本文選題：細胞增殖 + 宮頸癌��； 參考：《山東大學》2015年博士論文

【摘要】：研究目的與意義宮頸癌(cervical cancer)是全球女性發(fā)病率第二位的惡性腫瘤,僅次于乳腺癌,在某些局部地區(qū)和國家其發(fā)病率位居首位,每年大約有50萬的新發(fā)病例,差不多其中45%的病例最終死亡。我國每年新增病例約15萬,嚴重威脅女性的健康。近年來,我國加大了對宮頸癌的普查力度,使之早期發(fā)現(xiàn)、早期診斷、早期治療的水平有了很大的提升,加之衛(wèi)生條件的改善和保健意識的增強使宮頸癌發(fā)病率及死亡率不斷下降。近20年來盡管對宮頸癌的研究越來越深入,可是中晚期宮頸癌的療效未見明顯提高,治療方法的種類和特異性明顯落后。為了提高宮頸癌的療效,尋找治療宮頸癌的新途徑顯得尤為迫切。雷帕霉素靶蛋白(target of rapamyein, TOR)基因是最早在酵母中發(fā)現(xiàn)的一類高度保守的蛋白激酶家族,隨后在自然界其它物種中廣泛發(fā)現(xiàn)。在哺乳動物細胞中也存在結構和功能相似的TOR蛋白,命名為哺乳動物雷帕霉素靶蛋白(mammalian target of rapamyein, mTOR)。mTOR基因位于人類第1號染色體短臂的3區(qū)6帶2亞帶中,是絲／蘇氨酸蛋白激酶家族成員,mTOR基因的羧基端具有與磷脂酰肌醇3-激酶(P13K)高度同源的催化結構域,是經典信號傳導通路：磷脂酰肌醇3-激酶、蛋白激酶B和哺乳動物雷帕霉素靶蛋白(P13K-Akt-mTOR)的下游基因,是該信號通路接受上游刺激并向下傳遞信號的節(jié)點；多種腫瘤的發(fā)生與該信號通路活化及mTOR高表達密切相關,這在結腸癌、淋巴瘤、乳腺癌及鼻咽癌等多種惡性腫瘤中早已有報道。MicroRNAs (miRNAs)是一類非編碼調節(jié)性小RNA分子,由21到25個核苷酸組成,miRNA前體成熟后與輔酶因子結合成RNA誘導沉默復合體,與靶信使RNA (message RNA, mRNA)的3’UTR以堿基互補配對結合,導致mRNA降解或轉錄后水平的抑制,從而抑制靶基因mRNA的翻譯并參與調控細胞進程,如炎癥反應、應激反應、分化、凋亡及遷移等。目前已經發(fā)現(xiàn)miRNAs可以調控細胞周期、增殖、分化、凋亡及衰老；多數(shù)腫瘤中都存在miRNAs的異常表達,它與腫瘤的發(fā)生、進展、診斷、治療及預后密切相關,是當前腫瘤領域研究熱點。有報道稱miR-634過表達能直接通過調控線粒體內穩(wěn)態(tài)相關基因而激活線粒體凋亡通路、抗氧化能力及細胞自噬能力。同時在異種移植小鼠模型中,有研究發(fā)現(xiàn)miR-634抑制腫瘤的生長和增強紫杉醇的敏感性。有研究表明在惡性膠質瘤中miR-634過表達對mTOR途徑起負調節(jié)作用；這為我們提供了研究其它miRNAs家族成員在宮頸癌致病機理中發(fā)揮作用指明了方向。本研究擬探討miR-634是否可以通過調控mTOR基因的表達影響宮頸癌細胞的生物學行為,能否以miR-634為媒介逆轉細胞的自我保護流程,影響宮頸癌細胞的增殖、侵襲及凋亡,從而提高宮頸癌化學治療及靶向治療的療效,為調控宮頸癌細胞的信號傳導通路及抑制腫瘤細胞的生長和促進腫瘤細胞的凋亡提供新的思路。研究方法與結果第一部分MiR-634在宮頸癌組織中的表達及其作用研究研究方法：1.自2013年8月至2013年12月收集煙臺毓璜頂醫(yī)院證實為宮頸鱗狀細胞癌標本15例,所有患者術前均未進行放、化療及其它治療。應用Real-time PCR技術檢測miR-634在宮頸癌組織和癌旁正常組織中的差異表達。2.培養(yǎng)HeLa及Siha細胞,通過構建miR-634過表達質粒,并檢測所構建質粒的有效性及轉染效率；然后將過表達質粒分別轉染宮頸癌HeLa及Siha細胞中,通過WST-8實驗、克隆形成實驗、劃痕實驗和Transwell實驗,研究過表達miR-634及封閉后對HeLa及Siha細胞的增殖、遷移和侵襲的影響。3.流式細胞儀檢測miR-634過表達和封閉后對HeLa及Siha細胞凋亡的影響。研究結果：1.應用Real-time PCR技術檢測發(fā)現(xiàn)與癌旁正常組織相比mi R-634在宮頸癌組織表達明顯降低。2.通過構建好的miR-634過表達質粒轉染HeLa及Siha細胞后發(fā)現(xiàn),過表達miR-634的HeLa及Siha細胞其增殖、遷移及侵襲能力減弱,封閉后結果相反。3.流式細胞分析儀結果顯示miR-634過表達誘導了HeLa及Siha細胞凋亡率的增加,而封閉miR-634時降低了HeLa及Siha細胞的凋亡率。第二部分MiR-634在宮頸癌細胞中與mTOR基因的相互關系研究方法：1.培養(yǎng)HeLa及Siha細胞,應用Real-time PCR方法及Western blot技術檢測mTOR基因mRNA和蛋白在HeLa及Siha細胞中的水平。2.構建含mTOR基因3’-UTR段和含mTOR基因3'-UTR-mut段雙熒光素酶報告基因載體,并驗證其活性；使用lipofectamine 2000轉染試劑將不同的質�；蛑亟M質粒轉染至HeLa及Siha細胞中,測定熒光素酶活性,分析miR-634對mTOR基因有無調控作用。3.通過Real-time PCR方法及Western blot技術檢測miR-634過表達和封閉后HeLa及Siha細胞中mTOR mRNA的表達及蛋白水平的變化。研究結果：1.通過雙熒光素酶報告載體實驗證明mTOR是miR-634的靶基因之一,其3’非翻譯區(qū)包含miR-634的結合位點；miR-634能夠通過作用于mTOR基因的3’非翻譯區(qū)(3’UTR)的特定位點,對其進行負性調節(jié)。2.通過Real-time PCR和Western blot技術檢測發(fā)現(xiàn)過表達miR-634后HeLa及Siha細胞中mTOR基因的mRNA和蛋白水平均明顯降低,封閉后結果相反。第三部分mTOR基因在宮頸癌組織中的表達及其作用研究研究方法：1.應用Real-time PCR技術檢測mTOR基因在宮頸癌組織和癌旁正常組織中的差異表達。2.培養(yǎng)HeLa及Siha細胞,構建pSilencer/si-mTOR質粒,利用RNA干擾技術封閉HeLa及Siha細胞中靶基因mTOR。3.采用Western blot方法驗證pSilencer/si-mTOR質粒的有效性。4.pSilencer/si-mTOR質粒轉染HeLa及Siha細胞后通過WST-8實驗、克隆形成實驗、劃痕實驗和Transwell實驗檢測細胞生長特性的變化。研究結果：1.應用Real-time PCR技術檢測發(fā)現(xiàn)與癌旁正常組織相比mTOR基因mRNA的表達水平在宮頸癌組織中明顯升高。2.WST-8實驗、克隆形成實驗、劃痕實驗和Transwell實驗結果顯示,將mTOR基因封閉后HeLa及Siha細胞的增殖、遷移及侵襲能力均下降。研究結論1.miR-634能夠抑制HeLa及Siha細胞的增殖侵襲和遷移,起到抑癌基因的作用。2.將mTOR基因封閉后,HeLa及Siha細胞增殖能力減弱,細胞集落形成和侵襲能力也明顯減弱。3.在HeLa及Siha細胞中,高表達的miR-634對mTOR基因表達水平的抑制能力增強,使mTOR基因的促進增殖活性減弱,造成細胞生長速度減慢。4.通過闡明miR-634在宮頸癌中的具體作用機制及其靶基因的研究有利于我們對宮頸癌的發(fā)生發(fā)展進行深入理解,也為臨床上宮頸癌的診斷和治療提供一條新的途徑。
