microRNA-142-3p对TET2基因的调控以及对卵巢癌细胞SKOV-3增殖的影响

发布时间：2018-01-08 01:24

本文关键词：microRNA-142-3p对TET2基因的调控以及对卵巢癌细胞SKOV-3增殖的影响　出处：《河北医科大学》2017年硕士论文　论文类型：学位论文

更多相关文章： microRNA-142-3p TET2 卵巢癌 SKOV-3

【摘要】：背景:卵巢癌作为女性系统最常见的恶性肿瘤之一,其发病率列居第三位,仅次于子宫颈癌和子宫内膜癌,死亡率为妇科肿瘤首位。卵巢作为重要的内分泌器官,具有复杂的生理功能和特殊的组织结构,这就导致卵巢恶性肿瘤,在早期诊断和恶性程度鉴别上存在很大的困难,在发现患病之后经常已经是晚期,失去了最好的治疗时机。因此,各种关于卵巢癌的临床基础研究备受关注。目前关于卵巢癌和miRNAs研究已成为大家关注的热点,例如:miR-9,miR-203,miR-30a-5p在卵巢癌细胞中呈明显高表达,miR-96-5p和miR-200c-3p呈明显的低表达。但是关于miRNAs对卵巢癌影响的具体研究还没有太深入的报道。在卵巢癌病程中,miRNAs不仅可以影响癌细胞的增殖、分化等,对其耐药及基因治疗方面也存在作用。通过大量的文献检索,网络数据库及生物信息学分析,发现miRNA-142-3p在卵巢癌中呈明显的高表达。且有大量的文献报道,miR-142-3P在众多癌症的发病过程中都发挥着重要作用。目的:构建能够过表达miR-142-3p小分子的慢病毒载体,研究其对TET2基因的调控作用,探讨其对卵巢癌细胞株SKOV-3细胞增殖的影响,阐述miR-142-3p在卵巢癌发病过程中的分子机制,为临床卵巢癌的治疗方面的研究提供理论依据和新的思路。miRNAs对基因调控的理论研究和技术相对比较成熟,通过研究miRNA-142-3p对TET2的调控作用,来研究其对卵巢癌的发病机制的调控作用,在理论和实际操作上都是可行的。本课题采用的实验方法和技术如:载体构建,慢病毒包装,Real-Time PCR,Western blot等,都是国内外广泛采用的实验技术,并且都有相应的试剂盒。实验室具备该课题操作的所有仪器支持。方法:1根据人miR-142-3p基因序列,化学合成含有miR-142-3p单链小分子序列,同时带有茎环序列的碱基链,退火合成双链后插入到病毒包装所需要的载体上,应用双酶切和碱基序列测序两种实验方法验证构建结果,利用脂质体转染法进行慢病毒的包装,采用第二代慢病毒载体转染三质粒的方式构建,并共转染到HEK-293细胞系中,包装成功后测定病毒的滴度;另外合成miR-142-3p的inhibitor试剂。2应用micro RNA、miRanda及Target Scan等网络数据库信息,预测可与miR-142-3p碱基互补,且可能存在靶向作用关系的TET2作为研究的靶基因,针对TET2的3’非翻译区(3’UTR)设计特异性扩增引物,并将扩增好的序列构建到p MIR-Report表达载体中,经酶切及测序方法验证成功后,利用脂质体法将构建好的p Sico R-miR-142-3p与p MIR-ReportTET2共转染到293细胞中,同时设计空白对照组及抑制组,应用双荧光素酶报告基因检测系统,以海蜃荧光素酶作为内参对照,进行荧光素酶的活性检测,以验证miR-142-3p与TET2之间的靶向作用关系。3将包装成功的LV-miR-142-3p慢病毒及miR-142-3p inhibitor试剂分别侵染SKOV-3细胞系,24h后观察侵染结果,并收集细胞及上清液备后续实验。分别提取各组细胞的m RNA及总蛋白,应用RT-q PCR方法检测每组间miR-142-3p及靶基因的m RNA的表达情况,应用Western blot法检测TET2蛋白表达水平,同时用分度仪检测蛋白灰度值,应用MTT法检测miR-142-3p对SKOV-3细胞的增殖的影响,采用SPSS20.0软件进行统计学分析。结果:1成功构建了p Sico R-miR-142-3p的慢病毒载体,并成功包装了可过表达miR-142-3p的慢病毒颗粒,同时测定的病毒滴度为2.66×105 pfu/m L。2 miRNA相关的网站内容显示miR-142-3p和TET2的3’UTR存在碱基互补,可能存在相互作用关系,因此可通过双荧光素酶报告基因系统检测荧光表达情况来证实二者间是否存在靶向作用关系,结果发现将miR-142-3p过表达后,靶基因的荧光表达情况明显改变:野生型与突变型相比,表达量明显下调,下降约4.09倍(P0.05);转染inhibitor试剂后,靶基因TET2的荧光表达活性,其野生型比突变型有所上调。在各组野生型之间的比较可以看出,过表达组与空白对照组相比,明显降低,约4.52倍(P0.05);而过表达组较inhibitor组的表达值,又呈明显的上升趋势,升高可达5.18倍(P0.05)。3病毒侵染细胞系后,小RNA的表达情况显示:过表达组明显高于SKOV-3原始细胞组,升高约17.5倍(P0.05),而靶基因TET2 m RNA的检测结果显示过表达组与SKOV-3原始细胞组相比下调近7.8倍(P0.05);当miR-142-3p被抑制后,miR-142-3p的表达情况明显降低,与SKOV-3原始细胞组的小RNA表达情况相比,下调约18倍(P0.05),与此同时靶基因TET2的表达水平上调,约3.9倍(P0.05)。4 Western blot结果显示:过表达组较未处理组相比,条带明显减弱,转染miR-142-3p inhibitor后,TET2的条带明显增粗,即表达量升高,蛋白的灰度值检测更为直观的展现了miR-142-3p病毒刺激后TET2蛋白的表达水平。5 MTT实验证实,miR-142-3p可显著抑制SKOV-3的增殖。结论:成功构建了可过表达miR-142-3p的慢病毒颗粒,进一步证实过表达miR-142-3p可有效抑制SKOV-3细胞中TET2基因m RNA和蛋白水平的表达,并抑制SKOV-3细胞的增殖。为今后卵巢癌的基因治疗研究提供新的思路和研究靶点。
[Abstract]:Background: one of the ovarian cancer as the most common female malignant tumor, its incidence ranks the third, second only to cervical cancer and endometrial cancer, mortality rate of gynecological malignancies. The ovary is an important endocrine organ, has the structure of complicated physiological function and special group, which leads to the existence of malignant ovarian tumor great difficulty in early diagnosis and malignant degree of identification, found after the illness is often late, lost the best treatment time. Therefore, a variety of clinical and basic research on ovarian cancer has attracted much attention. At present