本文選題：宮頸癌 + microRNA��； 參考：《吉林大學(xué)》2017年博士論文

【摘要】：目的:越來越多的證據(jù)表明,包括宮頸癌在內(nèi)的多種危害較大的惡性腫瘤的發(fā)生、發(fā)展和轉(zhuǎn)移與micro RNA的失調(diào)密切相關(guān)。從宮頸癌前病變發(fā)展到宮頸癌遠(yuǎn)處轉(zhuǎn)移,疾病的每個階段都有相應(yīng)的micro RNA參與。micro RNA可以通過對癌基因、抑癌基因以及相關(guān)蛋白表達(dá)的表觀遺傳學(xué)調(diào)控來影響宮頸癌的發(fā)生和發(fā)展。據(jù)多個研究報道,micro RNA家族中的mi R-138參與了腎癌、非小細(xì)胞肺癌、黑色素瘤、神經(jīng)膠質(zhì)瘤、食管鱗狀細(xì)胞癌、鼻咽癌和肝癌的發(fā)生和發(fā)展。但是mi R-138是否參與宮頸癌的發(fā)生和發(fā)展及其具體機(jī)制還未闡明。本研究旨在探討mi R-138在宮頸癌發(fā)生、發(fā)展以及轉(zhuǎn)移中的變化規(guī)律和作用靶點(diǎn),為臨床診斷和治療宮頸癌提供理論依據(jù)。方法:本研究收集了2010年10月到2015年9月在吉林大學(xué)第一醫(yī)院行宮頸癌手術(shù)治療的59例宮頸癌患者的臨床資料和標(biāo)本。首先,通過RT-PCR的方法證明mi R-138在宮頸癌發(fā)生發(fā)展中的變化規(guī)律;其次,通過流式細(xì)胞術(shù)和細(xì)胞生物學(xué)技術(shù)等方法闡明了mi R-138對宮頸癌細(xì)胞增殖、克隆、凋亡、遷移和侵襲等細(xì)胞生物學(xué)行為的影響;并且通過生物信息學(xué)比對、RT-PCR和western blot等方法證實(shí)h TERT可能是mi R-138的直接調(diào)控靶點(diǎn);最后,在體移植轉(zhuǎn)染了mi R-138的宮頸癌He La細(xì)胞,建立裸鼠人宮頸癌轉(zhuǎn)移瘤模型,通過評價小鼠體內(nèi)移植瘤的大小和重量推斷mi R-138在宮頸癌發(fā)展過程中的作用。結(jié)果:分為以下5部分:1.mi R-138在人源宮頸癌細(xì)胞系和宮頸癌組織中表達(dá)顯著下調(diào)通過熒光實(shí)時定量PCR(RT-PCR)的方法分析mi R-138在人源宮頸癌細(xì)胞系He La和Si Ha的表達(dá)情況,結(jié)果顯示,與對照細(xì)胞系即與人的永生化無HPV感染的表皮細(xì)胞系Ha Ca T細(xì)胞相比,mi R-138在宮頸癌細(xì)胞系He La細(xì)胞和Si Ha細(xì)胞中的表達(dá)水平明顯下調(diào),結(jié)果有統(tǒng)計學(xué)差異(p0.01),而與Si Ha細(xì)胞相比,mi R-138在He La細(xì)胞中的表達(dá)水平下調(diào)更加明顯;通過RT-PCR的方法觀察mi R-138在59例患者宮頸癌組織中的表達(dá)情況,結(jié)果顯示,與癌旁正常宮頸組織相比,mi R-138在59例宮頸癌患者癌組織中的表達(dá)明顯下調(diào),mi R-138的相對表達(dá)值下調(diào)83.3%,相差約2.1倍,有統(tǒng)計學(xué)差異(p0.01)。mi R-138的表達(dá)水平與宮頸癌分期明顯相關(guān),宮頸癌分期越晚,mi R-138的表達(dá)水平越低,宮頸癌FIGO(International Federation of Gynecology and Obstetrics,國際婦產(chǎn)科聯(lián)盟)分期Ib1-Ib2的21例患者mi R-138的相對表達(dá)值為:0.53±0.11,FIGO分期Ⅱa1-Ⅱa2的38例患者mi R-138的相對表達(dá)值為:0.35±0.06,有統(tǒng)計學(xué)差異(p0.05),mi R-138的表達(dá)水平還與宮頸癌是否發(fā)生淋巴轉(zhuǎn)移密切相關(guān),45例沒有淋巴轉(zhuǎn)移的患者mi R-138的相對表達(dá)值為:0.49?±?0.08,14例有淋巴轉(zhuǎn)移的患者mi R-138的相對表達(dá)值為:0.21±0.06,有統(tǒng)計學(xué)差異(p0.01)。但是mi R-138的表達(dá)水平與宮頸癌患者年齡、腫塊大小(是否為局部晚期)以及組織學(xué)分級無明顯相關(guān)性,沒有統(tǒng)計學(xué)差異。2.mi R-138可抑制宮頸癌He La細(xì)胞的增殖、克隆和抗凋亡能力通過脂質(zhì)體在培養(yǎng)的宮頸癌細(xì)胞系He La中轉(zhuǎn)染化學(xué)合成的mi R-138以及對照mi R-NC,通過RT-PCR的方法來檢測轉(zhuǎn)染后細(xì)胞內(nèi)mi R-138的表達(dá)情況,結(jié)果顯示,與對照組(轉(zhuǎn)染mi R-NC)相比,mi R-138組(轉(zhuǎn)染mi R-138)He La細(xì)胞mi R-138的表達(dá)水平明顯上調(diào),并有統(tǒng)計學(xué)差異(p0.01);MTT實(shí)驗(yàn)檢測mi R-138對He La細(xì)胞增殖能力的影響,結(jié)果提示,與對照組(轉(zhuǎn)染mi R-NC)相比,He La細(xì)胞系轉(zhuǎn)染mi R-138后,He La細(xì)胞的增殖活力明顯減弱,并有統(tǒng)計學(xué)差異(p0.01);在He La細(xì)胞系中分別轉(zhuǎn)染mi R-138及對照mi R-NC后,檢測He La細(xì)胞的克隆能力,通過細(xì)胞計數(shù)發(fā)現(xiàn),與對照組相比,He La細(xì)胞系轉(zhuǎn)染mi R-138后,He La細(xì)胞的克隆形成能力明顯減弱,并有統(tǒng)計學(xué)差異(p0.01);通過流式細(xì)胞技術(shù)檢測mi R-138對He La細(xì)胞凋亡的影響,結(jié)果證實(shí),與對照組相比,He La細(xì)胞轉(zhuǎn)染mi R-138后細(xì)胞凋亡明顯增加,并有統(tǒng)計學(xué)差異(p0.01)。3.在宮頸癌細(xì)胞系He La中過表達(dá)mi R-138可明顯抑制細(xì)胞的遷移與侵襲通過Transwell實(shí)驗(yàn)驗(yàn)證過表達(dá)mi R-138對宮頸癌細(xì)胞遷移及侵襲能力的影響。Transwell細(xì)胞體外遷移實(shí)驗(yàn)結(jié)果證實(shí),與過表達(dá)對照mi R-NC相比,在He La細(xì)胞中過表達(dá)mi R-138可以明顯抑制細(xì)胞的遷移,并有統(tǒng)計學(xué)差異(p0.01)。Transwell細(xì)胞體外侵襲實(shí)驗(yàn)結(jié)果證實(shí),與過表達(dá)對照mi R-NC相比,在He La細(xì)胞中過表達(dá)mi R-138可以明顯抑制細(xì)胞的侵襲能力,并有統(tǒng)計學(xué)差異(p0.01)。4.mi R-138可通過調(diào)控h TERT表達(dá)來抑制宮頸癌發(fā)生發(fā)展通過生物數(shù)據(jù)庫Targetscan6.2和mi RWalk分析與mi R-138結(jié)合的靶基因,并通過文獻(xiàn)比對和分析,推測人端粒酶逆轉(zhuǎn)錄酶(human Telomerase Reverse Transcriptase,h TERT)可能是mi R-138的直接作用靶點(diǎn)。