【作者】 刘玲；

【导师】 李力；

【作者基本信息】 广西医科大学 ， 肿瘤学， 2014， 博士

【摘要】 卵巢癌在女性生殖道恶性肿瘤发病率中占第三位，但是死亡率高居第一。化疗是治疗卵巢上皮癌重要手段之一，但多药耐药的产生又是导致化疗失败的中心环节。卵巢上皮癌多药耐药机制十分复杂，目前研究认为可能涉及肿瘤细胞内化疗药物的外排、DNA损伤与修复、细胞凋亡异常和信号传导通路障碍等分子机制，但是至今仍未找到安全有效的逆转耐药靶点。本研究拟通过运用miRNA芯片技术和基因芯片技术，以及生物信息学等多样化研究手段，从临床到基础研究，然后回归临床的研究桥梁，阐述卵巢上皮癌多药耐药分子机制，为寻找安全有效的逆转卵巢上皮癌多药耐药治疗靶点提供理论基础。研究目的：筛选卵巢上皮癌多药耐药相关miRNA表达谱。材料和方法：采用Agilent miRNA芯片，检测5例卵巢上皮癌耐药组织、5例敏感组织、5例卵巢良性肿瘤和5例正常卵巢组织的miRNA表达情况，运用Fc差异倍数，获得了卵巢上皮癌耐药组和敏感组差异大于2倍的上调或下调miRNA。用多个在线软件分别预测上调和下调差异miRNA对应的靶基因，并运用GO分析和Pathway通路富集等生物信息学方法分析预测到的靶基因潜在的生物学功能和参与的信号通路。用QRT-PCR验证芯片结果，并扩大样本量检测目的miRNA的表达情况，并分析miRNA表达水平与临床病理因素关系。结果：miRNA芯片筛选出与卵巢上皮癌多药耐药相关的差异表达miRNA共62个，其中有42个miRNA在卵巢上皮癌耐药组织中上调、20个miRNA下调，上调miRNA预测到1391个靶基因，下调miRNA预测到1231个靶基因。GO分析结果显示：上调表达的miRNA预测到的靶基因主要参与了细胞蛋白质代谢过程和RNA聚合酶II启动子的转录调控过程，下调表达的miRNA预测的靶基因主要参与转录调控、蛋白酶参与的细胞蛋白质分解代谢过程以及细胞蛋白质分解代谢等过程。KEGG Pathways通路分析提示上调表达的miRNA预测的靶基因主要参与TGF-beta信号通路和粘着连接信号通路。疾病富集分析部分靶基因与卵巢上皮癌或卵巢肿瘤相关。QRT-PCR验证miRNA芯片结果，差异表达的miR-200a-3p、miR-382-5p、miR-1、miR-152和miR-299-5p与芯片表达趋势一致，扩大临床样本验证亦得到了一致的结果，其中miR-200a-3p在卵巢癌耐药组织中下调0.31倍，差异有统计学意义（P＜0.01）；miR-382-5p、miR-152和miR-299-5p分别上调3.53倍、2.69倍和2.70倍，差异有统计学意义（P＜0.01）；miR-1上调1.14倍，差异无统计学意义（P＞0.05）。结论：我们通过miRNA芯片技术筛选出了跟卵巢上皮癌耐药相关miRNA表达谱，同时靶基因预测提示我们一个miRNA可以调控多个靶基因，而一个靶基因又可能同时受几个miRNA的调控，可见miRNA的调控机理是十分复杂的。生物信息学分析提示差异表达miRNA预测到的靶基因广泛参与多个生物学过程，我们可以推测这些异常表达的miRNA可能通过调控特定功能的靶基因，参与重要信号传导通路，在肿瘤的生长及肿瘤耐药产生过程中发挥重要角色。后期需要进一步研究以认识这些差异miRNA参与卵巢上皮癌耐药发生的相关分子机制。研究目的：筛选卵巢上皮癌多药耐药相关mRNA表达谱。材料和方法：采用Agilent lncRNA芯片，检测5例卵巢上皮癌耐药组织、5例敏感组织、5例卵巢良性肿瘤和5例正常卵巢组织的mRNA表达情况，获得与卵巢上皮癌耐药组和敏感组差异大于2倍的上调或下调mRNA。运用GO分析和Pathway通路富集等生物信息学方法分析差异表达mRNA潜在的生物学功能和参与的信号通路。用QRT-PCR验证芯片结果，并扩大样本量检测目的mRNA的表达情况，并分析mRNA表达水平与临床病理因素关系。结果：mRNA芯片筛选出于卵巢上皮癌多药耐药相关差异表达mRNA共855个，其中在卵巢上皮癌耐药组织中上调表达的mRNA598个，下调表达的257个。生物信息学分析结果显示上调mRNA主要参与细胞粘附、生物黏附、调节细胞增殖、免疫反应以及血管发育等31个生物学过程，下调mRNA未富集到显著相关生物学过程。KEGGPathway分析结果显示上调mRNA主要参与了粘着斑和ECM-受体相互作用的信号传导途径。QRT-PCR验证芯片结果，KIT、HGF、CSF1R、PDGFRB和KIAA1804与芯片表达趋势一致，扩大临床样本验证亦得到了一致的结果，其中KIT、HGF、CSF1R、PDGFRB基因在EOC耐药组织中分别上调3.81倍、2.74、2.03倍和2.92倍，差异有统计学意义（P﹤0.05），而KIAA1804基因则下调0.42倍，差异有统计学意义（P﹤0.05）。结论：利用基因芯片基础筛选出了跟卵巢上皮癌多药耐药相关的mRNA表达谱，丰富了目前已知的跟耐药相关的靶基因数据。生物信息学提示差异表达mRNA多参与细胞粘附、调节细胞增殖、免疫反应等生物学过程，以及部分mRNA被富集在粘着斑和ECM-受体相互作用的信号通路上，提示这些差异基因可能通过多种途径参与卵巢上皮癌多药耐药的发生，后期需要进一步研究以认识这些差异miRNA参与卵巢上皮癌耐药发生的相关分子机制。研究目的：构建与卵巢上皮癌多药耐药相关miRNA-mRNA调控网络。材料和方法：对筛出的卵巢上皮癌多药耐药相关的miRNA差异表达谱和mRNA差异表达谱信息运用MAGIA软件进行整合分析，并选取miRNA跟mRNA负相关的miRNA-mRNA，构建卵巢上皮癌耐药相关miRNA-mRNA调控网络图，并用QRT-PCR方法验证关键位点miRNA和mRNA的表达，并分析miRNA-mRNA表达水平与临床病理因素关系。结果：miRNA表达谱和mRNA表达谱关联后，共得到53对负关联的miRNA/miRNA，其中包括10个miRNA和43个mRNA，被富集到的靶基因最多的是miR-429和miR-381，对应靶基因数目分别为16个和8个，而被最多miRNA关联调控的mRNA分别是ERBB4、PRKCE和PPP1R9A，印证了一个miRNA调控多个靶基因，而一个靶基因可受到多个miRNA调控。扩大临床样本QRT-PCR验证结果：相比于卵巢癌敏感组，miR-429、miR-373-5p在卵巢耐药组中分别下调0.35倍和0.39倍，差异有统计学意义（P＜0.01）；对应靶基因KANK2、ZEB2和CTSD分别上调2.56倍、3.15倍和1.89倍，差异有统计学意义（P＜0.05）；miR-381、miR-495和miR-410在耐药组中分别上调2.36倍、2.46倍和2.42倍，差异有统计学意义（P＜0.01）；对应的靶基因ERBB4、TIAL1、FGFRL1和MPPED2分别下调0.46倍、0.38倍、0.29倍和0.43倍，差异有统计学意义（P＜0.05）。结论：经过QRT-PCR验证以及文本挖掘，我们认为构建与卵巢上皮癌多药耐药相关的miRNA-mRNA调控网络图比较可靠，提示了在卵巢癌耐药组织中miRNA和mRNA复杂的调控网络关系，亦从多基因多靶点角度阐述了卵巢上皮癌多药耐药的发生机制。而关键位点的miRNA及其负调控mRNA具体以何种方式和途径参与EOC多药耐药机制的发生，需要后期做进一步深入的功能学研究，以期找到确实可靠的逆转卵巢上皮癌MDR被关键靶点，指导临床更好得防治卵巢上皮癌多药耐药的发生，提高患者预后和生活质量。更多还原

