Ⅰ型子宫内膜腺癌miRNA-mRNA调控网络分析及CPEB1对其生物学行为的影响

发布时间：2018-05-18 04:33

本文选题：Ⅰ型子宫内膜腺癌 + miRNA-mRNA网络　；参考：《南方医科大学》2016年博士论文

【摘要】：研究背景：子宫内膜癌(endometrial carcinoma, EC)是子宫内膜上皮细胞来源的一种常见的女性生殖系统恶性肿瘤,是妇科三大恶性肿瘤之一,发病率仅次于宫颈癌,位于第二位,占妇科肿瘤癌症的30%左右。虽然相对于其它女性生殖系统恶性肿瘤患者,子宫内膜癌患者大多数五年的生存率不低,预后程度也较好,但在我国有逐年上升趋势。严重危害女性健康。作为一种病因尚不明确的疾病,子宫内膜癌中有85%-90%为Ⅰ型子宫内膜癌；目前肿瘤疾病相关的研究依旧不能很好的解释子宫内膜癌的发病过程。子宫内膜癌的高发人群主要是绝经后妇女,年龄多为50-60岁,约占总发病人数的75%左右。近年来,在国内外的相关研究报道中,子宫内膜癌的发病率和死亡率在全世界范围内呈明显年轻化和迅速上升的趋势。美国癌症协会的评估报告显示,2005年美国新发现的子宫内膜癌40880例,其中7310例患者死亡,2010年,有新增病例43470例,死亡7950例,而最新的流行病学调查资料则显示,在2015年美国女性所有新发的癌症中,子宫内膜癌发病率明显上升,仅排在乳腺癌、肺癌和结肠癌之后位居第4位,并且预计因子宫内膜癌死亡的人数将达到10170之多。在我国,目前还没有大规模完全的子宫内膜癌统计学资料报道,但据不完全的调查统计结果显示,我国子宫内膜癌的发病率逐年上升的趋势已非常明显。根据肿瘤病理形态特征不同,子宫内膜癌分为两种类型：即Ⅰ型雌激素依赖和Ⅱ型非雌激素依赖,两者的发生发展的分子机制、形态学特征以及预后等存在明显的差别。Ⅰ型与雌激素过度刺激相关,主要为子宫内膜过度生长的基础上发展形成的子宫内膜样腺癌,占子宫内膜癌的80-90%；Ⅱ型病理类型主要为透明细胞癌,浆液性乳头状癌,粘液腺癌以及腺鳞癌等,临床以雌激素依赖的Ⅰ型为主[9],因此本研究主要针对Ⅰ型的子宫内膜样腺癌进行研究,后面提到的子宫内膜癌均指代Ⅰ型子宫内膜样腺癌。子宫内膜癌临床早期表现为阴道不规则流血症状,可有助于早期诊断,并且80%子宫内膜癌患者的病灶会局限在子宫体内,因此,相对于其它的女性生殖系统恶性肿瘤患者,子宫内膜癌患者大多数五年的生存率不低,预后程度也较好,有研究报道,早期子宫内膜癌患者的5年无病存活率可达98%,5年总生存率可达93%。然而,仍然有部分子宫内膜癌患者的病理分化程度低,侵袭转移率高,预后效果差,通常会因为肿瘤的转移和浸润生长,导致病情的深度恶化而死亡。目前,对于子宫内膜癌的发病机理尚未完全清楚,对于病理分期高、发生复发性转移的患者目前也没有确切有效的治疗措施,因此,深入研究子宫内膜癌的生物学特点,搜寻子宫内膜癌相关标记物并分析其与子宫内膜癌的分化、转移和侵袭存在的不同差异及其分子机制,对提高子宫内膜癌病程的判断预测、临床治疗的指导及患者预后的改善具有重要的意义。微小RNA (micro RNAs, miRNA)是一种由22-25个核苷酸组成的非编码单链RNAs,目前认为在哺乳动物体内,miRNA与靶基因作用,使靶基因的mRNA在转录后的翻译过程中被抑制或直接降解,导致靶基因的蛋白表达水平降低。通过这种方式,miRNA参与到包括生长发育、造血、器官形成、细胞增殖、凋亡乃至肿瘤发生等多种生命活动中。据研究表明,每个miRNA可以调控200个靶基因,同样一个靶基因可以被多个miRNA调控。生物信息学则是利用miRNA与靶基因mRNA可以完全或部分互补的原则,通过各自的碱基序列预测出可能与特定miRNA(或mRNA)作用的所有mRNA(或niRNA),以缩小研究范围,进一步通过实验验证二者的确切关系。miRNA-mRNA调控网络定义为在相似生物过程中的一组miRNAs和一组mRNAs。一个调控模块中包含的miRNAs和靶基因mRNAs都具有高度的关联性,他们被认为具有相似的生物功能。目前,较为广泛应用的是MRMs,这种方法就是利用多种异构数据源miRNA-mRNA碱基互补配对信息、miRNAs和mRNAs表达信息来构建miRNA-mRNA调控网络,他们他们所发现的miRNA-mRNA对具有较高的置信度并且miRNAs与mRNAs的表达模式高度相关。因此,通过挖掘miRNA-mRNA调控模块可以在分子层次上了解疾病的生物过程；其次,可以了解包括癌症或肿瘤在内的疾病生物机理；另外,还可以为基因诊断和治疗提供参考。胞质型多聚腺苷酸化原件结合蛋白(cytoplasmic polyadenylation element binding protein, CPEBs)是一组高度保守的RNA结合蛋白家族。在其与肿瘤关系的研究中,以CPEB1和CPEB4研究较为广泛。但结合前期工作基础,子宫内膜癌高通量测序提示CPEBs家族中的CPEB1与子宫内膜癌的发生更为密切。已有文献研究,CPEBs人类乳头瘤病毒(human papilloma virus, HPV)的感染有关[14]。HPV持久的感染和蛋白质E5、E6和E7的表达是宫颈癌发生的先决条件,HPVE5、E6、E7 mRNA含有3'不翻译区域(3'UTR)特定序列(富含U的胞质型聚腺苷酸化原件,CPE区),它们是CPEB1作用的特异性靶点,其多聚腺苷酸化、翻译受到CPEB1的调控。CPEB1在HPV阳性肿瘤发展中可能具有重要的调节功能,将CPEB mRNA作为生殖器官癌症的标记物应该非常有用,因此探究CPEB1在子宫内膜癌的发生中的作用有重要意义。研究目标：本研究旨在通过采用网络生物信息学理论,从子宫内膜癌相关miRNA文献中挖掘Traits associated miRNA,结合已知基因功能和miRNA靶基因预测数据库,构建miRNA和靶基因的综合调控关系网络,通过与前期研究工作想结合并初步验证,筛选出新的与子宫内膜癌高度相关的靶基因CPEB1及以靶基因CPEB1为中心的关系调控网络；在此基础上再以获得性与缺失性功能试验明确所筛选靶基因在子宫内膜癌细胞中的生物学功能,并初步探求CPEB1在子宫内膜癌发生发展中可能的分子机制。方法：使用自由检索词,以FACTA工具和Pubmed数据库进行文献挖掘子宫内膜癌相关miRNAs;4种不同的预测方法(TargetScan、miRanda、miRDB和Starbase)预测所挖掘出来每个miRNA的作用靶基因获得miRNA-mRNA对；从miRNA-mRNA对中选取至少5个以上miRNA共同靶向的mRNA为子宫内膜癌相关基因,构建子宫内膜癌的miRNA-mRNA网络；利用QRT-PCR法验证调控网络中综合排名靠前基因mRNA表达情况,筛选出特异性差异表达的基因CPEB1为目的基因进行深入研究。利用QRT-PCR法检测CPEB1在子宫内膜癌组织和细胞中的表达情况；构建CPEB1过表达载体,通过体外转染CPEB1过表达载体过表达CPEB1,以MTT、克隆形成、流式细胞术、划痕实验、transwell小室等实验方式体外检测CPEB1对子宫内膜癌细胞株生物学行为的影响；构建稳定过表达CPEB1的子宫内膜癌细胞株,以裸鼠成瘤实验体内分析CPEB1的生物学功能；通过转染CPEB1过表达载体,观察细胞形态变化,应用western blot分析细胞凋亡相关蛋白以及EMT标记关键蛋白分子的表达,初步探讨CPEB1参与子宫内膜癌细胞生物学行为调控的分子机制。