本文關鍵詞： HOTAIR 上皮性卵巢癌 q PCR SKOV3 A2780 增殖 侵襲 轉移 HOTAIR PIK3R3 機制 免疫組化　出處：《重慶醫(yī)科大學》2016年博士論文　論文類型：學位論文

【摘要】：卵巢癌是繼宮頸癌和子宮內膜癌的三大惡性腫瘤之一,其病死率高居婦科惡性腫瘤之首,由于起病隱匿、早期缺乏特異性癥狀與體征及有效的醫(yī)學篩查手段,約70%卵巢癌患者就診時已發(fā)生局部或遠處轉移,失去了手術的最佳時期,結果導致5年的總體生存率欠佳。早期卵巢癌(Ⅰ期和Ⅱ期)的遠期生存率可達到80%~95%,相比之下,晚期卵巢癌(Ⅲ期和Ⅳ期)的遠期生存率只有10%~30%。因此卵巢惡性腫瘤存在“兩個70%”:超過70%的患者確診時已屬晚期;約70%的患者在兩年內復發(fā)。故由此可見,尋找早期診斷及有效的治療方法對于卵巢癌患者的生存率至關重要。近幾年,學者們一直在尋找具有特異性的方法,以提高卵巢癌的生存率,保證患者的有效預后。另外一種學說“二元論模型”認為,卵巢癌分為高級別和低級別兩類。高級別卵巢上皮性癌占卵巢癌發(fā)病的75%,占卵巢癌病死率的90%,其發(fā)病迅速,侵襲性極強,常伴發(fā)廣泛的盆腹腔轉移和種植,預后極差。而低級別卵巢上皮性癌發(fā)病緩慢,多經(jīng)歷良性、交界性和低度惡性的發(fā)展過程,多為早期,預后較好。因此高級別卵巢上皮性癌由于極易發(fā)生侵襲和轉移,是威脅婦女健康的惡性腫瘤。然而其發(fā)生侵襲和轉移的機制不明。近來,繼mi RNA的大量研究之后,被稱為長鏈非編碼RNA(long non-coding RNA,lnc RNA)的一類分子量大于200nt的非編碼RNA被發(fā)現(xiàn)并逐漸受到越來越多的研究。因其對靶基因表達的調控作用,lnc RNA參與了許多癌癥發(fā)生、發(fā)展的過程。近年來的研究表明lnc RNA參與了轉錄激活、染色質修飾、轉錄干擾和核內運輸多種重要的調控。圖1 Lnc RNA的調控我們的前期研究,對44例卵巢癌組織及14例正常卵巢組織樣本采用實時熒光定量PCR(RT-PCR)的方法檢測長鏈非編碼RNA HOTAIR m RNA的表達。結果卵巢癌組織中HOTAIR m RNA表達高于正常卵巢組織[(1.26士0.27 vs.(0.14士0.09)],差異有統(tǒng)計學意義(P㩳0.05),病理類型為低度分化及未分化的卵巢癌組織中HOTAIR m RNA表達高于中-低、中一高及高度分化組織⺌(1.65士0.4 1)vs(0.39士0.l4)⺗,差異有統(tǒng)計學意義(P㩳0.05)。結論卵巢癌組織中HOTA IR的表達增高,且癌組織分化越低,HOTA IR表達越高。提示HOTA IR有可能作為發(fā)現(xiàn)卵巢癌,并判斷其惡性程度的一個分子標志,可能在卵巢癌的發(fā)生、發(fā)展中扮演重要的角色。但機制不清。因此本研究擬在前期基礎上進一步研究Hotair在卵巢癌中的作用及其機制。為Hotair在卵巢癌中的作用以及精準治療提供理論依據(jù)和基礎。第一部分HOTAIR促進卵巢上皮性癌惡性生物學行為及其機制的研究目的研究沉默HOTAIR后對上皮性卵巢癌惡性生物學行為的影響。方法(1)q PCR檢測在HOTAIR在卵巢癌細胞株SKOV3,OVCAR3和A2780的表達情況。(2)沉默HOTAIR后,劃痕實驗檢測卵巢癌細胞轉移能力,transwell檢測卵巢癌細胞侵襲能力,Ed U檢測卵巢癌細胞增殖能力。結果(1)HOTAIR在SKOV3和OVCAR3細胞中表達高于A2780細胞;(2)沉默HOTAIR后,卵巢癌SKOV3細胞轉移、侵襲和增殖能力降低;結論沉默HOTAIR可以抑制卵巢癌SKOV3細胞惡性生物學行為。第二部分HOTAIR通過mi R-214和mi R-217調控PIK3R3參與卵巢癌惡性生物學行為目的探索HOTAIR影響卵巢癌惡性生物學行為的分子機制。方法(1)生物信息學預測HOTAIR可能調控的ce RNA;(2)q PCR檢測沉默HOTAIR后,PIK3R3的表達情況;同時檢測沉默PIK3R3后HOTAIR的表達情況;(3)q PCR檢測沉默HOTAIR或PIK3R3后,mi R-214和mi R-217表達情況;(4)q PCR檢測mi R-214和mi R-217以及mi R-214和mi R-217inhibitor對HOTAIR和PIK3R3的影響;(5)雙熒光素酶報告基因檢測HOTAIR或PIK3R3是否是mi R-214和mi R-217的直接靶點。(6)免疫組化檢測PIK3R3在卵巢癌組織的表達情況;(7)沉默PIK3R3后,檢測卵巢癌細胞的增殖、轉移和侵襲能力。結果:(1)生物信息學預測HOTAIR和PIK3R3互為ce RNA,HOTAIR和PIK3R3是mi R-214和mi R-217的潛在靶點;(2)沉默HOTAIR后,PIK3R3表達降低;沉默PIK3R3后,HOTAIR表達下調(P0.05);(3)沉默HOTAIR或PIK3R3后,mi R-214和mi R-217表達上調(P0.05);(4)轉染mi R-214和mi R-217 mimics后,HOTAIR或PIK3R3表達降低;轉染mi R-214和mi R-217 inhibitor后,HOTAIR或PIK3R3表達增加;(5)和對照組比較,共轉染mi R-214 mimics和p MIR-HOTAIR3’UTR后,熒光素酶活性明顯下降(P0.05);和對照組比較,共轉染mi R-214 mimics和MIR-HOTAIR 3’UTRm后,熒光素酶活性無明顯改變(P0.05);共轉染mi R-217 mimics和p MIR-HOTAIR3’UTR后,熒光素酶活性明顯下降(P0.05);和對照組比較,共轉染mi R-217 mimics和MIR-HOTAIR 3’UTRm后,熒光素酶活性無明顯改變(P0.05);(6)免疫組化結果顯示,PIK3R3在卵巢漿液性癌和粘液性癌中表達高于正常卵巢組織;(7)沉默PIK3R3后,SKOV3細胞增殖能力、轉移能力和侵襲能力均受到抑制。結論:卵巢癌SKOV3細胞株沉默HOTAIR基因表達后,細胞增殖、轉移和侵襲能力降低;HOTAIR通過mi R-214和mi R-217靶向PIK3R3調控卵巢癌SKOV3細胞的惡性生物學行為。第三部分沉默HOTAIR影響SKOV3體內成瘤能力目的探究HOTAIR在體內對卵巢癌SKOV3細胞生長的影響方法(1)慢病毒載體構建HOTAIR沉默的穩(wěn)定細胞株,然后移植到裸鼠左側腋窩,觀察移植瘤生長情況;(2)采用免疫組織化學檢測沉默HOTAIR基因后,裸鼠移植瘤中PIK3R3的表達情況。結果(1)和對照組比較,沉默HOTAIR后,移植瘤生長緩慢,體積和重量降低(P0.05)(2)免疫組化結果顯示,卵巢癌SKOV3細胞株感染慢病毒攜帶的HOTAIR基因sh RNA后,沉默組和對照組比較,PIK3R3的表達降低(P0.05)。結論沉默HOTAIR后,卵巢癌致瘤能力降低。
[Abstract]:Ovarian cancer is one of the three malignant tumors of the cervical cancer and endometrial cancer, the mortality rate in gynecologic cancers, because of insidious onset, the lack of specific symptoms and signs and effective screening early, about 70% of patients with ovarian cancer have local or distant metastases, lost the best time for operation as a result, the overall 5 year survival rate is poor. Early ovarian cancer (stages I and II) the survival rate can reach 80%~95%, compared with advanced ovarian cancer (stage III and IV) the long-term survival rate of ovarian malignant tumor is only 10%~30%. so