通過熒光素酶報告實(shí)驗(yàn)證實(shí),與過表達(dá)含h TERT3’端(3’UTR)突變位點(diǎn)的質(zhì)粒相比,過表達(dá)h TERT質(zhì)粒后熒光吸收值更高,并有統(tǒng)計學(xué)差異(p0.01);western blot實(shí)驗(yàn)從蛋白水平證實(shí),轉(zhuǎn)染mi R-138以后,He La細(xì)胞中h TERT的蛋白表達(dá)被明顯抑制。5.mi R-138可在體抑制裸鼠人宮頸癌移植瘤的發(fā)展我們在He La細(xì)胞中轉(zhuǎn)染mi R-138,并將過表達(dá)mi R-138的He La細(xì)胞移植到小鼠體內(nèi),構(gòu)建裸鼠過表達(dá)mi R-138人宮頸癌細(xì)胞移植瘤模型,然后通過不同時間點(diǎn)觀察小鼠體內(nèi)移植瘤的大小在體評估m(xù)i R-138對宮頸癌發(fā)展的影響,結(jié)果提示,移植轉(zhuǎn)染了mi R-138的He La細(xì)胞的小鼠,其體內(nèi)移植瘤體積明顯小于對照組,其移植瘤重量也明顯輕于對照組,并有統(tǒng)計學(xué)差異(p0.01);最后我們將小鼠體內(nèi)移植瘤取出并通過western blot實(shí)驗(yàn)在體檢測h TERT的蛋白表達(dá),實(shí)驗(yàn)結(jié)果提示,與對照組相比,移植過表達(dá)mi R-138的He La細(xì)胞后,移植瘤中h TERT的蛋白表達(dá)水平明顯降低,并有統(tǒng)計學(xué)差異(p0.01)。結(jié)論與創(chuàng)新點(diǎn):本研究首次通過體外和在體實(shí)驗(yàn)初步說明了mi R-138在宮頸癌發(fā)生、發(fā)展中的作用以及可能機(jī)制,即mi R-138可能通過與h TERT結(jié)合進(jìn)而抑制h TERT的蛋白表達(dá)來抑制宮頸癌細(xì)胞的增殖、克隆,并抑制宮頸癌細(xì)胞的抗凋亡能力,減弱宮頸癌細(xì)胞的遷移與侵襲能力,從而減慢宮頸癌的發(fā)生、發(fā)展和轉(zhuǎn)移。主要創(chuàng)新點(diǎn):1.本研究表明,宮頸癌組織中mi R-138的表達(dá)水平變化可以實(shí)時提示宮頸癌的是否發(fā)生以及是否發(fā)生轉(zhuǎn)移,通過檢測宮頸癌組織總mi R-138的表達(dá)或許可監(jiān)測宮頸癌的發(fā)生和是否發(fā)生轉(zhuǎn)移,所以mi R-138未來有可能作為臨床篩查和診斷宮頸癌并判斷其預(yù)后的生物學(xué)標(biāo)記物;2.通過在體實(shí)驗(yàn)證實(shí),上調(diào)mi R-138的表達(dá)可以明顯抑制裸鼠人宮頸癌移植瘤的發(fā)展,從而為臨床治療宮頸癌提供可參考的藥物設(shè)計靶點(diǎn)和理論依據(jù)。
[Abstract]:Objective: more and more evidence shows that the development and metastasis of a variety of malignant tumors, including cervical cancer, are closely related to the maladjustment of micro RNA. From cervical precancerous lesions to distant metastasis of cervical cancer, each stage of the disease has a corresponding micro RNA involved in.Micro RNA through the oncogene and tumor suppressor It is reported that MI R-138 in the micro RNA family is involved in the occurrence and development of renal cancer, non small cell lung cancer, melanoma, glioma, glioma, esophageal squamous cell carcinoma, nasopharyngeal carcinoma and liver cancer, but whether mi R-138 is involved in cervical cancer, according to a number of reports. This study aims to explore the changes and targets of MI R-138 in the occurrence, development and metastasis of cervical cancer, and to provide a theoretical basis for the clinical diagnosis and treatment of cervical cancer. Methods: This study collected the operation of cervical cancer in No.1 Hospital of Jilin University from October 2010 to September 2015. The clinical data and specimens of 59 cases of cervical cancer were treated. First, the changes of MI R-138 in the occurrence and development of cervical cancer were proved by RT-PCR method. Secondly, the cell biological behavior of MI R-138 on the proliferation, Kuron, apoptosis, migration and invasion of cervical cancer cells was elucidated by flow cytometry and cell biology techniques. And through bioinformatics comparison, RT-PCR and Western blot and other methods confirmed that h TERT may be the direct target of MI R-138; finally, the MI R-138 cervical cancer He La cells were transfected in body transplantation, and the metastatic tumor model of human cervical cancer in nude mice was established, and the size and weight of the transplanted tumor in the mice was evaluated for cervical cancer. The following 5 parts: the expression of 1.mi R-138 in human cervical cancer cell lines