毒Ad-siRNA-XIAP经鉴定构建成功,转染293细胞后可见绿色荧光表达,步骤简明并获得了较高的病毒滴度;
3.pAdEasy-siRNA-XIAP转染对卵巢癌细胞的生长有抑制作用,透射电镜发现转染后卵巢癌细胞超微结构发生了典型的凋亡样改变;而在空白对照及空载体对照组细胞中则未观察到相同的细胞超微结构改变,细胞结构基本正常,很少有细胞出现凋亡征象。AO染色及流式细胞仪检测细胞及细胞周期变化,发现转染后凋亡细胞的比率逐渐增加,较对照组有显著差异(P＜0.05);DNA凝胶电泳检测结果显示,总DNA被降解产生成180～200bp整数倍的片断,形成较明显的DNA“梯形”条带。。
4.pAdEasy-siRNA-XIAP转染能够显著提高A2780/DDP细胞对顺铂的敏感性,顺铂联合pAdEasy-siRNA-XIAP作用能够明显提高化疗效果,增加肿瘤细胞的凋亡。从结果看,pAdEasy-siRNA-XIAP转染对肿瘤细胞化疗敏感性的增强作用与肿瘤细胞内XIAP基因的状态与功能有关;
5.在pAdEasy-siRNA-XIAP载体转染前后A2780/DDP细胞中出现差异表达的基因功能群,初步研究结果发现pAdEasy-siRNA-XIAP载体转染A2780/DDP细胞前后,存在的大量差异表达基因涵盖了多种不同的功能群落,涉及基因包括信号与蛋白传递、癌基因与原癌基因、免疫和发育相关基因、凋亡基因,以及DNA结合转录和转录因子等。
结论:
1.实验中采用pSilencer载体系统成功构建了干扰质粒pSilencer-XIAP1、pSilencer-XIAP2和pSilencer-XIAP3,分别转染卵巢癌顺铂耐药细胞A2780/DDP,敲除其XIAP mRNA,敲除效率均较高,其中以pSilencer-XIAP1效果更佳;
2.采用pSilencer-XIAP1和腺病毒pAdEasy系统成功构建了重组腺病毒pAdEasy-siRNA-XIAP,步骤简明并获得了较高的病毒滴度,从而将RNA聚合酶Ⅲ和启动子H1的特点和优势合而为一;
3.重组腺病毒pAdEasy-siRNA-XIAP可显著抑制人卵巢癌顺铂耐药细胞A2780/DDP中XIAP的表达,对A2780/DDP细胞XIAP mRNA的敲除效率明显高于干扰质粒pSilencer-XIAP1;
4.腺病毒介导的RNA干扰技术能够显著抑制人卵巢癌顺铂耐药细胞A2780/DDP的生长、增殖、克隆形成能力,显著提高细胞对顺铂的敏感性,XIAP有望成为逆转卵巢癌多药耐药,诱导卵巢癌细胞自发凋亡的新的有希望的基因治疗靶点;
5.基因芯片和RNA干扰技术是分析基因功能的有力工具,成功获得XIAP基因表达抑制细胞中差异表达的基因谱,明确XIAP调控细胞凋亡、周期改变及多药耐药产生通路中的可能定位,为进一步明确卵巢肿瘤细胞耐药机制、为逆转卵巢癌多药耐药的发生提供了良好的实验基础。
Introduction
Ovarian carcinoma is among the most lethal of all malignancies in women.Cisplatin-centered chemotherapy is the currently preferred treatment modality in human ovarian carcinoma,yet chemoresistance severely limits treatment success.Recent evidence suggests that deregulation of key pro- and anti-apoptotic pathways is a key factor in the onset and maintenance of chemoresistance.Furthermore,the discovery of novel interactions between these pathways suggests that chemoresistance may be multi-factorial.
