子宫内膜癌中差异表达microRNA的筛选及miR-944对子宫内膜癌生物学行为影响的实验研究

发布时间：2018-06-24 04:02

本文选题：子宫内膜癌 + microRNA　；参考：《广西医科大学》2015年博士论文

【摘要】：子宫内膜癌(endometrial carcinoma, EC)是最常见的妇科恶性肿瘤之一,严重威胁妇女健康。近年由于环境、平均寿命延长、激素替代疗法应用等各种因素的影响,其发病率逐年上升。美国癌症协会统计在2012年全球大概有47130例新发子宫内膜癌患者,已经跃居发达国家女性生殖道恶性肿瘤首位。尽管目前对子宫内膜癌的治疗方法和技术不断发展,但对晚期和复发的子宫内膜癌的治疗仍为棘手。进一步研究和寻找子宫内膜癌的新的治疗手段尤为重要。国内外研究证实肿瘤发生发展是一个复杂的分子调控失控过程,与抑癌基失活、癌基因的激活从而导致细胞生长失控,细胞基质间黏附及细胞信号传导异常密切联系。因此,探索子宫内膜癌发生的分子学基础,寻找安全可靠的切入点抑制子宫内膜癌细胞生长,转移,对预防子宫内膜癌的发生和改善子宫内膜癌患者的预后,保护我国妇女健康具有重大意义。MicroRNA(miRN A)是近年来发现的一类保守的长度为19～24个碱基的非编码RNA分子,广泛存在真核生物体内,其通过与靶基因mRNA3’非翻译区(3’UTR)相对应的靶序列完全或不完全的互补结合,抑制或促进mRNA降解,调节基因,这些仅占人类基因1%的miRNA分子,却调控着人类三分之一以上基因的表达、修饰、转录和翻译的过程,对细胞增殖、分化及凋亡等生物学功能起着重要的调控作用。miRNA是肿瘤研究领域中的热点,其包括miRNA表达谱筛选,分子结构,调控机制,靶基因预测等研究。越来越多的研究证实,niRNA在多种恶性肿瘤中存在表达异常,在肿瘤中高表达的niRNA可能发挥癌基因的作用,而低表达的miRNA发挥抑癌基因作用。miRNA的发现可能成为极有应用价值的分子标志物,为恶性肿瘤的早期诊断和预后评估提供新方向。miR-944是目前学者关注的新焦点,目前国内外对关于miR-944功能的报道还比较少,初步研究发现：miR-944在子宫内膜癌、宫颈癌及胰腺癌等恶性肿瘤中表达显著增高,我们推测其在肿瘤中发挥癌基因的作用。但迄今为止,对子宫内膜癌组织进行差异表达miRNA的筛选及分析研究甚少。本课题旨在阐明子宫内膜癌组织中的miRNA表达谱及miR-944对子宫内膜癌生物学行为影响及调控作用的机制研究。研究内容包括以下四部分：第一部分子宫内膜癌组织中差异表达miRNA的筛选研究目的：筛选出子宫内膜癌组织中的差异表达的miRNA分子,获得子宫内膜癌miRNA表达谱,寻找新的诊断标志物。方法：收集3例子宫内膜癌患者的癌变内膜组织和3例正常对照组子宫内膜组织,提取RNA,应用丹麦Exiqon公司的miRCURYTM LNA miRNA芯片,筛选出子宫内膜癌组织中的差异表达的miRNA分子,采用实时定量逆转录聚合酶链反应(qRT-PCR)在样本中进行验证。结果：1.3例癌组织和3例正常内膜组织的总RNA抽取及质检,结果显示,所提取的总RNA的电泳可见带28S和18s条带,260／A280的值都介于1.8-2.1之间,检测RNA检测结果符合杂交芯片实验要求。2. miRNA芯片结果显示：子宫内膜癌组织与正常子宫内膜组织存在有显著差异表达的miRNA:有14个miRNA表达上调,6个miRNA表达下调。3.采用qRT-PCR方法对上调的miR-944、miR-373及下调的miR-548、miR-885进行验证,结果显示：子宫内膜癌中miR-944、miR-373上调的倍数为3.13倍和4.2倍,1niR-548、miR-885下调倍数为2.08倍及3.84倍,与芯片结果比较,趋势一致。结论miRNA表达谱芯片可用于分析子宫内膜癌组织miRNA表达谱；通过miRCURYTMLNA miRNA芯片筛选结合qRT-PCR验证,共得到14个显著上调6个显著下调的miRNA,为下一步开展子宫内膜癌组织niRNA的研究及功能学研究提供实验基础。第二部分子宫内膜癌组织中miR-944表达及其与临床病理特征的关系目的：探讨miR-944在子宫内膜癌组织中的表达,并分析其与临床病理特征的关系。方法：收集2012年3月—2014年4月在广西医科大学第一附属医院进行手术治疗的子宫内膜癌患者的癌变内膜组织40例,收集同期因子宫肌瘤等良性病变行子宫切除术的正常子宫内膜组织25例作为对照。采用qRT-PC R方法进行miR-944表达水平的相对定量检测,并分析其与患者临床病理特征的关系。结果：1.miR-944在子宫内膜癌组织及正常子宫内膜组织中的表达子宫内膜癌组miR-944的表达水平高于及正常内膜组(P0.05)。2.miR-944与临床病理特征的关系miRNA-944的表达与组织学分级、病理类型、淋巴转移、肌层浸润深度及FIGO分期因素相关,均有统计学意义(P0.05),与年龄无相关性。结论：miR-944在子宫内膜癌组织中高表达,提示miR-944可能参与子宫内膜癌发生及发展；miR-944的表达与组织学分级、病理类型、淋巴转移、肌层浸润深度及FIGO分期因素相关,可能可以预测子宫内膜癌的恶性程度及不良预后。第三部分 miR-944对子宫内膜癌细胞生物学行为的影响目的：探讨miR-944对子宫内膜癌Ishikawa细胞和KLE细胞生物学行为的影响。方法：人工合成miR-944的模拟物mimics及抑制物inhibitor,利用阳离子脂质体法,分别转染至人子宫内膜癌Ishikawa细胞和KLE细胞中。设置阴性对照组及空白对照组。采用Cell Counting Kit-8(CCK-8)法对比转染前后细胞增殖生长能力的区别；流式细胞学对比转染前后细胞凋亡情况的区别；Transwell小室法对比转染前后细胞迁移和侵袭能力的区别。结果：1.细胞转染效果荧光倒置显微镜观察转染后的miR-944 mimics的转染效率,通过荧光镜下视野与光镜下视野对比,Ishikawa细胞和KEL细胞的转染效率大约为85%。2. miR-944-mimics及miR-944-inhibitor(?)子宫内膜癌Ishikawa细胞KLE细胞生物学行为的影响。(1)CCK8法检测细胞增殖实验分别在(24h、48h、72h、96h)四个时间点行CCK8检测Ishikawa和KLE细胞的OD值,结果显示,相对于对照组细胞,转染了miR-944mimics的Ishikawa细胞和KLE细胞增殖活性均增强,转染了niR-944inhibitor的Ishikawa细胞和KLE细胞增殖活性均降低(P0.05)。(2)流式细胞术检测细胞凋亡对miR-944转染48h后的Ishikawa细胞进行流式细胞术细胞凋亡分析,结果显示,B组、NC组、miR-944inhibitor组和miR-944mimics组凋亡率分别为0.58±0.06%、0.66±0.07%、11.74±0.19%、0.68±0.03%, miR-944inhibitor组凋亡率明显高于B组(P0.05),niR-944mimics组凋亡率与B组差别无统计学意义(P0.05)。对miR-944转染48h后的KLE细胞进行流式细胞术细胞凋亡分析,结果显示,B组、NC组、miR-944 inhibitor组和miR-944mimics组凋亡率分别为2.55±0.23%、2.53±0.10%、5.13±0.14%、2.99±0.18%,miR-944inhibitor凋亡率明显高于B组(P0.05),miR-944mimics组凋亡率与B组差别无统计学意义(P0.05)。(3)细胞侵袭实验检测转染48h后各组KLE细胞迁移能力,对穿膜细胞进行拍照和计数,结果显示,转染miR-944 mimics的KLE细胞穿膜数量较B组明显增多(P0.05)；转染miR-944 inhibitor的KLE细胞穿膜数量较B组明显减少(P0.05)。Ishikawa细胞过膜后铺展性不好,拍照后无法计数,CCK8方法检测过膜细胞,结果显示：相对于对照组细胞,转染了miR-944 mimics的 Ishikawa细胞OD值高,间接反映了mimics组的穿膜数量多；转染miR-944 inhibitor 的 Ishikawa细胞OD低,间接反映了inhibitor组的穿膜数量少(P0.05)。结论：miR-944可促进子宫内膜癌细胞的增殖,侵袭与转移能力,降低miR-944水平可促进细胞凋亡,表明miR-944可能发挥癌基因的作用参与子宫内膜癌的发生发展。