there is a "two 70%": more than 70% patients are diagnosed at advanced stage; approximately 70% of patients within two years. Therefore, thus, for early diagnosis and effective treatment for patients with ovarian cancer survival rate is very important. In recent years, scholars have been looking for a specific The method to improve the survival rate of ovarian cancer, ensure effective prognosis. Another theory of "dualism model" that ovarian cancer is divided into high grade and low grade two. High grade ovarian cancer ovarian cancer accounted for 75%, accounting for 90% of the death rate of ovarian cancer, the disease rapidly that is highly invasive, often accompanied by extensive abdominal metastasis and poor prognosis. The cultivation of epithelial ovarian cancer has low level of slow, benign, and malignant tumor at the junction of the development process, for the early, the prognosis is good. So the high level of epithelial ovarian cancer as prone to invasion and metastasis that is a healthy women. However, the threat of malignant tumor invasion and metastasis mechanism is unknown. Recently, after extensive research of MI RNA, called the long chain of non RNA (long non-coding RNA encoding, LNC encoding RNA RNA) non a molecular weight greater than 200nt was found And they have attracted more and more research. Because of the effect of regulation on the expression of target genes, LNC RNA is involved in many cancers, the process of development. Recent studies have shown that LNC RNA is involved in transcriptional activation, chromatin modification, transcriptional interference and nuclear transport several important regulation. Early regulation of figure 1 Lnc RNA the US, in 44 cases of ovarian carcinoma and 14 cases of normal ovarian tissue samples by real-time fluorescent quantitative PCR (RT-PCR) method to detect the expression of RNA encoding HOTAIR m long chain non RNA. The expression of HOTAIR in ovarian cancer m RNA higher than that of normal ovarian tissues [(1.26 + 0.27 vs. (0.14 + 0.09)], the difference was statistically significant (P? 0.05), the pathological types were poorly differentiated and undifferentiated ovarian carcinoma HOTAIR m RNA was higher than that in low and high, in a highly differentiated tissue? (1.65 + 0.41) vs (0.39 + 0.l4)?, the difference was statistically significant (P 0.? 05). Conclusions the increased expression of HOTA in ovarian cancer IR, and the differentiation of the carcinoma is lower, the higher the expression of IR HOTA suggest that HOTA IR may be found in ovarian cancer, and to judge the malignant degree of a molecular marker, in ovarian cancer, plays an important role in the development of it. The mechanism is not clear. Therefore the study effect and mechanism in the early stage on the basis of further study of Hotair in ovarian cancer. The role of Hotair in ovarian cancer and accurate treatment and provide a theoretical basis and foundation. Influence on the biological behavior of malignant epithelial ovarian cancer the first part of the research on HOTAIR to aim to study the silencing of HOTAIR in epithelial ovarian cancer for the malignant biological behavior and its mechanism. Methods (1) Q PCR in the detection of HOTAIR in ovarian cancer cell line SKOV3, the expression of OVCAR3 and A2780. (2) after silencing HOTAIR, metastasis detection of ovarian cancer cell scratch test, t The invasion ability of ovarian cancer cell ranswell detection, Ed detection of U ovarian cancer cell proliferation. Results (1) the expression of HOTAIR in SKOV3 and OVCAR3 cells than A2780 cells; (2) after silencing HOTAIR, SKOV3 