本文關(guān)鍵詞： microRNA miR-93 宮頸癌 宮頸上皮內(nèi)瘤變 microRNA miR-93 宮頸癌 增殖 凋亡 miR-93 RAB11FIP1蛋白 靶基因 慢病毒　出處：《浙江大學(xué)》2015年博士論文　論文類型：學(xué)位論文

【摘要】：官頸癌(Cervical Cancer, CC)是全球女性最常見的婦科惡性腫瘤之一。隨著官頸癌篩查的普及以及對于官頸癌前病變的及時治療,使得宮頸癌的發(fā)生率在發(fā)達(dá)地區(qū)顯著下降,但在欠發(fā)達(dá)地區(qū)由于篩查未開展或不規(guī)范,官頸癌的發(fā)生率仍然保持較高的水平。同時宮頸癌也是死亡率較高的女性惡性腫瘤,嚴(yán)重威脅廣大婦女的健康。 業(yè)已公認(rèn),高危人乳頭瘤病毒(High Risk Human papillomavirus,HR-HPV)感染是官頸癌發(fā)病的必需因素,但不是惟一因素,除病毒感染以外,宿主因素也制約了疾病的發(fā)生和發(fā)展。研究這些因素在在官頸癌發(fā)生與進展過程中的作用與機制,對制定宮頸癌防控新策略具有十分重要的意義。 miRNA是一類非編碼小分子單鏈RNA,可以與靶基因mRNA3'非翻譯區(qū)結(jié)合,引起靶基因mRNA降解或翻譯抑制,在轉(zhuǎn)錄后水平調(diào)節(jié)基因的表達(dá)。最近的研究發(fā)現(xiàn),miRNAs的表達(dá)異常與腫瘤的發(fā)生發(fā)展關(guān)系密切,但niRNA在不同的組織和細(xì)胞中發(fā)揮的生物學(xué)功能和機制并不相同,具有組織與細(xì)胞特異性和時效性的特點。另外,同一個niRNA可以有多個不同的靶基因,多個不同的miRNA也可以同時作用于一個靶基因,從而造成不盡相同的生物學(xué)功能。因此,miRNA及其靶分子構(gòu)成了一個巨大的錯綜復(fù)雜的生物調(diào)控網(wǎng)絡(luò)。 已有研究發(fā)現(xiàn),miRNAs在腫瘤的發(fā)生發(fā)展過程中的發(fā)揮了重要作用。迄今已發(fā)現(xiàn)多種miRNA在宮頸癌中表達(dá)下降,并通過各自不同的靶基因調(diào)控官頸細(xì)胞的生物學(xué)行為,參與宮頸癌的發(fā)生、侵襲、轉(zhuǎn)移等過程。闡明miRNA在官頸癌發(fā)生發(fā)展過程中的作用及機制對探索宮頸癌防治新策略有重要意義。 本課題組前期通過官頸癌相關(guān)miRNA芯片篩查發(fā)現(xiàn),在官頸癌組織表達(dá)改變的眾多miRNA中,miR-93表達(dá)升高。在此基礎(chǔ)上,本研究首先驗證miR-93在宮頸癌、高級別CIN和正常組織中的表達(dá)差異,并比較分析miR-93的表達(dá)水平與宮頸癌臨床不良預(yù)后因素的相關(guān)性。再通過基因功能實驗,探尋miR-93的異常表達(dá)對宮頸癌細(xì)胞生物學(xué)行為的可能作用并確定其發(fā)揮作用的靶基因。旨在為探尋官頸癌發(fā)生與進展新機制及宮頸癌防控新策略提供科學(xué)依據(jù)。 第一部分官頸癌及高級別CIN組織中miR-93表達(dá)及意義 目的： 驗證miR-93在宮頸癌及高級別CIN組織中表達(dá)上調(diào),并探討miR-93表達(dá)與宮頸癌不良預(yù)后因素之間的相關(guān)性。 方法： 收集宮頸鱗癌168例、CIN2-360例和正常上皮48例組織標(biāo)本,采用Stem-loop RT-PCR和Real time PCR法檢測miR-93表達(dá)水平,并收集官頸癌患者的臨床病理資料,分析miR-93的表達(dá)水平與官頸癌臨床不良預(yù)后因素的相關(guān)性。 結(jié)果： 1.miR-93在宮頸鱗癌和CIN2-3組織中的表達(dá)水平與宮頸正常上皮組織相比顯著上調(diào),但兩者間無顯著差異。 2.miR-93表達(dá)與宮頸癌FIGO分期、盆腔淋巴結(jié)轉(zhuǎn)移、深間質(zhì)浸潤、陰道壁累及以及脈管浸潤無關(guān)。 結(jié)論： 1.宮頸癌組織中miR-93表達(dá)水平顯著高于宮頸正常組織,但與宮頸癌不良預(yù)后因素?zé)o顯著相關(guān)性。 2.高級別CIN組織中miR-93表達(dá)水平顯著高于宮頸正常組織,而高級別CIN組織與癌組織相比miR-93表達(dá)水平無差異,提示miR-93表達(dá)改變在宮頸癌發(fā)生發(fā)展進程中可能是一個早期事件。 第二部分miR-93對宮頸癌細(xì)胞生物學(xué)行為的影響 目的： 明確miR-93對宮頸癌細(xì)胞增殖、凋亡、侵襲等生物學(xué)行為的調(diào)控作用。 方法： 在宮頸癌細(xì)胞株Siha和CaSki中,利用細(xì)胞轉(zhuǎn)染技術(shù)抑制miR-93的表達(dá),利用MTT法檢測細(xì)胞增殖能力,流式細(xì)胞技術(shù)檢測細(xì)胞凋亡及細(xì)胞周期分布,Transwell侵襲試驗檢測細(xì)胞的侵襲能力。 結(jié)果： 1.在宮頸癌細(xì)胞中,下調(diào)miR-93表達(dá)抑制Siha和CaSki細(xì)胞的增殖,促進細(xì)胞的凋亡。 2.在宮頸癌細(xì)胞中,下調(diào)miR-93表達(dá)不改變細(xì)胞周期和侵襲力。