about ovarian cancer and miRNAs research has become the focus of attention, such as: miR-9, miR-203, miR-30a-5p were significantly high expression in ovarian cancer cells, miR-96-5p and miR-200c-3p expression was low obviously. But the specific research on the effects of miRNAs on ovarian cancer has not been reported in too deep into the egg. In the course of ovarian cancer, miRNAs can not only affect cancer cell proliferation, differentiation, but also the existence of the drug resistance and gene therapy. Through literature retrieval, analysis of network database and bioinformatics, that was significantly higher miRNA-142-3p expression in ovarian cancer. And there is a lot of literature reports, miR-142-3P play plays an important role in the pathogenesis of many cancers. Objective: to construct a lentiviral vector expressing miR-142-3p had small molecules, study its role in the regulation of TET2 gene, and to investigate its effect on the proliferation of ovarian cancer cell line SKOV-3, describes the molecular mechanism of miR-142-3p in the pathogenesis of ovarian cancer, to study the clinical treatment ovarian cancer and provide a theoretical basis and new ideas of.MiRNAs theory and technology of gene regulation is relatively mature, through the study of miRNA-142-3p's effects on TET2, to Study on the regulation of ovarian cancer pathogenesis, it is feasible in theory and actual operation. Experimental methods and techniques used in this paper such as: vector, lentivirus packaging, Real-Time PCR, Western blot, is the experimental technology widely used at home and abroad, and have the corresponding kit this subject has all the instruments. The laboratory operations support. Methods: 1 according to the sequence of human miR-142-3p gene and chemical synthesis of single stranded molecule containing miR-142-3p sequence, chain with stem loop sequence, carrier annealing of synthetic double stranded inserted into the virus packaging required, application of double enzyme digestion and sequencing of two experimental bases the construction method to verify the results, lentivirus by liposome transfection packaging, constructed by the second generation lentivirus vector transfected three plasmid, and were transfected into HEK-293 cell line, after the success of packaging The titer of virus; also the synthesis of miR-142-3p inhibitor using micro RNA miRanda reagent.2, Target and Scan network database information, forecasting can be complementary with miR-142-3p bases, and there may be targeting the relationship between TET2 as a target gene of TET2, according to the 3 'untranslated region (3' UTR) design specific amplification primer sequence and amplification well constructed into P expression vector MIR-Report. After enzyme digestion and sequencing method after successful verification, using liposome method to construct P Sico R-miR-142-3p and P MIR-ReportTET2 were transfected into 293 cells, while the design of the blank control group and inhibition group, using the dual luciferase reporter assay system. The sea mirage luciferase as internal control, active detection of luciferase, to verify the miR-142-3p and TET2 targeting.3 packaging LV-miR-142-3p lentivirus successfully