【Abstract】 Ovarian cancer accounts for third place in the female reproductive tract cancer incidence,but the mortality rate ranks first.Chemotherapy is an important treatment for epithelial ovariancancer(EOC).But multidrug resistance of EOC is a vital link which lead to Chemotherapyfailure. Present study suggests those molecular mechanisms of multidrug resistance of EOCmight involve intracellular chemotherapeutic drug efflux,DNA damage and repair, abnormalMolecular mechanisms of apoptosis and signal transduction pathway disorders. But has yet tofind a safe and effective drug target for reversal multidrug resistance.This study intends todiversify through the use of research tools miRNA microarray technology and gene chiptechnology, and bioinformatics, from clinical studies to basic research and finally returnclinical strategies.Looking for multi-drug resistance molecular mechanism in ovarian cancerfor finding security effective reversal of multidrug resistance. Objective Construction miRNA exprssion profiles in epithelial ovarian cancer multidrugresistance.Materials and Methods Using Agilent miRNA chip,detect the miRNAs expressionbetween the five cases of epithelial ovarian cancer tissues with5cases of drug-resistant tissueand5sensitive one,5cases of benign ovarian tumors and5cases of normal ovarian tissue.Ttest and Fc differences was be used to find greater different than2-fold up-regulation ordown-regulation in miRNAs. We mined the data of microRNA-target genes via multiplesonline software.We analysised the target gene of up-regulation or down-regulation miRNAsby GO enrichment analysis and Pathway bioinformatics analysis to predict potential targetgenes and signaling pathways involved in the biological functio. And then we validate themicroarray results using QRT-PCR analysis and expand the sample size to detect theexpression of purpose miRNA.And analyze the relationships between miRNA expressionlevels and clinicopathological factors.Results A total of62microRNAs expressed differentially related to EOCchemoresistance,there were42miRNAsincreased in ovarian cancer resistance tissue and20ones decreased.And1391target genes were predicted by up-regulation miRNAs,1231targetgenes were predicted by down-regulation miRNAs.GO analysis showed:miRNAs ofup-regulated predicted target genes are involved in regulation of cellular protein metabolicprocess and regulation of transcription from RNA polymerase II promoter.Down-regulateddifferentially predicted target genes are involved in regulation of transcription, proteolysisinvolved in cellular protein catabolic process, cellular protein catabolic process and otherclass of biological process-es.Pathways pathway analysis is enriched to TGF-beta signalingpathway and Adherens junction a signaling pathway for up-regulated predicted target genes.Enrichment for disease analysis showed that part of the target genes associated with epithelialovarian cancer or ovarian cancer.We screened four upregulated genes of miR-382-5p,miR-1,miR-152and miR-299-5p and one down-regulated miR-200a-3p carried by QRT-PCRmicroarray validation. The result matched with the microarray results,expand clinical sampleobtained consistent results,which miR-200a-3p was downregulated0.31-fold in ovariancancer drugresistant tissue via drugsensitive tissue,the difference was statistically significant (P<0.01);miR-382-5p,miR-152and