and cervical cancer tissues was significantly reduced by fluorescence real-time quantitative PCR (RT-PCR) method to analyze the expression of MI R-138 in human cervical cancer cell line He La and Si Ha. Compared with the Ha Ca T cells infected by V, the expression level of MI R-138 in the He La cells and Si Ha cells in the cervical cancer cell line was obviously down, and the results were statistically different (P0.01). The expression level of MI R-138 in the cells was significantly lower than that in the Si cells. The expression in the cervical cancer tissue showed that the expression of MI R-138 in the cancer tissues of 59 patients with cervical cancer was significantly down, the relative expression value of MI R-138 decreased by 83.3%, and the difference was about 2.1 times, and the level of.Mi R-138 was significantly correlated with the stage of cervical cancer, the late stage of cervical cancer was, the later the stage of cervical cancer was, The lower the expression level of MI R-138, the relative expression value of MI R-138 in 21 cases of cervical cancer FIGO (International Federation of Gynecology and Obstetrics, international gynaecology and obstetrics alliance) was 0.53 + 0.11, and the relative value was 0.35 + 0.06. The expression level of MI R-138 in 45 patients without lymphatic metastasis was relative expression value: the relative expression value of MI R-138 in 0.49? 0.08,14 patients with lymphatic metastasis was 0.21 + 0.06, with statistical difference (P0.01). But the expression level of MI R-138 and the age of cervical cancer patients, mass There is no significant correlation between size (whether locally advanced) and histological classification. There is no statistical difference in.2.mi R-138 to inhibit the proliferation of He La cells in cervical cancer. The cloning and anti apoptotic ability can be transfected by chemically synthesized mi R-138 and control mi R-NC in the He La of the cultured cervical cancer cell line, and the conversion is detected by RT-PCR method. The expression of MI R-138 in the infected cells showed that compared with the control group (transfected mi R-NC), the expression level of MI R-138 in the MI R-138 group (transfected mi R-138) was obviously up and there was a statistical difference. After transfection of a cell line to MI R-138, the proliferation activity of He La cells was significantly weakened, and there was a statistical difference (P0.01). The cloning ability of the cells was detected in He La cell lines and after the control of MI R-NC, the cloning ability of the cells was detected by cell count, and the clone formation ability of the cells was compared with the control group. The effect of MI R-138 on the apoptosis of He La cells was detected by flow cytometry (P0.01). The results showed that compared with the control group, the apoptosis of He La cells was significantly increased after MI R-138 (MI R-138), and there was a statistical difference (P0.01).3. in the cervical cancer cell line. Migration and invasion test the effect of MI R-138 on the migration and invasion ability of cervical cancer cells by Transwell test, the results of in vitro migration of.Transwell cells confirmed that the overexpression of MI R-138 in He La cells could significantly inhibit cell migration in He La cells, and there