结果：1.通过多步分析,结合现有近五年来41篇相关文献,初步筛选出208个子宫内膜癌相关miRNA及其靶基因；经过4个不同数据库比对分析,筛选同时存在于两个或两个以上数据库,并至少有两篇研究报道的miRNA为目标miRNA,最终获得35个子宫内膜癌相关miRNA。2.通过4种预测方法对35个子宫内膜癌相关miRNA进行靶基因预测,获得110995个miRNA-mRNA对,去除出现次数少于3的miRNA-mRNA对,最终获得由27个miRNAs和3082个基因组成的7919个miRNA-mRNA对,其中至少5个miRNA同时靶向的基因457个。3.分析457个候选基因的蛋白间的相互作用,有61个基因至少存在5个互作蛋白,14个基因至少有10个互作蛋白。信号通路分析这61个候选基因富集的调控网络,大部分通路为癌相关通路。4.从61个候选基因中挑选互作蛋白数量靠前的10个基因进行QRT-PCR实验验证,有3个基因表达失调,其中CDC25A和IGF1R在子宫内膜癌中表达上调,CPEB1表达下调,CDC25A和IGF1R已有文献报道与子宫内膜癌密切相关,筛选CPEB1为目标基因进行功能验证。5.分别检测CPEB1在20对子宫内膜癌组织及配对癌旁组织、3株子宫内膜癌细胞和1株正常子宫内膜上皮细胞的表达水平,结果显示CPEB1在子宫内膜癌组织和子宫内膜癌细胞株中表达显著下调,提示CPEB1可能作为子宫内膜癌发生的分子标记。6. CPEB1体外功能研究结果显示：MTT、克隆形成实验证实CPEB1能显著抑制子宫内膜癌细胞的体外增殖能力；流式细胞凋亡检测发现CPEB1能促进子宫内膜癌细胞的凋亡；划痕实验证实CPEB1过表达抑制子宫内膜癌细胞的迁移；transwell实验证实CPEB1能显著抑制子宫内膜癌细胞的侵袭能力；小鼠体表皮下成瘤实验及HE、IHC检测证实,CPEB1能显著抑制肿瘤在体内的形成。7. CPEB1在ISK细胞株中极低表达,CPEB1-siRNA实验未能成功构建CPEB1低表达干扰载体用于后续实验。8.通过生物信息学分析及数据库的预测,提示CPEB1可能通过细胞凋亡通路及EMT相关通路参与肿瘤疾病的发生；凋亡相关分子检测结果显示,过表达CPEB1后,子宫内膜癌细胞ISK中细胞凋亡相关分子P53、Bax、Caspase-3表达上调,Bcl-2表达下调；提示CPEB1可能参与P53介导子宫内膜癌细胞凋亡调控。9.EMT相关分子检测结果显示,过表达CPEB1后,细胞发生上皮化的形态学改变。子宫内膜癌细胞ISK中上皮-间质转化特征标志物和相关转录因子E-cadherin、β-catenin的表达明显上调,Vimentin、SMA、Snail的表达显著下调,Twist不发生明显变化,由此证实,CPEB1高表达促进细胞发生间质-上皮化改变(MET)。结论1.成功构建子宫内膜癌特异miRNA-mRNA调控网络,筛选CPEB1为子宫内膜癌密切相关基因。2.证实CPEB1在子宫内膜癌组织及细胞中特异性低表达,CPEB1低表达可能是子宫内膜癌发生及恶性转移的分子标记。3. CPEB1作为抑癌因子,具有抑制子宫内膜癌细胞的生长和增殖、促进子宫内膜癌细胞的凋亡、抑制侵袭和转移,抑制小鼠皮下成瘤肿瘤形成的生物学功能。4. CPEB1影响子宫内膜癌细胞中细胞凋亡相关蛋白P53、Bax、Bcl-2. caspase-3的表达,推测CPEB1可能通过P53介导的信号通路参与子宫内膜癌细胞凋亡的调控,参与子宫内膜癌的发生。5. CPEB1影响子宫内膜癌细胞中EMT特异标记物E-cadherin、Vimentin、 SMA、β-catenin及促EMT转录因子Snail的表达,推测CPEB1可能通过下调子宫内膜癌细胞EMT转录因子的表达,促进肿瘤发生MET,抑制肿瘤细胞的侵袭。
[Abstract]:Background: endometrial carcinoma (EC) is a common malignant tumor of female reproductive system from endometrium epithelial cells. It is one of the three most malignant gynecologic malignancies. The incidence is second only to cervical cancer, which is located in the second place, accounting for about 30% of the cancer of gynecologic cancer. The five year survival rate of the patients with endometrial cancer is not low and the prognosis is good, but it is increasing year by year in our country. It is serious harm to women's health. As a disease that is not clear, 85%-90% is type I endometrium cancer in endometrial cancer, and the research of tumor related diseases is still not very good. The high incidence of endometrial carcinoma is mainly the postmenopausal women, the age of which is 50-60 years old and about 75% of the total incidence of endometrium. In recent years, the incidence and mortality of endometrial cancer are becoming more and more young and rapidly rising in the world. In 2005, 40880 newly discovered endometrial cancer cases were found in the United States, of which 7310 patients died. In 2010, there were 43470 new cases and 7950 deaths. The latest epidemiological data showed that in all new American women's cancers, the incidence of endometrial