第四部分 miR-944对子宫内膜癌细胞生物学行为调控的靶基因预测及鉴定目的：预测并实验证实miR-944的靶基因,初步探讨miR-944的生物学机制。方法：登陆1nicroRN A库及靶基因预测的网站,在线检测或下载靶基因预测软件,综合运用TargetScan(http://genes.mit.edu/targetscan)、Mirbase (http://mirbase.org)、miRanda (http://www.microrna.org)生物信息筛选miR-944最可能的靶基因。Western blot (WB)对比转染前后细胞表达靶基因的区别。构建最可能的靶基因的3’UTR双荧光素酶报告载体,检测荧光素的活性以期在基因水平上验证靶基因上存在miR-944的作用靶点。结果：1. 预测miR-944的靶基因通过预测软件miRanda、Mirbase及TargetScan预测miR-944的靶基因,三个软件交集的niRNA-944靶基因共70个；登陆基因库,查询以上靶基因功能,尤其注意与恶性肿瘤侵袭转移密切相关的功能基因。最后选择非受体型蛋白酪氨酸磷酸酶14(protein tyrosine phosphatase non-receptor type 14,PTPN14)作为miR-944的可能靶基因来进一步研究。应用Targetscan预测miR-944与PTPN14基因的结合位点。2.蛋白水平验证PTPN14是否为miR-944的靶基因miR-944mimics组的KLE细胞的PTPN14蛋白表达量与B组比较下降,差异有统计学意义(P0.05)；niR-944inhibitor组则高于B组,差异有统计学意义(P0.05)表明miR-944模拟物能降低KLE细胞中PTPN14蛋白的表达,B组与NC组比较无统计学意义。在Ishikawa细胞中,miR-944mimics组,miR-944 inhibitor组的PTPN14蛋白表达量与B组均无差异(P0.05)。3. miR-944与PTPN14的结合结合靶点验证将niR-944mimics与连接了PTPN14基因3'-UTR区的pmirGLO载体共转染KLE细胞,实验设立阳性对照组及阴性对照组,重组报告质粒的荧光素酶活性,结果显示与对照组比较,实验组的荧光素酶活性显著下调,差别有统计学意义(P0.05)结论：PTPN14是miR-944是其中一个靶基因,抑制PTPN14的表达,可能是miR-944促进子宫内膜癌细胞增殖及侵袭转移的作用机制之一。
[Abstract]:Endometrial carcinoma (EC) is one of the most common gynecologic malignancies. It is a serious threat to women's health. In recent years, the incidence of the cancer is increasing year by year because of the influence of environment, prolonged life expectancy, and hormone replacement therapy. The American Cancer Association combined with 47130 new endometrium cancers worldwide in 2012. Patients have been in the first place in women's reproductive tract cancer in developed countries. Although the treatment and techniques for endometrial cancer are developing continuously, the treatment of advanced and recurrent endometrial cancer is still difficult. Further research and search for new treatment hand Duan You for endometrial cancer are important. Domestic and foreign studies have confirmed the occurrence of tumor. Development is a complex process of uncontrolled molecular regulation, which is associated with the inactivation of tumor suppressor base, the activation of the oncogene and the close relationship between cell growth out of control, cell matrix adhesion and abnormal cell signal transduction. Shift, to prevent endometrial cancer and improve the prognosis of endometrial cancer patients, the protection of the health of women in China is of great significance.MicroRNA (miRN A) is a class of conserved non coded RNA molecules with 19~24 bases in length. There is a wide range of eukaryotes, and it passes through the target gene mRNA3 'non translation zone (3' UTR). A complete or incomplete combination of corresponding target sequences, inhibiting or promoting mRNA degradation and regulating genes, which only occupy 1% of the human gene miRNA, regulate the expression, modification, transcription and translation of more than 1/3 of the human genes, and play an important role in regulating biological functions such as cell proliferation, differentiation and apoptosis,.Mi RNA is a hot spot in the field of cancer research, which includes the screening of miRNA expression spectrum, molecular structure, regulation mechanism and target gene prediction. More and more studies have proved that niRNA has abnormal expression in a variety of malignant tumors, and the high expression of niRNA in the tumor may play the role of cancer based cause, and the low expression miRNA plays the role of tumor suppressor gene. The discovery of miRNA may be a highly valuable molecular marker, providing new direction for the early diagnosis and prognosis evaluation of malignant tumor..miR-944 