ovarian cancer cell metastasis, reduce proliferation and invasion; conclusion silencing of HOTAIR can inhibit the malignant biological behavior of ovarian cancer cell line SKOV3 the second part of the HOTAIR. Through the MI R-214 and MI R-217 PIK3R3 is involved in the regulation of malignant biological behavior of ovarian cancer objective to explore the molecular mechanism of HOTAIR affecting the malignant biological behavior of ovarian cancer. Methods (1) bioinformatics prediction CE HOTAIR may regulate the RNA; (2) HOTAIR Q PCR after the detection of silence, the expression of PIK3R3 and expression; HOTAIR silencing of PIK3R3; (3) Q PCR detection of silencing HOTAIR or PIK3R3, MI R-214 and MI R-217 expression; (4) Q PCR and MI R-214 detection of MI R-217 and MI R-214 and MI R-217inhibitor The effects on HOTAIR and PIK3R3; (5) dual luciferase reporter gene assay of HOTAIR or PIK3R3 is a direct target of MI R-214 and MI R-217. (6) the expression of immunohistochemical detection of PIK3R3 in ovarian cancer tissues; (7) after silencing PIK3R3, detection of ovarian cancer cell proliferation and invasion, metastasis results: (1) forecasting of biological information HOTAIR and PIK3R3 are CE, RNA, HOTAIR and PIK3R3 is a potential target for MI R-214 and MI R-217; (2) after HOTAIR was silenced, the decreased expression of PIK3R3; PIK3R3 silencing, downregulation of HOTAIR (P0.05); (3) the silencing of HOTAIR or PIK3R3, up regulation the expression of MI R-214 and MI R-217 (P0.05); (4) mi R-214 and MI R-217 transfected with mimics, HOTAIR or PIK3R3 decreased expression of R-214 and MI R-217; transfection of MI inhibitor, HOTAIR or PIK3R3 expression increased; (5) and the control group, mimics and P were transfected into mi R-214 MIR-HOTAIR3 'UTR, luciferase activity obviously Drop (P0.05); and the control group, mimics and MIR-HOTAIR were transfected into mi R-214 3 'UTRm, luciferase activity had no significant change (P0.05); MI R-217 and P mimics were transfected into MIR-HOTAIR3 UTR, luciferase activity was significantly decreased (P0.05); and the control group, mimics and R-217 were transfected into mi MIR-HOTAIR 3 "UTRm, luciferase activity had no significant change (P0.05); (6) immunohistochemistry showed that the expression of PIK3R3 in ovarian serous carcinoma and mucinous carcinoma was higher than that in normal ovarian tissues; (7) after PIK3R3 was silenced, SKOV3 cell proliferation, metastasis and invasion ability was inhibited. Conclusion: the expression of silence HOTAIR gene, cell proliferation of ovarian cancer cell line SKOV3, reduce the metastasis and invasion ability; the malignant biological behavior of HOTAIR PIK3R3 by Mi R-214 to control MI and R-217 target of ovarian cancer SKOV3 cells. The third part effects of SKOV3 silencing HOTAIR in vivo tumorigenicity Objective to explore the effects of HOTAIR on the ability of in vivo on growth of ovarian cancer cell line SKOV3 (1) to construct a lentiviral vector of HOTAIR silencing stable cell lines, and then transplanted into nude mice to observe the tumor growth of the left armpit; (2) immunohistochemical detection of HOTAIR gene silencing by PIK3R3, expression of tumor in nude mice. Results (1) compared with the control group, after HOTAIR was silenced, tumor growth is slow, the volume and weight reduction (P0.05) (2) immunohistochemistry results showed that SKOV3 of ovarian cancer cell lines infected with lentivirus HOTAIR sh RNA, the silent group and the control group, decreased the expression of PIK3R3 (P0.05) conclusion silencing of HOTAIR after ovarian cancer tumorigenicity decreased.