cancer was significantly increased in 2015, only in line with the incidence of endometrial cancer. The number of breast cancer, lung cancer and colon cancer is the fourth place, and the number of deaths due to endometrial cancer is expected to reach more than 10170. In China, there are no statistical reports on large scale endometrial cancer in China. However, according to the incomplete survey statistics, the trend of the incidence of endometrial cancer in China has been increasing year by year. According to the pathological features of the tumor, endometrial carcinoma is divided into two types: type I estrogen dependence and type II non estrogen dependence. There are obvious differences in the molecular mechanism, morphological characteristics and prognosis of the two. Type I is associated with over stimulation of estrogen, mainly the overgrowth of endometrium. Endometrioid adenocarcinoma, which is based on the development of endometrioid adenocarcinoma, accounts for 80-90% of endometrial carcinoma. Type II pathological types are mainly transparent cell carcinoma, serous papillary carcinoma, mucous adenocarcinoma, and adenosscale carcinoma. The main clinical type of estrogen dependent type I is [9]. Therefore, this study mainly focuses on type I endometrioid adenocarcinoma. Endometrial carcinoma of the endometrium refers to type I endometrioid adenocarcinoma. The early clinical manifestations of endometrial carcinoma are irregular vaginal bleeding, which can help early diagnosis, and 80% of the endometrium cancer patients are localized in the uterus. Therefore, most of the patients with endometrial cancer are compared to other women with reproductive system cancer. The survival rate of the five years is not low and the prognosis is good. The 5 year disease free survival rate of the patients with early endometrial cancer can reach 98%, the total survival rate of 5 years can reach 93%., however, there are still some endometrium cancer patients with low degree of pathological differentiation, high invasion and metastasis rate and poor prognosis, usually because of tumor metastasis and infiltration. At present, the pathogenesis of endometrial cancer is not completely clear. There is no effective and effective treatment for patients with high pathological stages and recurrent metastasis. Therefore, the biological characteristics of endometrial cancer are studied, and the related markers of endometrial cancer are searched and analyzed. The differentiation, metastasis and invasion of endometrial carcinoma have different differences and their molecular mechanisms. It is of great significance to predict the course of endometrial cancer, guide the clinical treatment and improve the prognosis of the patients. RNA (micro RNAs, miRNA) is a non coding single chain RNAs consisting of 22-25 nucleotides, which is currently considered to be feeding. In milk moving objects, miRNA and target genes act to inhibit or directly degrade the target gene mRNA in the post transcriptional