was a statistically significant difference (P0.01).Transwell cell body. Compared with overexpression of MI R-NC, the overexpression of MI R-138 in He La cells could significantly inhibit the invasion of cells, and there was a statistically significant difference (P0.01).4.mi R-138 (P0.01).4.mi R-138 could be used to inhibit the development of cervical cancer by regulating h TERT expression. The target gene of the human telomerase reverse transcriptase (human Telomerase Reverse Transcriptase, H TERT) may be the direct target of MI R-138 through the literature comparison and analysis. Through the luciferase reporter experiment, it is proved that the fluorescence absorption of the plasmid is over expressed after the plasmid containing the H TERT3 'end (3' UTR) mutation site. The Western blot experiment showed that the protein expression of H TERT in He La cells was significantly inhibited after MI R-138, and.5.mi R-138 could inhibit the development of cervical cancer transplanted in nude mice after transfection of MI R-138. In the mice, the MI R-138 human cervical cancer cell transplantation tumor model was overexpressed in nude mice. Then the effect of MI R-138 on the development of cervical cancer was evaluated in vivo by observing the size of the transplanted tumor in the mice in vivo at different time points. The results suggested that the mice transplanted with He La cells of MI R-138 were significantly smaller than the control group, and the transplantation tumor volume was significantly smaller than that of the control group. The weight of the tumor was also significantly lighter than that in the control group (P0.01). Finally, we removed the transplanted tumor in the mice and tested the protein expression of H TERT through the Western blot test. The results showed that the protein expression level of H TERT in the transplanted tumor was significantly lower than that of the control group after the He La cells expressed mi R-138. Statistical difference (P0.01). Conclusions and innovation points: This study first demonstrated the role and possible mechanism of MI R-138 in the development of cervical cancer in vitro and in vivo, that is, MI R-138 may inhibit the proliferation, cloning, and inhibition of cervical cancer by combining with H TERT to inhibit the protein expression of H TERT. The anti apoptosis ability of the cells reduces the migration and invasiveness of cervical cancer cells and slows down the occurrence, development and metastasis of cervical cancer. 1. the main innovation points are: 1. this study shows that the changes in the expression level of MI R-138 in cervical cancer tissue can prompt the occurrence of cervical cancer in real time and whether it may occur or not, by detecting the total Mi of cervical cancer tissue The expression or permission of R-138 can monitor the occurrence and metastasis of cervical cancer, so mi R-138 may be used as a biological marker to screen and diagnose cervical cancer and determine its prognosis in the future. 2. the expression of MI R-138 can obviously inhibit the development of cervical cancer in nude mice by in vivo experiment, and thus for clinical treatment. It provides a reference point and theoretical basis for drug treatment of cervical cancer.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R737.33