【學位授予單位】：重慶醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R737.31

【相似文獻】

相關期刊論文 前10條

1 宋水勤;二次腫瘤細胞減滅術治療復發(fā)性卵巢上皮性癌[J];實用婦產(chǎn)科雜志;2002年05期

2 程琪輝,張喜平,曾小瀾,程穎波;血清腫瘤相關物質在卵巢上皮性癌診斷中的臨床價值[J];實用癌癥雜志;2002年04期

3 程憲珍,陳真,李剛,楊步榮;中晚期卵巢上皮性癌26例治療分析[J];腫瘤研究與臨床;2002年04期

4 苗華艷,王蓁,戴淑真,王言奎;卵巢上皮性癌患者血清可溶性細胞間粘附分子-1檢測的臨床意義[J];中華婦產(chǎn)科雜志;2003年01期

5 楊嫣紅;卵巢上皮性癌綜合治療43例分析[J];中國生化藥物雜志;2003年02期

6 李力;如何正確處理復發(fā)性卵巢上皮性癌[J];中國婦產(chǎn)科臨床;2003年06期

7 李力;如何正確處理復發(fā)性卵巢上皮性癌[J];廣西醫(yī)學;2004年10期

8 周艷芳;李黎;;晚期卵巢上皮性癌治療的探討[J];實用癌癥雜志;2006年01期

9 蔣維洪;陳士民;;晚期卵巢上皮性癌與血小板計數(shù)增高相關性分析[J];腫瘤學雜志;2006年03期

10 籍海虹;鄭曙民;;卵巢上皮性癌132例臨床分析[J];山西醫(yī)藥雜志(下半月刊);2007年06期

相關會議論文 前10條

1 胡蓉;惠寧;歐俊;吳冬;;三種化療方案對晚期卵巢上皮性癌的療效比較[A];第四屆長三角婦產(chǎn)科學術論壇暨浙江省2009年婦產(chǎn)科學術年會論文匯編[C];2009年

2 陳蘭芳;;中晚期卵巢上皮性癌的治療與預后影響因素[A];中國抗癌協(xié)會婦科腫瘤專業(yè)委員會第六次全國學術會議論文匯編[C];2001年

3 溫宏武;姚曉玲;邵愛玲;;晚期卵巢上皮性癌近20年預后變化[A];中國抗癌協(xié)會婦科腫瘤專業(yè)委員會第七次全國學術會議論文匯編[C];2003年

4 薛金玲;;晚期卵巢上皮性癌生存率20年變化研究[A];第八次全國婦產(chǎn)科學學術會議論文匯編[C];2004年

5 何成章;浦紅;呂蓓;董若凡;邱旭軍;;復發(fā)性/難治性卵巢上皮性癌45例臨床分析[A];江蘇省抗癌協(xié)會婦科腫瘤專業(yè)委員會第四次腫瘤學術研討會暨無錫市婦產(chǎn)科年會論文匯編[C];2005年