and mi R-142-3p inhibitor kit respectively infected SKOV-3 cells, observation of infection after 24h, and collect the cells and supernatant by subsequent experiments. M cells were RNA and total protein were extracted, the expression of M RNA using RT-q PCR method to detect each group between miR-142-3p and target genes, the expression level of TET2 protein was detected by Western blot method, at the same time protein gray detection instrument index value, using MTT method to study the effect of miR-142-3p on proliferation of SKOV-3 cells, using SPSS20.0 software for statistical analysis. Results: 1 we successfully constructed P Sico R-miR-142-3p slow virus vector, and the successful packaging of the over expression of miR-142-3p lentivirus particles, simultaneous determination of virus titer was 2.66 * 105 pfu/m L.2 miRNA related web content display miR-142-3p and TET2 3 UTR are complementary base, there may be interactions, so by dual luciferase The fluorescence expression of reporter gene system to confirm whether targeting relationship exists between the two, the results will be found after overexpression of miR-142-3p significantly alters the expression of target genes: fluorescence compared to the wild type and mutant, the expression decreased significantly, decreased about 4.09 times (P0.05); inhibitor transfection reagent, fluorescent target gene the expression of TET2 activity than the wild type of mutant increased. Compared between groups of wild type showed that over expression group compared with the control group, decreased about 4.52 times (P0.05); and the expression of group than in inhibitor group, and showed a rising trend, rising up to 5.18 times (P0.05) of.3 virus infected cell lines, showed expression of small RNA: over expression group was significantly higher than that of the original SKOV-3 cell group, increased about 17.5 times (P0.05), TET2 m RNA and target gene detection results showed that overexpression of SKOV-3 and the original group The cell group reduced compared to nearly 7.8 times (P0.05); when miR-142-3p is inhibited, the expression of miR-142-3p decreased significantly, and the original SKOV-3 small cell group RNA expression compared by about 18 times (P0.05), at the same time the up-regulated expression of the target gene of TET2, about 3.9 times (P0.05).4 Western blot showed that over expression group than in untreated group compared with transfection of miR-142-3p significantly decreased after inhibitor, TET2 bands were obviously thickening, namely protein expression increased, the gray value of detection to confirm that the expression level of.5 MTT experiment shows the miR-142-3p virus after stimulation of TET2 protein, miR-142-3p can significantly inhibit SKOV-3 proliferation. Conclusion: we have successfully constructed a lentivirus expressing miR-142-3p particles, further confirmed that overexpression of miR-142-3p can effectively inhibit TET2 gene in SKOV-3 cells m RNA and protein levels, and inhibit SKOV-3 cell growth It provides new ideas and targets for the study of gene therapy for ovarian cancer in the future.

【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.31

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