miR-299-5p were raised3.53times,2.69times and2.70times, the differences were statistically significant (P<0.01),miR-1was raised1.14times,butthe difference was not statistically significant (P>0.05).Conclusion We screened out miRNA expression profiling associated with epithelialovarian cancer multidrug-resistance by miRNA microarray.the result of predicting targetgeneprompted that miRNA can regulate multiple target genes, and a target gene may also beregulated by several of miRNA.Visiblely miRNAregulatory network is very complex.Bioinformatics analysis showed differential expression of miRNA target genes predicted tobroad participation in a number of biological processes,we speculated that these abnormalexpression of miRNA expression is likely through regulatedof target gene take part in cellproliferation and apoptosis related factors,and involved in several signal transductionpathways,may play an important role in tumor growth and cell prognosis.There aesneed forfurther indepth study of these differentially expressed miRNA to identify potential Molecularmechanisms underlying drug resistance in ovarian cancer,as well as clinical diagnosis andtreatment of individual markers, even to effectively reverse the resistance of epithelial ovariancancer treatment provide new ideas. Objective Construction mRNA exprssion profiles in epithelial ovarian cancer multidrugresistance.Materials and Methods Using Agilent lncRNA chip,detect themRNAs expressionbetween the five cases of epithelial ovarian cancer group with5cases of drug-resistant tissueand5sensitive one,5cases of benign ovarian tumors and5cases of normal ovarian tissue.Ttest and Fc differences was be used to find greaterdifferent than2-fold up-regulation ordown-regulationin mRNAs.We analysised the target gene of up-regulation or down-regulationmiRNAs by GO enrichment analysis and Pathway bioinformatics analysis to predict thebiological functions and involved inwhich signaling pathways of the differential expression genes. And then we validatethe microarray results using QRT-PCR analysis and expand thesample size to detect the expression of purpose mRNA.And analyze the relationships betweenmiRNA expression levels and clinicopathological factors.Results A total of855microRNAsexpressed differentially related to EOCchemoresistance,there were598mRNA increased in ovarian cancer resistance tissue and257ones were decreased.Bioinformatics analysis showed that: up-regulation mRNA involved incell adhesion,biological adhesion,regulation of cell prolifera-tion,immune response,bloodvessel development, and other class of biological processes.Down-regulation mRNA is notinvolved in significantly enrichment related biological processes.KEGG Pathway analysisshowed upregulated mRNA related pathways involved in Focal adhesion and ECM-receptorinteraction.We screened four upregulated genes of KIT,HGF,CSF1R,PDGFRB and onedown-regulated ERBB4gene carried by QRT-PCR microarray validation,the results matchedwith the microar-ray results,expand clinical sample obtained consistent results,whichKIT,HGF,CSF1R, PDGFRB genes in EOC drugresistance group were raised3.81times and2.92times2.74,2.03times via drugsensitive group,the difference was statistically significant(P<0.05), while the KIAA1804gene is