[Abstract]:The purpose and significance of cervical cancer (cervical cancer) is a second place malignant tumor in the world. Second only to breast cancer, the incidence of the disease is the first in some local areas and countries, with about 500 thousand new cases each year, about 45% of them die out. The number of new cases in China is about 150 thousand a year, and it is a serious threat to women. In recent years, China has increased the survey of cervical cancer, making early detection, early diagnosis, the level of early treatment has been greatly improved, combined with the improvement of health conditions and health awareness, the incidence and mortality of cervical cancer have declined continuously. In recent 20 years, although the research of cervical cancer has become more and more in-depth, but In order to improve the curative effect of cervical cancer, it is very urgent to find a new way to treat cervical cancer. The target of rapamyein (TOR) gene is the first kind of highly conserved protein kinase family found in yeast. It is found widely in other species in nature. In mammalian cells, there are also TOR proteins with similar structure and function. The mammalian target of rapamyein, mTOR.MTOR gene is located in the 6 band 2 subband of the short arm of the human chromosome first, and is a member of the family of silk / threonine protein kinase. The carboxyl terminal of the mTOR gene has a catalytic domain that is highly homologous to the phosphatidylinositol 3- kinase (P13K). It is the classic signal transduction pathway: the downstream genes of phosphatidyl inositol 3- kinase, protein kinase B and mammalian rapamycin target protein (P13K-Akt-mTOR), which are the nodes of the signal through the upstream stimulation and the downward transmission of signals. The occurrence of the tumor is closely related to the activation of the signal pathway and the high expression of mTOR. It has been reported that.MicroRNAs (miRNAs) is a class of non coding and regulatory small RNA molecules in colon cancer, lymphoma, breast cancer and nasopharyngeal carcinoma, which is composed of 21 to 25 nucleotides, and miRNA precursor is combined with coenzyme factors into RNA induction. The silencing complex, combined with the 3 'UTR of the target messenger RNA (message RNA, mRNA), is combined with base pairs to lead to the degradation of mRNA or inhibition of post transcriptional levels, which inhibits the translation of the target gene mRNA and participates in the regulation of cell processes, such as inflammation, stress response, differentiation, withering and migration and so on. The cell cycle has been regulated by miRNAs. Proliferation, differentiation, apoptosis and senescence; most of the tumors have abnormal expression of miRNAs. It is closely related to the occurrence, progression, diagnosis, treatment and prognosis of tumors. It is a hot spot in the field