新疆维吾尔族宫颈癌患者癌组织中HPV-DNA与miRNA相互作用的研究

Human papilloma virus (HPV) is considered to be the main factors of cervical cancer,high risk HPV state of integration is a feature of cerical cancer gene which is closely related with the progress of the cervical cancer, and at the same time, research shows that small RNA (miRNA) may get the adjusting control of the high risk HPV , the exact mechanism which the miRNA and HPV is mutual regulation of gene expression is unclear, prompted the researchers further research. Through the gene chip, This study aims to reseach miRNA expression spectrum in xinjiang uygur cervical cancer patients with high-risk HPV negative, positive carcinoma tissues and normal tissues, probe into the patients of the xinjiang Uygur miRNA whether have expression differences, wether presence of racial diversity research. The application of nested PCR to detect cervical cancer and normal tissue HPV - 16 DNA, and analyse the difference of HPV-DNA detection rate in the organization of different groups patients and the correlation between the detection rate and cervical cancer clinical stage, analysing the relationship between HPV - DNA and miRNA, and understand relationship between HPV -DNA and miRNA in the cervical cancer tissues and find the miRNA molecular associated with high risk HPV infection and its related target genes,perhaps as the early screening of cervical cancer biomarkers for the prevention and treatment has a certain significance. We want to verify that wether there are mutual regulation between miRNA and HPV in vitro experiments.

人乳头瘤病毒感染（HPV）是宫颈癌的主要因素，其整合状态是宫颈癌的一个特征，与宫颈癌的进展密切相关，同时研究显示微小RNA（miRNA）可能受到高危HPV的调控，两者是否相互调控基因表达的机制还不清楚，促使研究者研究。本研究通过基因芯片对新疆维吾尔宫颈癌患者中高危HPV阴性、阳性癌组织及正常组织中miRNA表达谱研究，探讨不同组患者miRNA是否有表达差异性，有无种族差异性的研究，应用巢式PCR检测宫颈癌及正常组织中HPV-16DNA，分析各组患者组织中HPVDNA检出率的差异，以及检出率与宫颈癌临床分期的相关性，分析HPV-DNA与miRNA之间的相关性,了解宫颈癌组织中HPV与miRNA之间的相关性，找到与高危HPV感染相关的miRNA 分子及其相关的靶基因，作为宫颈癌的早期筛查的生物标记可能对于宫颈癌的预防及治疗有一定的意义.在体外试验中验证miRNA与HPV是否存在相互调控的作用。

1）探讨高危人乳头瘤病毒(HR-HPV)在新疆维吾尔族宫颈鳞癌患者中的感染状态，分析其表达状态与宫颈鳞癌的相关性。2) 研究miRNA在新疆维吾尔族正常宫颈( normal cervical epithelium，NCE)与高危HPV感染的侵袭性宫颈癌( invasive carcinoma of cervix，ICC)中的表达水平差异，及其在侵袭性宫颈癌中与各临床指标间的相关性。3)在体外细胞实验中研究验证miRNA在HPV阳性宫颈鳞癌细胞中的生物学功能。结果;1）宫颈鳞癌(SCC)患者HPV-16阳性率为96.7%，与HC2检出率差异无统计学意义(p﹥0.05)； HPV-16DNA在宫颈鳞癌组织中以整合态存在，其整合率为89.7%；HPV-16DNA的表达水平与宫颈鳞癌患者的年龄、有无淋巴结转移无关（P﹥0.05）， 但在病理分级高的患者中表达高于病理分级低的患者（P﹤0.05），临床分期高的患者中表达高于低分期患者（P﹤0.05）；2）基因芯片检测显示在新疆维吾尔族正常宫颈组织及HPV阳性的宫颈鳞癌组织中有64个上调的miRNA，76个下调的miRNA，经RT-PCR方法在组织中验证基因芯片结果显示，在组织中11个表达上调的miRNA的表达与基因芯片结果一致，表达下调的miRNA其中3个与基因芯片结果一致，5个与基因芯片结果相反, 基因芯片符合率达73.8%。3）miRNA96、17、196、21、15等在宫颈鳞癌组织的表达水平与患者临床分期、病理分级和间质浸润深度有关（P<0.01）,在级别高的肿瘤患者中表达高于级别低的患者，而与患者年龄、肿瘤直径大小和绝经与否均无明显关系（P>0.05）。4)HPV16-E6及HPV16-E7基因沉默后显著降低siha细胞的HPV16-E6-mRNA及HPV16-E7-mRNA表达（P<0.05）；HPV16-E6及HPV16-E7蛋白的表达量显著下降；HPV16-E6及HPV16-E7基因沉默后显著抑制了siha细胞的生长（P<0.05）；siha细胞的凋亡显著增加（P<0.01）；细胞样本验证的5个mRNA中hsa-miR-30c-5p、hsa-miR-140hsa-miR-574-5p是E7沉默的细胞表达有显著差异，hsa-miR-1290、hsa-miR-130b-3p是E6和E7都沉默的细胞中表达有差异。