HOTAIR促进卵巢上皮性癌恶性生物学行为及其机制的研究

发布时间：2018-02-28 12:12

本文关键词： HOTAIR 上皮性卵巢癌 q PCR SKOV3 A2780 增殖侵袭转移 HOTAIR PIK3R3 机制免疫组化　出处：《重庆医科大学》2016年博士论文　论文类型：学位论文

【摘要】：卵巢癌是继宫颈癌和子宫内膜癌的三大恶性肿瘤之一,其病死率高居妇科恶性肿瘤之首,由于起病隐匿、早期缺乏特异性症状与体征及有效的医学筛查手段,约70%卵巢癌患者就诊时已发生局部或远处转移,失去了手术的最佳时期,结果导致5年的总体生存率欠佳。早期卵巢癌(Ⅰ期和Ⅱ期)的远期生存率可达到80%~95%,相比之下,晚期卵巢癌(Ⅲ期和Ⅳ期)的远期生存率只有10%~30%。因此卵巢恶性肿瘤存在“两个70%”:超过70%的患者确诊时已属晚期;约70%的患者在两年内复发。故由此可见,寻找早期诊断及有效的治疗方法对于卵巢癌患者的生存率至关重要。近几年,学者们一直在寻找具有特异性的方法,以提高卵巢癌的生存率,保证患者的有效预后。另外一种学说“二元论模型”认为,卵巢癌分为高级别和低级别两类。高级别卵巢上皮性癌占卵巢癌发病的75%,占卵巢癌病死率的90%,其发病迅速,侵袭性极强,常伴发广泛的盆腹腔转移和种植,预后极差。而低级别卵巢上皮性癌发病缓慢,多经历良性、交界性和低度恶性的发展过程,多为早期,预后较好。因此高级别卵巢上皮性癌由于极易发生侵袭和转移,是威胁妇女健康的恶性肿瘤。然而其发生侵袭和转移的机制不明。近来,继mi RNA的大量研究之后,被称为长链非编码RNA(long non-coding RNA,lnc RNA)的一类分子量大于200nt的非编码RNA被发现并逐渐受到越来越多的研究。因其对靶基因表达的调控作用,lnc RNA参与了许多癌症发生、发展的过程。近年来的研究表明lnc RNA参与了转录激活、染色质修饰、转录干扰和核内运输多种重要的调控。图1 Lnc RNA的调控我们的前期研究,对44例卵巢癌组织及14例正常卵巢组织样本采用实时荧光定量PCR(RT-PCR)的方法检测长链非编码RNA HOTAIR m RNA的表达。结果卵巢癌组织中HOTAIR m RNA表达高于正常卵巢组织[(1.26士0.27 vs.(0.14士0.09)],差异有统计学意义(P㩳0.05),病理类型为低度分化及未分化的卵巢癌组织中HOTAIR m RNA表达高于中-低、中一高及高度分化组织⺌(1.65士0.4 1)vs(0.39士0.l4)⺗,差异有统计学意义(P㩳0.05)。结论卵巢癌组织中HOTA IR的表达增高,且癌组织分化越低,HOTA IR表达越高。提示HOTA IR有可能作为发现卵巢癌,并判断其恶性程度的一个分子标志,可能在卵巢癌的发生、发展中扮演重要的角色。但机制不清。因此本研究拟在前期基础上进一步研究Hotair在卵巢癌中的作用及其机制。为Hotair在卵巢癌中的作用以及精准治疗提供理论依据和基础。第一部分HOTAIR促进卵巢上皮性癌恶性生物学行为及其机制的研究目的研究沉默HOTAIR后对上皮性卵巢癌恶性生物学行为的影响。方法(1)q PCR检测在HOTAIR在卵巢癌细胞株SKOV3,OVCAR3和A2780的表达情况。(2)沉默HOTAIR后,划痕实验检测卵巢癌细胞转移能力,transwell检测卵巢癌细胞侵袭能力,Ed U检测卵巢癌细胞增殖能力。结果(1)HOTAIR在SKOV3和OVCAR3细胞中表达高于A2780细胞;(2)沉默HOTAIR后,卵巢癌SKOV3细胞转移、侵袭和增殖能力降低;结论沉默HOTAIR可以抑制卵巢癌SKOV3细胞恶性生物学行为。第二部分HOTAIR通过mi R-214和mi R-217调控PIK3R3参与卵巢癌恶性生物学行为目的探索HOTAIR影响卵巢癌恶性生物学行为的分子机制。方法(1)生物信息学预测HOTAIR可能调控的ce RNA;(2)q PCR检测沉默HOTAIR后,PIK3R3的表达情况;同时检测沉默PIK3R3后HOTAIR的表达情况;(3)q PCR检测沉默HOTAIR或PIK3R3后,mi R-214和mi R-217表达情况;(4)q PCR检测mi R-214和mi R-217以及mi R-214和mi R-217inhibitor对HOTAIR和PIK3R3的影响;(5)双荧光素酶报告基因检测HOTAIR或PIK3R3是否是mi R-214和mi R-217的直接靶点。(6)免疫组化检测PIK3R3在卵巢癌组织的表达情况;(7)沉默PIK3R3后,检测卵巢癌细胞的增殖、转移和侵袭能力。结果:(1)生物信息学预测HOTAIR和PIK3R3互为ce RNA,HOTAIR和PIK3R3是mi R-214和mi R-217的潜在靶点;(2)沉默HOTAIR后,PIK3R3表达降低;沉默PIK3R3后,HOTAIR表达下调(P0.05);(3)沉默HOTAIR或PIK3R3后,mi R-214和mi R-217表达上调(P0.05);(4)转染mi R-214和mi R-217 mimics后,HOTAIR或PIK3R3表达降低;转染mi R-214和mi R-217 inhibitor后,HOTAIR或PIK3R3表达增加;(5)和对照组比较,共转染mi R-214 mimics和p MIR-HOTAIR3’UTR后,荧光素酶活性明显下降(P0.05);和对照组比较,共转染mi R-214 mimics和MIR-HOTAIR 3’UTRm后,荧光素酶活性无明显改变(P0.05);共转染mi R-217 mimics和p MIR-HOTAIR3’UTR后,荧光素酶活性明显下降(P0.05);和对照组比较,共转染mi R-217 mimics和MIR-HOTAIR 3’UTRm后,荧光素酶活性无明显改变(P0.05);(6)免疫组化结果显示,PIK3R3在卵巢浆液性癌和粘液性癌中表达高于正常卵巢组织;(7)沉默PIK3R3后,SKOV3细胞增殖能力、转移能力和侵袭能力均受到抑制。结论:卵巢癌SKOV3细胞株沉默HOTAIR基因表达后,细胞增殖、转移和侵袭能力降低;HOTAIR通过mi R-214和mi R-217靶向PIK3R3调控卵巢癌SKOV3细胞的恶性生物学行为。第三部分沉默HOTAIR影响SKOV3体内成瘤能力目的探究HOTAIR在体内对卵巢癌SKOV3细胞生长的影响方法(1)慢病毒载体构建HOTAIR沉默的稳定细胞株,然后移植到裸鼠左侧腋窝,观察移植瘤生长情况;(2)采用免疫组织化学检测沉默HOTAIR基因后,裸鼠移植瘤中PIK3R3的表达情况。结果(1)和对照组比较,沉默HOTAIR后,移植瘤生长缓慢,体积和重量降低(P0.05)(2)免疫组化结果显示,卵巢癌SKOV3细胞株感染慢病毒携带的HOTAIR基因sh RNA后,沉默组和对照组比较,PIK3R3的表达降低(P0.05)。结论沉默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.

【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R737.31

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