結(jié)論： miR-93表達(dá)下調(diào)抑制宮頸癌細(xì)胞增殖并促進凋亡,但對G1期細(xì)胞比例和細(xì)胞侵襲力沒有影響,與miR-93主要在官頸癌發(fā)生階段發(fā)揮癌基因功能相符。 第三部分miR-93在宮頸癌細(xì)胞中靶向調(diào)控RAB11FIP1蛋白表達(dá) 目的： 確定miR-93的靶基因,以揭示miR-93促宮頸癌發(fā)生過程中的分子機制。 方法： 通過軟件預(yù)測miR-93靶基因。用miR-93inhibitor轉(zhuǎn)染官頸癌細(xì)胞株Siha和CaSki,用Real time RT-PCR檢測RAB11FIP1mRNA表達(dá),用Westen Blot檢測RAB11FIP1蛋白表達(dá)。利用雙熒光素酶報告基因的方法確認(rèn)miR-93與RAB11FIP1的靶向關(guān)系。同時在宮頸癌細(xì)胞株Siha和CaSki中,利用慢病毒轉(zhuǎn)染技術(shù)過表達(dá)RAB11FIP1蛋白,利用MTT法檢測細(xì)胞增殖能力,流式細(xì)胞技術(shù)檢測細(xì)胞的凋亡。用免疫組化的方法在宮頸組織中檢測RAB11FIP1蛋白表達(dá)。 結(jié)果： 1.抑制miR-93的表達(dá)上調(diào)官頸癌細(xì)胞RAB11FIP1蛋白水平。 2.miR-93與含RAB11FIP13'UTR區(qū)的雙熒光素酶報告質(zhì)粒共轉(zhuǎn)染,下調(diào)熒光素酶的活性,而不影響3'UTR區(qū)突變報告質(zhì)粒的熒光素酶活性。 3.在宮頸癌細(xì)胞中,上調(diào)RAB11FIP1蛋白的表達(dá)抑制Siha和CaSki細(xì)胞增殖,促進細(xì)胞的凋亡。 4.在宮頸癌及高級別CIN組織中, RAB11FIP1蛋白低表達(dá),且miR-93表達(dá)與RAB11FIP1蛋白水平呈負(fù)相關(guān),但與宮頸癌不良預(yù)后相關(guān)因素?zé)o關(guān)。 結(jié)論： 1.RAB11FIP1是miR-93的直接靶基因,miR-93通過靶向RAB11FIP1調(diào)控宮頸癌細(xì)胞的生物學(xué)功能。 2.上調(diào)RAB11FIP1表達(dá)抑制宮頸癌細(xì)胞增殖并促進凋亡；在高級別CIN和宮頸癌組織中RAB11FIP1蛋白表達(dá)降低,但與宮頸癌不良預(yù)后因素?zé)o關(guān),提示RAB11FIP1也在官頸癌發(fā)生的早期階段發(fā)揮抑癌作用。
[Abstract]:Cervical cancer (Cervical Cancer CC) is one of the most common female gynecologic malignant tumors. With the popularity of cervical cancer screening and timely treatment for cervical precancerous lesions, the incidence of cervical cancer decreased significantly in developed areas, but in less developed regions due to screening did not carry out or not, the occurrence of cervical cancer rates still remain high level. At the same time, cervical cancer is a high mortality of malignant tumors of the female, a serious threat to women's health.
It has been well known that human papillomavirus (High Risk Human papillomavirus, HR-HPV) infection is a necessary factor of cervical cancer, but not the only factor, in addition to virus infection, the host factors also restrict the occurrence and development of disease. The effect and mechanism of these factors in the occurrence and progression of cervical cancer in the that is very important to develop new strategies of cervical cancer prevention and control.
