本文關(guān)鍵詞：microRNA-142-3p對TET2基因的調(diào)控以及對卵巢癌細胞SKOV-3增殖的影響　出處：《河北醫(yī)科大學》2017年碩士論文　論文類型：學位論文

  更多相關(guān)文章： microRNA-142-3p TET2 卵巢癌 SKOV-3

【摘要】：背景:卵巢癌作為女性系統(tǒng)最常見的惡性腫瘤之一,其發(fā)病率列居第三位,僅次于子宮頸癌和子宮內(nèi)膜癌,死亡率為婦科腫瘤首位。卵巢作為重要的內(nèi)分泌器官,具有復雜的生理功能和特殊的組織結(jié)構(gòu),這就導致卵巢惡性腫瘤,在早期診斷和惡性程度鑒別上存在很大的困難,在發(fā)現(xiàn)患病之后經(jīng)常已經(jīng)是晚期,失去了最好的治療時機。因此,各種關(guān)于卵巢癌的臨床基礎研究備受關(guān)注。目前關(guān)于卵巢癌和miRNAs研究已成為大家關(guān)注的熱點,例如:miR-9,miR-203,miR-30a-5p在卵巢癌細胞中呈明顯高表達,miR-96-5p和miR-200c-3p呈明顯的低表達。但是關(guān)于miRNAs對卵巢癌影響的具體研究還沒有太深入的報道。在卵巢癌病程中,miRNAs不僅可以影響癌細胞的增殖、分化等,對其耐藥及基因治療方面也存在作用。通過大量的文獻檢索,網(wǎng)絡數(shù)據(jù)庫及生物信息學分析,發(fā)現(xiàn)miRNA-142-3p在卵巢癌中呈明顯的高表達。且有大量的文獻報道,miR-142-3P在眾多癌癥的發(fā)病過程中都發(fā)揮著重要作用。目的:構(gòu)建能夠過表達miR-142-3p小分子的慢病毒載體,研究其對TET2基因的調(diào)控作用,探討其對卵巢癌細胞株SKOV-3細胞增殖的影響,闡述miR-142-3p在卵巢癌發(fā)病過程中的分子機制,為臨床卵巢癌的治療方面的研究提供理論依據(jù)和新的思路。miRNAs對基因調(diào)控的理論研究和技術(shù)相對比較成熟,通過研究miRNA-142-3p對TET2的調(diào)控作用,來研究其對卵巢癌的發(fā)病機制的調(diào)控作用,在理論和實際操作上都是可行的。本課題采用的實驗方法和技術(shù)如:載體構(gòu)建,慢病毒包裝,Real-Time PCR,Western blot等,都是國內(nèi)外廣泛采用的實驗技術(shù),并且都有相應的試劑盒。實驗室具備該課題操作的所有儀器支持。方法:1根據(jù)人miR-142-3p基因序列,化學合成含有miR-142-3p單鏈小分子序列,同時帶有莖環(huán)序列的堿基鏈,退火合成雙鏈后插入到病毒包裝所需要的載體上,應用雙酶切和堿基序列測序兩種實驗方法驗證構(gòu)建結(jié)果,利用脂質(zhì)體轉(zhuǎn)染法進行慢病毒的包裝,采用第二代慢病毒載體轉(zhuǎn)染三質(zhì)粒的方式構(gòu)建,并共轉(zhuǎn)染到HEK-293細胞系中,包裝成功后測定病毒的滴度;另外合成miR-142-3p的inhibitor試劑。2應用micro RNA、miRanda及Target Scan等網(wǎng)絡數(shù)據(jù)庫信息,預測可與miR-142-3p堿基互補,且可能存在靶向作用關(guān)系的TET2作為研究的靶基因,針對TET2的3’非翻譯區(qū)(3’UTR)設計特異性擴增引物,并將擴增好的序列構(gòu)建到p MIR-Report表達載體中,經(jīng)酶切及測序方法驗證成功后,利用脂質(zhì)體法將構(gòu)建好的p Sico R-miR-142-3p與p MIR-ReportTET2共轉(zhuǎn)染到293細胞中,同時設計空白對照組及抑制組,應用雙熒光素酶報告基因檢測系統(tǒng),以海蜃熒光素酶作為內(nèi)參對照,進行熒光素酶的活性檢測,以驗證miR-142-3p與TET2之間的靶向作用關(guān)系。3將包裝成功的LV-miR-142-3p慢病毒及miR-142-3p inhibitor試劑分別侵染SKOV-3細胞系,24h后觀察侵染結(jié)果,并收集細胞及上清液備后續(xù)實驗。分別提取各組細胞的m RNA及總蛋白,應用RT-q PCR方法檢測每組間miR-142-3p及靶基因的m RNA的表達情況,應用Western blot法檢測TET2蛋白表達水平,同時用分度儀檢測蛋白灰度值,應用MTT法檢測miR-142-3p對SKOV-3細胞的增殖的影響,采用SPSS20.0軟件進行統(tǒng)計學分析。結(jié)果:1成功構(gòu)建了p Sico R-miR-142-3p的慢病毒載體,并成功包裝了可過表達miR-142-3p的慢病毒顆粒,同時測定的病毒滴度為2.66×105 pfu/m L。2 miRNA相關(guān)的網(wǎng)站內(nèi)容顯示miR-142-3p和TET2的3’UTR存在堿基互補,可能存在相互作用關(guān)系,因此可通過雙熒光素酶報告基因系統(tǒng)檢測熒光表達情況來證實二者間是否存在靶向作用關(guān)系,結(jié)果發(fā)現(xiàn)將miR-142-3p過表達后,靶基因的熒光表達情況明顯改變:野生型與突變型相比,表達量明顯下調(diào),下降約4.09倍(P0.05);轉(zhuǎn)染inhibitor試劑后,靶基因TET2的熒光表達活性,其野生型比突變型有所上調(diào)。在各組野生型之間的比較可以看出,過表達組與空白對照組相比,明顯降低,約4.52倍(P0.05);而過表達組較inhibitor組的表達值,又呈明顯的上升趨勢,升高可達5.18倍(P0.05)。3病毒侵染細胞系后,小RNA的表達情況顯示:過表達組明顯高于SKOV-3原始細胞組,升高約17.5倍(P0.05),而靶基因TET2 m RNA的檢測結(jié)果顯示過表達組與SKOV-3原始細胞組相比下調(diào)近7.8倍(P0.05);當miR-142-3p被抑制后,miR-142-3p的表達情況明顯降低,與SKOV-3原始細胞組的小RNA表達情況相比,下調(diào)約18倍(P0.05),與此同時靶基因TET2的表達水平上調(diào),約3.9倍(P0.05)。4 Western blot結(jié)果顯示:過表達組較未處理組相比,條帶明顯減弱,轉(zhuǎn)染miR-142-3p inhibitor后,TET2的條帶明顯增粗,即表達量升高,蛋白的灰度值檢測更為直觀的展現(xiàn)了miR-142-3p病毒刺激后TET2蛋白的表達水平。5 MTT實驗證實,miR-142-3p可顯著抑制SKOV-3的增殖。結(jié)論:成功構(gòu)建了可過表達miR-142-3p的慢病毒顆粒,進一步證實過表達miR-142-3p可有效抑制SKOV-3細胞中TET2基因m RNA和蛋白水平的表達,并抑制SKOV-3細胞的增殖。為今后卵巢癌的基因治療研究提供新的思路和研究靶點。
[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.

【學位授予單位】：河北醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R737.31

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相關(guān)期刊論文 前1條

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本文編號：1395065