Technologies for gene knockout,including antisense oligonucleotides and ribozyme,have been frequently used to explore new functions of genes, but their low frequencies have limited their applications.Fortunately,the emergence of gene ablation technologies,based upon the RNA interference (RNAi) phenomenon,has provided new opportunities for experimental biology.Its blocking action on gene expression has been successfully observed in invertebrate,plant,and mammalian cells,and the knockdown of genes in cells has been achieved.Effective and highly specific,RNAi has become a new technique in knocking down genes,and it plays an important role in the study of gene function and gene therapy of diseases.
To date,X-linked inhibitor of apoptosis protein(XIAP),a novel member of the inhibitors(IAP family),has been identified as the most potent one of caspases' inhibitors.Several recent reports have demonstrated that XIAP is an important regulator in apoptosis,triggered by various apoptotic stimulations, and that it is also widely recognized to play an important role in tumor formation and invasion/metastasis in both animal models and cancer patients. In a present study,XIAP has been shown to be one of the important regulators in cisplatin-induced apoptosis in ovarian cancer cells and that downregulation of XIAP sensitizes cells to cisplatin.The mechanisms of cisplatin resistance in ovarian carcinoma,however,remain poorly understood,and all the previous studies,including our work,are only in vitro.We hypothesized that knockdown of XIAP would inhibit ovarian tumor information and invasion/metastasis and that XIAP would be a good molecular target for cancer therapy.
In this study,we hypothesized that manipulation of short hairpin RNA(shRNA)-induced gene silencing of XIAP would induce therapeutic apoptosis and sensitize drug-resistant human ovarian cancer cells to chemotherapy.To examine our hypothesis,we silenced XIAP expression in human ovarian cancer cells,A2780/DDP,by stable expression of an XIAP-specific siRNA.The knockdown of XIAP expression by siRNA in A2780/DDP cells showed a marked increase in chemosensitivity,besides an obvious inhibition of cell proliferation,tumorigenicity and a notable induction of cell apoptosis.
Objective:To design small interfering RNA(siRNA) targeting to human XIAP gene,construct recombinant adenovirus(pAd-siRNA-XIAP) and transfer it to the human ovarian carcinoma cells A2780/DDP,and observe the effects of siRNA on the gene expression,proliferation,apoptosis, tumorigenicity and chemosensitivity.
Study design:
1.The psiRNA-XIAP expression plasmids targeting to human XIAP gene were designed,synthesized and identified:
mRNA sequences of XIAP gene were got from Genbank.According to the design principles of siRNA,three specific siRNA sequences were selected by BLAST.The template DNA was constructed with these sequences.
The recombinant plasmids psiRNA-XIAP1,2,3 were obtained by annealing and cloning the template DNA into a blank vector pSilencer.Then they were transferred into the strain DH5α,and identified by bacterial colonies PCR,restriction enzyme and sequencing of nucleic acid.
The recombinant plasmids psiRNA-XIAP1,2,3 were transferred into the A2780/DDP cells.Twenty-four hours later,the interference effects of them were identified by RT-PCR and Western blot.
2.The recombinant adenovirus pAd-siRNA-XIAP was constructed,identified and modified:
By digestion of XbaⅠand XholⅠ,the siRNA-XIAP fragment was derived from the best effective psiRNA-XIAP,and cloned into the shuttle plasmid pAdTrack.After the digestion and linearization of PmeⅠ, pAdTrack-siRNA-XIAP was transfected into the E.coli strain BJ5183,which has a adenovirus plasmid pAdEasy-1.Then the homologous recombinant plasmid pAd-siRNA-XIAP was screened by kanamycin resistance test,cut by enzyme BamHⅠ,and identified by gel electrophoresis.After an amplification of electroporation transformation and a digestion and linearization of PmeⅠ, pAd-siRNA-XIAP was transferred into the 293 ceils with liposome Lipofectamine 2000,to modify and construct the recombinant adenovirus Ad-siRNA-XIAP.After ultracentrifugation and purification,the titer of Ad-siRNA-XIAP was identified by dilution assay.