MiRNA is a kind of non encoding single stranded RNA molecules, and target gene mRNA3'untranslated region of target genes by mRNA degradation or translation inhibition, regulation of gene expression at the post transcriptional level. Recent studies have found that the occurrence and development is closely related to abnormal expression of miRNAs and tumor, but the biological function and mechanism of niRNA in the play different tissues and cells are not the same, has the characteristics of tissue and cell specificity and timeliness. In addition, the same niRNA can have a number of different target genes, multiple different miRNA can also be applied simultaneously to a target gene, resulting in biological function is not the same. Therefore, miRNA and the target molecules constitute a huge biological network perplexing.
It has been found that miRNAs in the process of tumor progression has played an important role. So far it has been found that many decreased expression of miRNA in cervical cancer, and the biological behavior of different target gene regulation of cervical cells, participate in the occurrence of cervical cancer, invasion, metastasis. To clarify the effect and mechanism of miRNA development in the process of cervical cancer has important significance to explore the prevention and treatment of cervical cancer new strategies.
Ourprevious by cervical cancer related miRNA microarray screening in cervical cancer tissues the expression of numerous miRNA, increase the expression of miR-93. On this basis, this study firstly verified miR-93 in cervical cancer, differentially expressed high level of CIN and normal tissues, and the correlation analysis of the expression of miR-93 and cervical cancer clinical adverse prognostic factors. Through the experiment of gene function, to explore the abnormal expression of miR-93 may effect on the biological behavior of cervical cancer cell line and to determine its role. To play the target gene to explore cervical cancer to provide a scientific basis for the occurrence and progress of the new mechanism and new strategies for cervical cancer prevention and control.
The expression and significance of miR-93 in the first part of cervical cancer and high grade CIN tissue
Objective:
The expression of miR-93 was up-regulated in cervical cancer and high grade CIN tissues, and the correlation between the expression of miR-93 and the adverse prognostic factors of cervical cancer was investigated.
Method錛，

本文編號：1508618