translation process, resulting in a decrease in the protein expression level of the target gene. By this way, miRNA participates in a variety of life activities including growth, hematopoiesis, organogenesis, cell proliferation, apoptosis and even tumorigenesis. The study shows that 200 target genes can be regulated by each miRNA, and the same target gene can be regulated by multiple miRNA. Bioinformatics uses the principle that miRNA and target gene mRNA can be fully or partially complementary, and all the mRNA (or niRNA) that may interact with a specific miRNA (or mRNA) can be predicted by their respective base sequences to reduce the scope of the study. Further experiments verify that the exact relationship between the two.MiRNA-mRNA regulatory networks is defined as a high correlation between a group of miRNAs in similar biological processes and the miRNAs and target gene mRNAs contained in a set of mRNAs. regulatory modules, and they are considered to have similar biological functions. At present, MRMs is widely used. The method is to use a variety of heterogeneous data sources miRNA-mRNA base complementary pairing information, miRNAs and mRNAs to construct the miRNA-mRNA regulatory network, they found that the miRNA-mRNA pairs have high confidence and the miRNAs and mRNAs expression patterns are highly correlated. Therefore, the miRNA-mRNA regulation module can be divided by mining the miRNA-mRNA regulation module. The biological processes of disease are understood at the sub level; secondly, we can understand the biological mechanisms of diseases including cancer or tumor; in addition, it can provide reference for genetic diagnosis and treatment. The cytoplasmic polyadenosine acidified original binding protein (cytoplasmic polyadenylation element binding protein, CPEBs) is a highly conserved RNA CPEB1 and CPEB4 are more widely used in the study of the association with the tumor. But combined with the early work basis, high throughput sequencing of endometrial cancer suggests that CPEB1 in the CPEBs family is more closely related to endometrial cancer. The infection of the CPEBs human mammary head tumor virus (human papilloma virus, HPV) has been studied. [14].HPV persistent infection and the expression of protein E5, E6 and E7 are the prerequisites for cervical cancer. HPVE5, E6, and E7 mRNA contain 3'untranslated region (3'UTR) specific sequence (rich in U cytoplasmic polyadenosine acidification original, CPE region). They are the specific targets of the action. The role of CPEB mRNA as a marker of reproductive organ cancer should be very useful in the development of positive tumor. Therefore, it is important to explore the role of CPEB1 in the occurrence of endometrial cancer. Traits associated miRNA was excavated, combined with the known gene function and the miRNA target gene prediction database, the comprehensive regulatory network of miRNA and target genes was constructed. Through the combination of previous research work and preliminary verification, the