is a new focus of attention at present. There are few reports about miR-944 function at home and abroad. The preliminary study found that miR-944 is in endometriosis, cervix and pancreatic cancer. There is a significant increase in the expression of the tumor in the tumor, we speculate that it plays the role of the oncogene in the tumor. But so far, the screening and analysis of differential expression of miRNA in endometrial carcinoma tissue is very few. The purpose of this study is to clarify the expression of miRNA in endometrial carcinoma and the effect of miR-944 on the biological behavior of endometrial carcinoma and its regulation and control. Research on the mechanism of action. The study includes the following four parts: the first part: the screening of differential expression of miRNA in endometrial carcinoma: screening the differentially expressed miRNA molecules in endometrial carcinoma, obtaining the miRNA expression profile of endometrial carcinoma and finding new diagnostic markers. Methods: 3 cases of endometrial carcinoma were collected. The endometrial tissue of the patients and the endometrium of 3 normal controls, RNA, and the miRCURYTM LNA miRNA chip of Danish Exiqon company were used to screen the differentially expressed miRNA molecules in endometrial carcinoma tissue, and the real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to verify the samples. Results: 1.3 cases of cancer tissue were found. The total RNA extraction and quality examination of 3 cases of normal endometrium showed that the extracted total RNA electrophoresis showed 28S and 18S bands, and the values of 260 / A280 were between 1.8-2.1. The detection results of RNA conformed to the requirements of the hybridization chip experiment. The results of.2. miRNA chip showed that there was a significant difference between endometrial carcinoma tissue and normal endometrium. The differential expression of miRNA: was up to 14 miRNA expressions, and 6 miRNA expression down-regulation.3. was verified by qRT-PCR method for up regulated miR-944, miR-373 and down regulated miR-548 and miR-885. The results showed that the multiplier of miR-944, miR-373 up-regulated in endometrial carcinoma was 3.13 times and 4.2 times, 1niR-548, 2.08 times and 3.84 times the down-regulation of 1niR-548. The results were the same. Conclusion the miRNA expression chip can be used to analyze the miRNA expression profile of endometrial carcinoma tissue. Through the miRCURYTMLNA miRNA chip screening and qRT-PCR verification, 14 significantly up regulation 6 down regulated miRNA are obtained, which can provide experimental basis for the next step in the study and functional study of endometrial carcinoma group niRNA. Second part of the expression of miR-944 in endometrial carcinoma and its relationship with the clinicopathological features: To explore the expression of miR-944 in endometrial carcinoma and to analyze its relationship with the clinicopathological features. Methods: to collect the uterus from March 2012 to April 2014 at the First Affiliated Hospital of Guangxi Medical University. 