【相似文獻(xiàn)】

相關(guān)期刊論文 前10條

1 趙紅曄;杜樾;;宮頸癌297例臨床病理分析[J];中國社區(qū)醫(yī)師(醫(yī)學(xué)專業(yè)半月刊);2008年15期

2 徐艷紅;;宮頸癌的致病因素及預(yù)防的研究綜述[J];中國實(shí)用醫(yī)藥;2009年25期

3 畢建軍;楊慧蘭;;單純皰疹病毒2型與宮頸癌相關(guān)性的研究進(jìn)展[J];實(shí)用醫(yī)學(xué)雜志;2011年03期

4 孟斐;;組織因子途徑抑制物-2在宮頸癌中表達(dá)情況的研究分析[J];中國實(shí)用醫(yī)藥;2013年05期

5 劉威;高文霞;;宮頸癌患者組織及血清血管內(nèi)皮生長因子的變化研究[J];中國藥物經(jīng)濟(jì)學(xué);2013年04期

6 拉萊·蘇祖克;表皮生長因子受體和轉(zhuǎn)化生長因子-α在宮頸癌中的表達(dá)[J];診斷病理學(xué)雜志;2000年04期

7 許爭峰,胡婭莉,王迅美,戴魯琴,韓克,束澤寶;宮頸組織標(biāo)本中端粒酶活性檢測及臨床意義[J];江蘇醫(yī)藥;2001年05期

8 鄒紅燕,姚安梅,楊栓雀,趙西俠;青年宮頸癌相關(guān)因素分析[J];陜西腫瘤醫(yī)學(xué);2001年03期

9 張忠福,李輝;宮頸癌合并妊娠的診斷與治療[J];中國實(shí)用婦科與產(chǎn)科雜志;2004年07期

10 小松;;宮頸癌已成女性殺手[J];中國醫(yī)藥指南;2004年03期

相關(guān)會議論文 前10條

1 譚文福;吳緒峰;陳惠禎;;年輕宮頸癌的臨床特征及防治[A];全國子宮頸癌暨湖北省婦科腫瘤專業(yè)委員會第五次婦科腫瘤學(xué)術(shù)會議論文匯編[C];2006年

2 王勒渝;;宮頸Ⅰ號栓阻斷宮頸癌發(fā)生的組織病理學(xué)、超微結(jié)構(gòu)及免疫組織化學(xué)的研究[A];第八屆全國中西醫(yī)結(jié)合腫瘤學(xué)術(shù)會議論文集[C];2000年

3 段微;;超高頻無線電波刀治療宮頸癌前病變的研究[A];2000全國腫瘤學(xué)術(shù)大會論文集[C];2000年

4 黃妙玲;陳R，

本文編號：1951266