of cancer. It is reported that miR-634 overexpression can activate mitochondrial apoptosis pathway directly by regulating the homeostasis related genes in mitochondria and the antioxidant energy can be activated. It is also found that miR-634 inhibits tumor growth and increases the sensitivity of taxol in xenotransplantation mice. Studies have shown that miR-634 overexpression in malignant gliomas can negatively regulate the mTOR pathway; this provides us with the study of the pathogenesis of its miRNAs family in the pathogenesis of cervical cancer. The purpose of this study is to indicate whether miR-634 can affect the biological behavior of cervical cancer cells by regulating the expression of mTOR gene, whether it can reverse the self protection process of cells with miR-634 as a medium, influence the proliferation, invasion and apoptosis of cervical cancer cells, so as to improve the treatment of cervical cancer and the treatment of targeted therapy. A new idea to regulate the signal transduction pathway of cervical cancer cells and to inhibit the growth of tumor cells and promote the apoptosis of tumor cells. Research methods and results of the first part of MiR-634 in cervical cancer tissue expression and its role: 1. collected from Yantai Yuhuangding Hospital from August 2013 to December 2013 proved to be 15 specimens of squamous cell carcinoma of the cervix were not performed, chemotherapy and other treatments were not performed before operation. The differential expression of miR-634 in the tissues of cervical cancer and normal tissues by Real-time PCR was used to detect the.2. culture of HeLa and Siha cells by constructing miR-634 overexpressed plasmids, and the effectiveness and transfection efficiency of the constructed plasmids were detected. The expression plasmids were then transfected into cervical cancer HeLa and Siha cells respectively. Through WST-8 experiment, clone formation, scratch test and Transwell experiment, the effects of miR-634 and miR-634 on the proliferation, migration and invasion of HeLa and Siha cells were studied..3. flow cytometry was used to detect miR-634 over expression and closed to HeLa and Siha cells. The effect of apoptosis. Results: 1. the Real-time PCR technique detected that the expression of MI R-634 in cervical cancer tissues was significantly lower than that of normal tissues adjacent to the carcinoma..2. was found to transfect HeLa and Siha cells through the constructed miR-634 overexpressed plasmid, and the proliferation of HeLa and Siha cells overexpressing miR-634, the ability to migrate and invasiveness were weakened. The result of reverse.3. flow cytometry showed that miR-634 overexpression induced the increase of apoptosis rate of HeLa and Siha cells, while blocking miR-634 decreased the apoptosis rate of HeLa and Siha cells. Second part MiR-634 in cervical cancer cells and mTOR gene interaction methods: 1. to cultivate HeLa and Siha cells, and to apply Real-time Methods and Western blot technique, the mTOR gene mRNA and protein were detected in the level.2. in HeLa