【摘要】：目的 卵巢癌（Ovarian cancer）是女性生殖系統(tǒng)常見的三大惡性腫瘤之一，但其致死率居各類婦科惡性腫瘤之首。由于其發(fā)病隱匿，缺乏早期診斷手段，約70%的卵巢癌患者確診時已屬晚期。研究發(fā)現(xiàn)卵巢癌的發(fā)生與遺傳因素、初潮和絕經(jīng)年齡、生殖及內(nèi)分泌等多種因素有關(guān)。近年來，一類非蛋白質(zhì)編碼microRNAs（miRNAs）的出現(xiàn)，為卵巢腫瘤的研究提供了新的思路。miRNAs是一類長度為20-22個核苷酸的非編碼小分子RNA，在動物體內(nèi)miRNA主要通過與靶基因3’-UTR區(qū)不完全配對，抑制靶基因mRNAs翻譯，，參與調(diào)控個體發(fā)育、細(xì)胞凋亡、增殖及分化等生命活動。隨著對miRNA研究的不斷深入發(fā)現(xiàn)miRNA在多種惡性腫瘤中扮演著重要角色，miRNA可作為抑癌基因或癌基因參與腫瘤的發(fā)生發(fā)展、轉(zhuǎn)移、耐藥等進(jìn)程。miR-125b是miRNA家族中的重要一員，它在多種腫瘤發(fā)生發(fā)展過程中發(fā)揮重要作用,特別是在卵巢癌的發(fā)生發(fā)展過程中。本研究擬通過在卵巢癌SKOV3細(xì)胞株中過表達(dá)miR-125b，檢測miR-125b對卵巢癌SKOV3細(xì)胞增殖、遷移及侵襲能力的影響，及其所調(diào)控的潛在靶基因進(jìn)行預(yù)測，以期明確miR-125b在卵巢癌發(fā)生發(fā)展中的作用。 方法 本研究通過過表達(dá)miR-125b慢病毒顆粒感染卵巢癌SKOV3細(xì)胞，嘌呤霉素篩選穩(wěn)定感染細(xì)胞株，建立穩(wěn)定過表達(dá)miR-125b的SKOV3細(xì)胞系。采用實(shí)時熒光定量PCR（qRT-PCR）檢測miR-125b在SKVO3細(xì)胞中的表達(dá)；MTT實(shí)驗(yàn)觀察miR-125b過表達(dá)后SKOV3細(xì)胞增殖能力的的改變；劃痕實(shí)驗(yàn)和Transwell侵襲實(shí)驗(yàn)分別觀察miR-125b過表達(dá)后SKOV3細(xì)胞遷移和侵襲能力的改變。采用iTRAQ多重化學(xué)標(biāo)記串聯(lián)質(zhì)譜（iTRAQ-LC/MS/MS）實(shí)驗(yàn)定性及定量分析miR-125b過表達(dá)前后SKOV3細(xì)胞內(nèi)蛋白質(zhì)的變化，篩選出miR-125b可能調(diào)控的潛在靶基因。再進(jìn)一步通過Western blot來驗(yàn)證篩選出的靶基因是否受miR-125b的影響。 結(jié)果 1. MTT實(shí)驗(yàn)顯示，miR-125b過表達(dá)后SKOV3細(xì)胞的增殖能力減弱；劃痕實(shí)驗(yàn)和Transwell侵襲實(shí)驗(yàn)顯示，miR-125b過表達(dá)后SKOV3細(xì)胞的遷移和侵襲能力明顯減弱。 2. iTQAR多重化學(xué)標(biāo)記串聯(lián)質(zhì)譜(iTQAR-LC/MS/MS)實(shí)驗(yàn)，結(jié)合miRNA候選靶基因預(yù)測軟件（TargetScan、Pictar等）分析篩選出61種可能受到miR-125b調(diào)控的差異表達(dá)蛋白質(zhì)，其中16種蛋白質(zhì)在表達(dá)水平上存在顯著差異。通過生物信息學(xué)對差異表達(dá)的蛋白質(zhì)進(jìn)行功能分析（整合NCBI、KEGG PATHWAY、Swiss-Prot等多個數(shù)據(jù)庫信息），我們選擇ERBB2基因作為miR-125b的一個潛在靶基因進(jìn)行進(jìn)一步研究。 3. Western blot結(jié)果顯示，miR-125b能抑制ERBB2基因的表達(dá)。 結(jié)論 本研究證明miR-125b能夠抑制SKOV3細(xì)胞的增殖、遷移和侵襲，并可能通過降低潛在靶基因ERBB2的表達(dá)而實(shí)現(xiàn)。
[Abstract]:Objective Ovarian cancer (Ovarian cancer) is one of the three most common malignant tumors in the female reproductive system, but its mortality is the highest among all kinds of gynecologic malignancies. Because of its hidden incidence and lack of early diagnosis, about 70% of patients with ovarian cancer are advanced when diagnosed. Genetic factors, menarche and menopausal age, reproductive and endocrine factors were found in the occurrence of ovarian cancer. In recent years, the emergence of a class of non-protein encoded microRNAs (miRNAs) provides a new idea for the study of ovarian tumors. MiRNAs are a kind of non-coding small molecule RNA, with a length of 20-22 nucleotides, which is mainly paired with the 3'-UTR region of the target gene in animals. Inhibition of target gene mRNAs translation, regulation of ontogeny, apoptosis, proliferation and differentiation, and other vital activities. With the further study of miRNA, it has been found that miRNA plays an important role in many kinds of malignant tumors. As a tumor suppressor