3.The Ad-siRNA-XIAP was transfected into A2780/DDP cells.Then the cell proliferation,cell cycle,ultrastructure,apoptotic rate and multidrug resistance were measured or observed by MTT assay,FCM,DNA Gel assay and AO stain assay.
4.The total RNA of ovarian carcinoma and normal tissue were extracted. Then the mRNA were isolated and purified,and were reverse-transcribed to construct a cDNA probe,which was marked with fluorescent molecule Cy3/Cy5.This probe was hybridized with a gene chip which contains 8464 cDNA genes.The genes,differently expressed after the transfection of Ad-siRNA-XINP to A2780/DDP cell,was identified and analyzed.
Results:
1.The recombinant plasmids psiRNA-XIAP1,2,3 were successfully constructed,which were proved by PCR,restriction enzyme and sequencing of nucleic acid.24h after the three siRNA expression plasmids were transfected into A2780/DDP cells;it was shown that the expression level of XIAP mRNA declined obviously.And results from Western blot assay indicated that the psiRNA-XIAP1 has the most remarkable effect,so it was used to construct the recombinant adenovirus.
2.The recombinant adenovirus Ad-siRNA-XIAP was identified to be successfully constructed.There was bright green fluorescence in the transfected 293 cells.And a high virus titer was got.
3.Transfected by the recombinant plasmid pAdEasy-siRNA-XIAP,the strain A2780/DDP could show some morph changes;its clonic grow could remarkably decrease;its cell cycle could be changed;its apoptosis rate could significantly increase;and the results of DNA Gel assay appear the typical "DNA ladder".
4.Transfected by the recombinant plasmid pAdEasy-siRNA-XIAP,the A2780/DDP cells were sensitized to cisplatin remarkably.Associated with the manipulation of pAdEasy-siRNA-XIAP,Cisplatin can obviously promote the effect of chemotherapy and induce therapeutic apoptosis of tumor cells. According to the research results,this potentiation of pAdEasy-siRNA-XIAP would be relevant to the status and function of XIAP gene in ovarian cancer cells.
5.Some genes were differently expressed after the transfection of Ad-siRNA-XIAP to A2780/DDP cell.After they were identified and analyzed, the research results showed that these genes belonged to different functional groups,including both signal and protein transmission,both oncogene and proto-oncogene,apoptosis genes,and so on.
Conclusion:
1.The recombinant plasmids pSilencer-XIAP1、pSilencer-XIAP2 and pSilencer-XIAP3 were successfully constructed.They could remarkably decrease the mRNA expression level of XIAP gene when transfected to A2780/DDP cells.Of all the three plasmids,pSilencer-XIAP1 has the most significant effect.
2.The recombinant adenovirus Ad-siRNA-XIAP was successfully constructed by pSilencer-XIAP1 and adenovirus pAdEasy.A high titer of virus was got by this concise method,in which merits of RNA polymeraseⅢand promoterH1 were combined.
3.The recombinant adenovirus Ad-siRNA-XIAP obviously inhibited the expression of XIAP gene in A2780/DDp cells.And it has more remarkable effect than the recombinant plasmid psiRNA-XIAP1.
4.Induced by the recombinant adenovirus,RNAi technology can remarkably increase the chemosensitivity of human ovarian cancer cells, A2780/DDP,to cisplatin.XIAP would be a good molecular target for reversing the multidrug resistance of ovarian carcinoma and inducing ovarian cancer cells to apoptosis.
5.Gene chip and RNAi technology are powerful tools to analyze functions of genes.Through them,we have successfully gotten the gene map of genes inhabited by XIAP gene's expression,and gotten the possible positions of XIAP gene's functional accesses.This will be very useful for further researches to clear the mechanisms of multidrug resistance in ovarian carcinoma.
引文
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