新疆维吾尔族高危HPV感染宫颈鳞癌组织中miRNA的初步研究

发布时间：2018-06-22 16:25

本文选题：宫颈鳞状细胞癌 + miRNA　；参考：《新疆医科大学》2014年博士论文

【摘要】：目的：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(invasivecarcinoma 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。方法：1）选择第二代杂交捕获(Hybrid capture,HC2)检测HR-HPV阳性的30例维吾尔族宫颈鳞癌患者，利用巢式PCR检测其组织标本中的HPV16-DNA的表达，多重Taqman探针荧光定量PCR检测HPV16-DNA在组织中的存在状态。2）应用miRNA基因芯片（Agilent Human miRNA8*60K）检测新疆维吾尔族正常宫颈及HPV阳性的宫颈鳞癌组织样本各6个，利用T检验的p值和倍数变化值进行差异miRNA筛选，筛选的标准为上调或者下调倍数变化值"g2.0且P值"f0.05，对差异miRNA进行聚类分析。3）根据基因芯片结果，筛选出19个差异表达的miRNA、5个差异表达的靶基因，进而采用RT-PCR的方法检测其在20例NCE和20例ICC 组织样本中miRNA的表达状况，并结合临床病理资料进行统计学分析；4）在siHa细胞中筛选表达丰度下调的miRNA，挑选出丰度下调的15个miRNA，，经慢病毒包被后经高通量筛选（HTS），筛选出有功能的miRNA，进一步推测其在发病机制中的作用及功能做准备。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㧐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㧐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㩳0.05），临床分期高的患者中表达高于低分期患者（P㩳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（P0.01）,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（P0.05）。4)筛选出siHa细胞中丰度下调的15个miRNA，经慢病毒包被后经高通量筛选，筛选出miRNA-1224，Let7c明显抑制肿瘤细胞的增殖。结论：1）HR-HPV感染是导致宫颈癌的主要因素，而HPV-16是新疆维吾尔族宫颈鳞癌主要的病毒类型，其在组织中多以整合态存在，在宫颈癌发病中起主要作用，HPV-DNA整合状态与宫颈细胞的恶性转化密切相关。2）miRNA-196，miRNA-21， miRNA-17、miRNA-15在HPV阳性的宫颈鳞癌组织中表达较正常组织有统计学差异，并与病理分级、间质浸润深度、临床分期相关，可能在宫颈癌的发生与进展中起重要作用，是宫颈癌诊断和预后的潜在标记物。3)miRNA-1224， Let7c在体外细胞系中明显抑制肿瘤细胞的增殖。
[Abstract]:Objective: 1) to investigate the infection status of high risk human papillomavirus (HR-HPV) in the Uygur cervical squamous cell carcinoma (Uygur squamous cell carcinoma) in Xinjiang, and analyze the relationship between the expression status and cervical squamous cell carcinoma (.2) to study the invasive cervical cancer (invasivecarcinoma of cerv) in the normal cervical (normal cervical epithelium, NCE) and high-risk HPV infection of the Uygur in Xinjiang (invasivecarcinoma of cerv). The difference in expression level in IX, ICC, and its correlation with various clinical indicators in invasive cervical cancer.3) in vitro cell test to verify the biological function of miRNA in HPV positive cervical squamous cell carcinoma. Method: 1) select second generation hybrid capture (Hybrid capture, HC2) to detect 30 cases of Uygur cervical squamous cell carcinoma positive in HR-HPV The patients, using the nested PCR to detect the expression of HPV16-DNA in the tissue specimens, and the multiplex Taqman probe fluorescence quantitative PCR to detect the existence of HPV16-DNA in the tissue.2), the miRNA gene chip (Agilent Human miRNA8*60K) was used to detect 6 samples of the normal cervix of the Uygur and HPV positive cervical squamous cell carcinoma in Xinjiang. And multiplier variation values miRNA screening, screening criteria for up or down multiplier values "g2.0 and P value" f0.05, the difference miRNA clustering analysis.3) according to the gene chip results, screening 19 differentially expressed miRNA, 5 differentially expressed target genes, and using RT-PCR method to detect its in 20 cases NCE and 20 ICC of ICC.
The expression of miRNA in the tissue samples was analyzed in combination with the clinicopathological data. 4) screening the down-regulation of miRNA in siHa cells, selected 15 miRNA down-regulation, and screened the functional miRNA by high throughput screening (HTS) after the lentivirus envelope, and further speculated its role and function in the pathogenesis. Results: 1) 1) the positive rate of HPV-16 in cervical squamous cell carcinoma (SCC) was 96.7%, and there was no significant difference from HC2 (P? 0.05). The integration rate of HPV-16DNA in cervical squamous cell carcinoma was 89.7%, and the expression level of HPV-16DNA was not related to the age of cervical squamous cell carcinoma (P? 0.05), but in pathology, but in pathology The patients with high grade were higher than those with low pathological grade (P? 0.05), and the patients with high clinical stages were higher than those with low stage (P? 0.05); 2) gene chip detection showed that 64 up regulated miRNA, 76 down-regulated miRNA in the Xinjiang Uygur normal cervical tissues and HPV positive cervical squamous cell carcinoma tissues were detected by RT-PCR method in tissue The results showed that the expression of 11 up-regulated miRNA in the tissue was consistent with the gene chip results, and the expression of down regulated miRNA was consistent with the results of gene chip, 5 in contrast to the gene chip results, the coincidence rate of the gene chip was 73.8%.3) miRNA96,17196,21,15 and so on in the squamous cell carcinoma of the cervix and the patients. The clinical stage, pathological grade and interstitial infiltration depth (P0.01) were higher than those with low level in the patients with high level of tumor, and there was no significant relationship with the patient's age, tumor diameter and menopause (P0.05).4). The 15 miRNA in the siHa cell abundances was screened out, and the m was screened by high throughput screening and screened out by high throughput screening. IRNA-1224, Let7c obviously inhibits the proliferation of tumor cells. Conclusion: 1) HR-HPV infection is the main factor leading to cervical cancer, and HPV-16 is the main virus type of cervical squamous cell carcinoma in Xinjiang Uygur, which is mostly integrated in the tissues, plays a major role in the pathogenesis of cervical cancer, and the integration of HPV-DNA is closely related to the malignant transformation of cervical cells. Related.2) miRNA-196, miRNA-21, miRNA-17, miRNA-15 in HPV positive cervical squamous cell carcinoma tissues are significantly different from normal tissues, and are related to pathological classification, interstitial infiltration depth, clinical staging, and may play an important role in the development and progression of cervical cancer. It is a potential marker for the diagnosis and prognosis of cervical cancer,.3) miRNA-1224, Let7c The proliferation of tumor cells was markedly inhibited in vitro.
【学位授予单位】：新疆医科大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R737.33

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