down-regulated0.42-fold, difference was statisticallysignificance (P<0.05).Conclusions Microarray-based screening out mRNA expression profiles associated withepithelial ovarian cancer multidrug resistance,which enriching the current data known targetgenes associated with drug resistance.Bioinformatics analysis of the differentially expressedmRNA principalinvolved in cell adhesion,biological adhesion,regulation of cell proliferation,immune response, as well as part of the mRNA is enriched in Focal adhesion and ECM-receptor interaction, suggesting that these differentially expressed genes may be involved inepithelial ovarian cancer multi-drug resistant through a variety of ways. ObjectiveConstruction miRNA-mRNA regulatory networks associated with epithelialovarian cancer multidrug resistance.Materials and Methods To screen out the miRNA and mRNA expression profiling ofepithelial ovarian cancer multidrug resistance,we analysised the differences expressionmiRNA and mRNA in expression profiling information using MAGIA meta-analysis software,and select miRNA negatively correlated with mRNA of miRNA/mRNA,build miRNA-mRNAregulatory network diagram associated with epithelial ovarian cancer drug resistance, andverify that the key expression of miRNA and mRNA using QRT-PCR methods.Results Received a total of53pairs of miRNA-miRNA for negative association,including10miRNA and43mRNA.miR-429and miR-381were enriched by many targetgene, corresponding to the number of target genes was16and8,respectively.The mRNA ofERBB4,PRKCE and PPP1R9A were associated with several miRNA. It was confirmed thatmiRNA regulation a multiple target genes, which gene subject to more than one miRNAregulation. Expanded clinical samples QRT-PCR validation results: compared to ovariancancer sensitive group, miR-429, miR-373-5p in ovarian resistance group were down0.35-fold and0.39-fold,the difference was statistically significant (P<0.01); Thecorresponding target genes KANK2, ZEB2and CTSD were raised2.56times,3.15times and1.89times, the difference was statistically significant (P<0.05); miR-381, miR-495andmiR-410in the drug group were raised2.36times,2.46times and2.42times,the differencewas statistically significant (P<0.01); corresponding target genes ERBB4, TIAL1, FGFRL1and MPPED2were down0.46times,0.38times,0.29times and0.43times, the differencewas statistically significant (P<0.05).Conclusion After QRT-PCR validation and text mining, we believe that to buildmiRNA-mRNA regulatory network diagram with epithelial ovarian cancer multidrugresistance associated with more reliable.It was display that the miRNA and mRNA complexregulatory network relationshipsin ovarian cancer resistance tissues,also from the perspectiveof multiple genes and multi-targets mechanisms of epithelial ovarian cancer multidrugresistance.The key miRNAand its negative regulation of mRNA in which specific ways andmeans to be involved in multidrug resistance mechanisms EOC, needs to make furtherin-depth functional studiesin order to find a really reliable way that can reversal the epithelialovarian cancer MDR,and happened to better guide clinical Chemical therapy and a effectiveprevention avoid multidrug resistance in ovarian cancer patient,expec-ting improve theprognosis and quality of life for EOC patients.更多还原