40 cases of endometrial carcinoma in endometrial carcinoma, 25 cases of normal endometrium with hysterectomy and hysterectomy were collected for the same period as control. The relative quantitative detection of miR-944 expression was carried out by qRT-PC R method, and the relationship with the patients' clinicopathological characteristics was analyzed. Results: 1.miR-944 was in endometrial carcinoma. The expression level of endometrial carcinoma group miR-944 in tissue and normal endometrium was higher than that of normal endometrium (P0.05).2.miR-944 and clinicopathological features. The expression of miRNA-944 was related to histological classification, pathological type, lymphatic metastasis, depth of myometrium infiltration and FIGO staging, and was statistically significant (P0.05). Conclusion: miR-944 is highly expressed in endometrial carcinoma, suggesting that miR-944 may be involved in the occurrence and development of endometrial carcinoma, and the expression of miR-944 is related to histological classification, pathological type, lymphatic metastasis, depth of myometrium infiltration and FIGO staging, which may predict the malignancy and poor prognosis of endometrial carcinoma. The effect of the three part of miR-944 on the biological behavior of endometrial carcinoma cells: To explore the effect of miR-944 on the biological behavior of endometrial carcinoma Ishikawa cells and KLE cells. Methods: artificial synthesis of miR-944 simulants, mimics and inhibitor inhibitor, were transfected to human endometrial carcinoma Ishikawa cells by cationic liposome method. And KLE cells. Set negative control group and blank control group. Cell Counting Kit-8 (CCK-8) method was used to compare the proliferation and growth ability of cells before and after transfection; flow cytology compared the difference of cell apoptosis before and after transfection; Transwell cell method compared the difference of cell migration and invasion before and after transfection. Results: 1. cells Transfection effect with fluorescence inverted microscope to observe transfection efficiency of miR-944 mimics after transfection, the transfection efficiency of Ishikawa cells and KEL cells is about 85%.2. miR-944-mimics and miR-944-inhibitor (?) Ishikawa cell KLE cell biological behavior of 85%.2. miR-944-mimics and miR-944-inhibitor (?) endometrial carcinoma. (1) CCK8 method CCK8 detection of Ishikawa and KLE cells at four time points (24h, 48h, 72h, 96h) detected the OD values of Ishikawa and KLE cells respectively. The results showed that the proliferation activity of Ishikawa cells and KLE cells transfected with miR-944mimics was enhanced compared with that of the control group, and both niR-944inhibitor Ishikawa cells and cell proliferation activities were reduced. (2) flow cytometry was used to detect apoptosis in Ishikawa cells transfected with 48h by flow cytometry. The apoptosis rate of B group, NC group, miR-944inhibitor group and miR-944mimics group was 0.58 + 0.06%, 0.66 + 0.07%, 11.74 + 0.19% and 0.68 + 0.03%, respectively. The apoptosis rate of miR-944inhibitor group was significantly higher than that of the B group (P0.05). There was no significant difference in apoptosis rate between group niR-944mimics and group B (P0.05). The apoptosis of KLE cells after miR-944 transfected with 48h showed that the apoptosis rate of B group, NC group, miR-944 inhibitor group and miR-944mimics group was 2.55 + 0.23%, 2.53 + 0.10%, 5.13 + 0.14%, 2.99 + 0.18%, and the apoptotic rate of miR-944inhibitor was obvious Higher than group B (P0.05), there was no significant difference in the apoptosis rate between group miR-944mimics and group B (P0.05). (3) cell invasion test was used to detect the migration ability of KLE cells in each group after transfection of 48h and to take pictures and count of membrane cells. The results showed that the number of membrane transfected on miR-944 mimics KLE cells was significantly higher than that of B group (P0.05). The number of LE cells was significantly less than that in the B group (P0.05), the spread