new target based on endometrial carcinoma was screened by CPEB1 and the target gene CPEB1 as the center. On this basis, the biological function of the screened target gene in endometrial cancer cells is identified by the acquired and missing function test, and the possible molecular mechanism of CPEB1 in the development of endometrial cancer is preliminarily explored. Methods: using free retrieval words, using FACTA tool and Pubmed database to carry out the literature mining in the uterus. Membrane cancer related miRNAs; 4 different prediction methods (TargetScan, miRanda, miRDB and Starbase) predicted that each miRNA target gene was excavated to obtain miRNA-mRNA pairs; at least 5 more miRNA common targets were selected from miRNA-mRNA pairs for endometrial cancer related basis, and the miRNA-mRNA network of endometrial cancer was constructed; Q was used for Q. The RT-PCR method was used to verify the expression of mRNA in the regulatory network, and the specific gene CPEB1 was screened out for the target gene. QRT-PCR was used to detect the expression of CPEB1 in endometrial carcinoma tissues and cells; to construct CPEB1 overexpressed carrier and transfect CPEB1 overexpression vector in vitro The effect of CPEB1 on the biological behavior of endometrial carcinoma cell lines was detected by MTT, cloned formation, flow cytometry, scratching experiment, and Transwell chamber. The biological function of the endometrial carcinoma cell line that stably overexpressed CPEB1 was constructed, and the biological function of CPEB1 in nude mice was analyzed in vivo of the tumor in nude mice; through transfection of CPEB1 over the table of CPEB1 The molecular mechanism of CPEB1 involvement in the regulation of biological behavior of endometrial cancer cells was preliminarily investigated by Western blot analysis of cell morphological changes. The molecular mechanism of CPEB1 involvement in the regulation of biological behavior of endometrial cancer cells was preliminarily discussed. Results: 1. through multistep analysis, 41 related documents have been screened in recent five years, and a preliminary screening was made. Endometrial carcinoma associated miRNA and its target genes were screened by 4 different database comparisons and at least two or more databases were screened at the same time, and at least two reported miRNA were targeted miRNA, and 35 endometrial cancer related miRNA.2. was finally obtained by 4 pretests for 35 endometrial cancer related miRNA. Target gene prediction, obtained 110995 miRNA-mRNA pairs, the removal of less than 3 of the miRNA-mRNA pair, and finally got 7919 miRNA-mRNA pairs of 27 miRNAs and 3082 genes, of which at least 5 miRNA targeted gene 457.3. analysis of the protein interaction between 457 candidate genes, 61 genes at least 5 interacts. Protein, 14 genes have at least 10 interacting proteins. The signaling pathway analyses the regulatory network of the 61 candidate genes, and most of the pathways are the cancer related pathway.4. from 