gene or oncogene, miR-125b is an important member of the miRNA family as tumor suppressor gene or oncogene is involved in tumorigenesis, metastasis, drug resistance and other processes. It plays an important role in the development of many kinds of tumors, especially in the development of ovarian cancer. The purpose of this study was to detect the effect of miR-125b on the proliferation, migration and invasion of ovarian cancer SKOV3 cells by overexpression of miR-125b, in ovarian cancer SKOV3 cell lines, and to predict the potential target genes regulated by miR-125b in order to clarify the role of miR-125b in the development and development of ovarian cancer. Methods in this study, ovarian cancer SKOV3 cells were infected by overexpression of miR-125b lentivirus particles, and stable infection cell lines were screened by purine mycin, and SKOV3 cell lines with stable expression of miR-125b were established. The expression of miR-125b in SKVO3 cells was detected by real-time fluorescence quantitative PCR (qRT-PCR). The proliferation of SKOV3 cells after miR-125b overexpression was observed by MTT assay, and the migration and invasion ability of SKOV3 cells after miR-125b overexpression was observed by scratch test and Transwell invasion assay, respectively. ITRAQ multiplex chemical labeling tandem mass spectrometry (iTRAQ-LC/MS/MS) assay was used to qualitatively and quantitatively analyze the changes of proteins in SKOV3 cells before and after miR-125b overexpression, and the potential target genes regulated by miR-125b were screened. Further, Western blot was used to verify whether the target gene was affected by miR-125b. Result 1. MTT assay showed that the proliferation ability of SKOV3 cells decreased after overexpression of miR-125b, the migration and invasion of SKOV3 cells were significantly weakened by scratch assay and Transwell invasion assay. 2. ITQAR multiple chemical labeling mass spectrometry (iTQAR-LC/MS/MS) assay. According to miRNA candidate target gene prediction software (TargetScan,Pictar et al), 61 differentially expressed proteins possibly regulated by miR-125b were screened, among which 16 proteins were significantly different in expression level. Using bioinformatics to analyze the function of differentially expressed proteins (integrating NCBI,KEGG PATHWAY,Swiss-Prot and other database information), we select the ERBB2 gene as a potential target gene of miR-125b for further study. 3. Western blot results showed that miR-125b could inhibit the expression of ERBB2 gene. Conclusion this study suggests that miR-125b can inhibit the proliferation, migration and invasion of SKOV3 cells, and may be achieved by reducing the expression of ERBB2, a potential target gene.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R737.31

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