of.Ishikawa cells was not good, and the number of membrane cells could not be counted after taking pictures. CCK8 method detected the membrane cells. The result showed that compared with the control group, the Ishikawa cells transfected with miR-944 mimics had high OD value, which indirectly reflected the number of membrane in the mimics group, and the transfection of miR-944 inhibitor. The Ishikawa cell OD is low, which indirectly reflects the small number of membrane in the inhibitor group (P0.05). Conclusion: miR-944 can promote the proliferation, invasion and metastasis of endometrial cancer cells, and reduce the level of miR-944 to promote cell apoptosis. It is suggested that miR-944 may play the role of oncogene in the development of endometrial carcinoma. The fourth part of miR-944 against the child. The target gene prediction and identification of the biological behavior regulation of endometrial carcinoma cells: predict and verify the target gene of miR-944 and preliminarily discuss the biological mechanism of miR-944. Methods: landing on the 1nicroRN A library and the site of target gene prediction, on-line detection or downloading target gene prediction soft parts, and using TargetScan (http://genes.mit.edu/ta) Rgetscan), Mirbase (http://mirbase.org), miRanda (http://www.microrna.org) biological information screening miR-944 the most likely target gene.Western blot (WB) to compare the difference between the target gene and the target gene before and after the transfection. The 3 'UTR double luciferase reporter vector of the most likely target gene is constructed to detect the activity of fluorescein at the gene level. The target genes of miR-944 were tested on the target gene. Results: 1. the target gene for predicting miR-944 was predicted by the software miRanda, Mirbase and TargetScan for the prediction of the target genes of miR-944, and 70 niRNA-944 target genes of the three software intersections; the landfall gene pool was used to query the function of the target gene, especially closely related to the invasion and metastasis of malignant tumor. Finally, we chose non receptor type protein tyrosine phosphatase 14 (protein tyrosine phosphatase non-receptor type 14, PTPN14) as a possible target gene for miR-944 to further study. Using Targetscan to predict the.2. protein of the binding site of miR-944 and PTPN14 genes to verify that PTPN14 is the target gene. The expression of PTPN14 protein in KLE cells in group imics was lower than that in group B (P0.05), and in group niR-944inhibitor, the difference was statistically significant (P0.05) showed that the miR-944 mimic could reduce the expression of PTPN14 protein in KLE cells, and there was no statistical significance in B group. In group miR-944, the expression of PTPN14 protein in group inhibitor was not different from that of B group (P0.05).3. miR-944 and PTPN14 binding target verifying that niR-944mimics was co transfected with pmirGLO carrier of 3'-UTR region of PTPN14 gene, and the experiment set up positive control group and negative control group. Compared with the control group, the luciferase activity of the experimental group was significantly reduced, and the difference was statistically significant (P0.05) conclusion: PTPN14 was one of the target genes, which inhibited the expression of PTPN14, which may be one of the mechanisms of miR-944 to promote the proliferation and invasion and metastasis of endometrial carcinoma cells.
【学位授予单位】：广西医科大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R737.33

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