61 candidate genes to select 10 genes from the 61 candidate genes to perform the QRT-PCR test, and 3 genes are dysfunctional, of which CDC25A and IGF1R are in endometrial cancer. The expression of CPEB1, CDC25A and IGF1R were closely related to endometrial cancer, and CPEB1 was selected as the target gene for functional verification..5. was used to detect CPEB1 in 20 endometrial and paracancerous tissues, 3 endometrial cancer cells and 1 normal endometrium epithelial cells. The expression of CPEB1 in endometrial carcinoma and endometrial carcinoma cell lines showed a significant downregulation, suggesting that CPEB1 may be a molecular marker of endometrial carcinoma in vitro. The results of the function of.6. CPEB1 in vitro showed: MTT, the clone formation experiment confirmed that CPEB1 could significantly inhibit the proliferation ability of endometrial carcinoma cells in vitro; flow cytometry It was found that CPEB1 could promote the apoptosis of endometrial cancer cells, and the scratch test confirmed that CPEB1 overexpression inhibited the migration of endometrial cancer cells, and the Transwell experiment confirmed that CPEB1 could significantly inhibit the invasion ability of endometrial cancer cells, the tumor formation experiment under the epidermis of the mice and the HE, IHC test evidence, CPEB1 could significantly inhibit the form of the tumor in the body. The expression of.7. CPEB1 was very low in ISK cell lines, and the CPEB1-siRNA experiment failed to construct CPEB1 low expression interference carrier for the follow-up experiment.8. through bioinformatics analysis and the prediction of database, suggesting that CPEB1 may participate in the occurrence of tumor disease through the apoptosis pathway and EMT related pathway; apoptosis related molecular detection results show that After overexpression of CPEB1, the apoptosis related molecules P53, Bax, Caspase-3, and Bcl-2 expression are up regulated in endometrial carcinoma cells, suggesting that CPEB1 may participate in the regulation of P53 mediated apoptosis of endometrial carcinoma cells and the results of.9.EMT related molecular detection show that after overexpression of CPEB1, the morphological changes of epithelial cells occur. Endometrial carcinoma is fine. The expression of epithelial mesenchymal transition in cell ISK and related transcription factor E-cadherin, the expression of beta -catenin was obviously up-regulated, the expression of Vimentin, SMA, Snail decreased significantly, and Twist did not change obviously. Thus, the high expression of CPEB1 promoted the change of cytoplasm epithelialization of cells (MET). Conclusion 1. successfully constructed the specific miRNA-mRNA of endometrial carcinoma. Regulatory network, screening CPEB1 for endometrial cancer closely related gene.2. confirmed that CPEB1 in endometrial cancer tissues and cells of low expression, CPE
【学位授予单位】：南方医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R737.33

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