6 陳亦樂;劉艷瓊;唐迪紅;褚超男;;晚期卵巢上皮性癌不同化療途徑臨床對比研究[A];中華醫(yī)學會第九次全國婦科腫瘤學術會議論文匯編[C];2006年

7 王莉英;;35歲以下卵巢上皮性癌患者29例臨床分析[A];中華醫(yī)學會第九次全國婦科腫瘤學術會議論文匯編[C];2006年

8 彭澎;沈鏗;楊佳欣;吳鳴;黃惠芳;潘凌亞;郎景和;;吉西他濱聯(lián)合化療在卵巢上皮性癌治療中的效應研究[A];中華醫(yī)學會第九次全國婦科腫瘤學術會議論文匯編[C];2006年

9 余冬青;李力;;卵巢上皮性癌腹膜后淋巴結清掃的臨床價值[A];中華醫(yī)學會第九次全國婦科腫瘤學術會議論文匯編[C];2006年

10 余冬青;李力;;卵巢上皮性癌腹膜后淋巴結清掃的臨床價值[A];中華醫(yī)學會第九次全國婦科腫瘤學術會議論文匯編[C];2006年

相關博士學位論文 前10條

1 董立軍;HOTAIR促進卵巢上皮性癌惡性生物學行為及其機制的研究[D];重慶醫(yī)科大學;2016年

2 楊波;循環(huán)無細胞DNA對卵巢上皮性癌患者的診斷、療效評價和病情監(jiān)測的價值[D];河北醫(yī)科大學;2008年

3 姚洪文;新的卵巢上皮性癌蛋白標志物的初步研究[D];中國協(xié)和醫(yī)科大學;2009年

4 楊建華;水通道蛋白在卵巢上皮性癌中的作用及其調節(jié)機制研究[D];浙江大學;2006年

5 趙素芬;破壁靈芝孢子粉提取物抑制人卵巢上皮性癌細胞生長、轉移及逆轉順鉑耐藥的研究[D];河北醫(yī)科大學;2010年

6 余立群;不良心理應激對人卵巢上皮性癌發(fā)生、發(fā)展影響的實驗和臨床研究[D];南昌大學;2008年

7 呂訥男;Septin-9和Clusterin在卵巢上皮性癌患者外周血和腫瘤組織中的蛋白水平及其臨床意義[D];北京協(xié)和醫(yī)學院;2011年

8 高軍;不良心理應激對人卵巢上皮性癌荷瘤裸鼠腫瘤影響的蛋白組學研究[D];南昌大學;2011年

9 孫亞楠;化療對于卵巢上皮性癌移植瘤中干細胞層級的調控[D];河北醫(yī)科大學;2015年

10 楊潔;HIF-1α與卵巢上皮性癌血管生成及生物學行為的關系[D];北京協(xié)和醫(yī)學院;2012年

相關碩士學位論文 前10條

1 周賢芳;超聲聯(lián)合CA125檢測在卵巢上皮性癌的早期診斷價值[D];遵義醫(yī)學院;2015年

2 耿欣;β-HCG、ERK1/2及MMP-2在卵巢上皮性癌中的表達及意義[D];石河子大學;2015年

3 李婭茹;Wnt1和Lgr5在卵巢上皮性癌組織中的表達及意義[D];河北醫(yī)科大學;2015年

4 郭歡歡;PITX2、β-catenin在卵巢上皮性癌中的表達及意義[D];鄭州大學;2015年

5 劉彥芳;β-HCG、TGF-β1和TβR-Ⅰ在卵巢上皮性癌中的表達及意義[D];石河子大學;2015年

6 沈慧敏;人附睪蛋白4、溶血磷脂酸和糖類抗原125在卵巢上皮性癌中的臨床診斷價值[D];新鄉(xiāng)醫(yī)學院;2015年

7 姜志軍;Cks2在卵巢上皮性癌中的表達及臨床意義[D];山東大學;2015年

8 楊蓉;卵巢上皮性癌中干擾素介導的跨膜蛋白1的表達意義[D];第四軍醫(yī)大學;2015年

9 徐小博;Bevacizumab靶向治療卵巢上皮性癌的臨床觀察和機制研究[D];第四軍醫(yī)大學;2015年

10 陳醇;hMSH2及Ki-67在卵巢上皮性癌中的表達及意義[D];昆明醫(yī)科大學;2015年

，

本文編號：1547257