and Siha cells to construct the 3 '-UTR segment containing mTOR gene and the 3'-UTR-mut segment double luciferase reporter gene carrier of mTOR gene, and to verify its activity. In the cell, the activity of luciferase was measured, and the effect of miR-634 on the mTOR gene was analyzed..3. was detected by Real-time PCR method and Western blot technique to detect the expression of mTOR mRNA in HeLa and Siha cells and the changes in protein level after miR-634's overexpression and closure. The results of the study: 1. through the double luciferase reporter carrier experiment proved that One of the target genes, its 3 'non translation region contains the binding site of miR-634, and miR-634 can negatively regulate it through a specific site that acts on the 3' non translation region (3 'UTR) of the mTOR gene, and.2. is detected by Real-time PCR and Western blot technique to detect the expression of the HeLa and protein water in Siha cells after miR-634 HeLa and Siha cells Third mTOR gene expression in cervical cancer tissue and its role in research methods: 1. the difference expression of mTOR gene in cervical cancer tissues and normal tissues adjacent to cancer by Real-time PCR technique,.2. culture HeLa and Siha cells, construction of pSilencer/si-mTOR plasmids, and RNA interference. Technology closed the target gene mTOR.3. in HeLa and Siha cells using Western blot method to verify the validity of pSilencer/si-mTOR plasmid,.4.pSilencer/si-mTOR plasmid transfected to HeLa and Siha cells after HeLa and Siha cells, clone formation experiment, scratch test and Transwell experiment to detect the changes of cell growth characteristics. Research results: 1. application Real-time The PCR technique detected the expression level of the mTOR gene mRNA compared with the normal tissues adjacent to the carcinoma. The.2.WST-8 experiment was significantly increased in the cervical cancer tissue, the cloning experiment, the scratch test and the Transwell experiment showed that the proliferation, migration and invasion ability of HeLa and Siha cells decreased after the mTOR gene was closed. Conclusion 1.miR-634 can be suppressed. The proliferation, invasion and migration of HeLa and Siha cells play a role in the tumor suppressor gene,.2. will close the mTOR gene, and the proliferation ability of HeLa and Siha cells is weakened, and the colony formation and invasion ability also weakens the.3. in HeLa and Siha cells. The inhibition ability of the high expression miR-634 to the mTOR gene expression is enhanced and the mTOR gene is promoted. The effect of.4. on the specific mechanism of miR-634 in cervical cancer and the study of its target genes will help us to understand the development of cervical cancer and provide a new way for the diagnosis and treatment of cervical cancer.
【學位授予單位】：山東大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R737.33

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相關期刊論文 前1條

1 陳軍瑩;姚德生;賀嬋娟;盧艷;歐婷瑜;;宮頸鱗癌MicroRNA差異表達的研究[J];實用醫(yī)學雜志;2014年01期

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本文編號：2096398