miR-195在子宫颈癌发生发展中的作用及分子机制研究

发布时间：2018-05-18 09:23

本文选题：子宫颈癌 + miR-195　；参考：《山东大学》2016年博士论文

【摘要】：研究背景：子宫颈癌是全世界女性最常见的生殖系统恶性肿瘤之一,发病率仅次于乳腺癌而位居第二位,死亡率为第五位,严重威胁人类生命和健康。据统计,全世界每年大约有50万新发子宫颈癌病例,占所有癌症新发病例的5%；近27万患者死亡。我国每年新发病例13-15万,大约占全球发病总数的三分之一；我国每年子宫颈癌死亡人数大约为5万。子宫颈癌的主要病理类型有鳞状上皮癌和腺上皮癌,其进展是一个相当复杂的过程,与多基因多阶段的异常调控有关,目前我们对子宫颈癌发生发展机制的了解仍不全面。研究已证实,高危型人乳头瘤病毒(HPV)持续感染是子宫颈癌明确的致病原因。然而,感染HPV的女性并不一定发展成为子宫颈癌；此外,并不是所有处于子宫颈上皮癌变的患者最终均能发展成为子宫颈浸润癌。这说明子宫颈癌的发生发展是个复杂的过程,除了人类HPV病毒之外,还有其他因素在子宫颈癌的发生发展中发挥重要作用。因此,子宫颈癌发病和进展的分子机制仍需深入研究。MicroRNAs (miRNAs)是由20-25个核苷酸组成的RNA,通过与靶基因的3’非翻译区(3’-UTR)结合进而促进mRNA降解或者抑制mRNA翻译而发挥作用。通过在转录后水平调控基因表达,miRNAs在很多生命活动中发挥其重要功能,包括细胞增殖、分化、凋亡、发育等。已有研究报道,miRNAs表达异常与许多疾病如糖尿病、心血管疾病、类风湿关节炎等的发病及进展有密切相关性。近年来研究表明, miRNAs在人类许多癌症中也发挥重要作用,参与了肿瘤的发生、发展以及转移等多个过程。研究发现,有些miRNAs发挥癌基因作用,另外一些miRNAs发挥抑癌基因作用。miRNAs与其调节的靶基因形成调控网络共同调节肿瘤的发生及发展。研究还证实,miRNAs在癌症患者血清及癌组织中表达异常与肿瘤的发病、进展、转移、侵袭及耐药有密切的关系,因此miRNAs可能成为肿瘤诊断的良好靶标分子。MicroRNA-195 (miR-195)是一重要的抑癌miRNA,在肺癌、乳腺癌等多种肿瘤中均发挥抑癌基因的作用。然而,miR-195在子宫颈癌中的作用尚不完全清楚。为明确miR-195在子宫颈癌发生发展中的作用,本学位论文第一部分首先从临床样本出发,检测miR-195在子宫颈癌组织及癌旁组织中的表达情况,分析其表达与子宫颈癌发生、发展、转移的相关关系。结果提示miR-195在子宫颈癌中可能发挥抑癌基因作用。肿瘤细胞的增殖和转移是子宫颈癌发生发展的关键环节。肿瘤细胞的增殖与肿瘤的生长密切相关,而肿瘤细胞的侵袭、迁移能力决定了其转移能力。为进一步明确miR-195对子宫颈癌发病的抑制作用,本学位论文第二部分在体外培养的子宫颈癌细胞(HeLa)中表达miR-195,检测miR-195对细胞增殖、侵袭、迁移能力的影响。在确定miR-195能抑制HeLa细胞增殖、侵袭及迁移能力的基础上,本学位论文第三部分深入研究了miR-195发挥作用的分子机制,筛选并鉴定了细胞周期蛋白D2 (CyclinD2, CCND2)和转录因子MYB为miR-195直接调控的靶基因。细胞增殖与细胞周期密切相关,而CCND2是一重要的细胞周期蛋白。CCND2通过与周期蛋白依赖的激酶4(CDK4)及周期蛋白依赖的激酶6(CDK6)结合,进而调节CDK4和CDK6的活性,从而促进细胞从G1期转化至S期。研究表明,CCND2异常表达与人脑组织胶质瘤、胃癌等多种肿瘤的发生具有密切相关性。MYB是广泛存在的转录因子,它可以通过调节基因表达从而在细胞的分化与增殖过程中起到重要作用。MYB在神经母细胞瘤及乳腺癌等多种肿瘤组织中呈现高表达状态,并且其表达水平与预后密切相关。此外还有研究表明,MYB蛋白和肿瘤侵袭及转移密切相关。可见,CCND2和MYB是两个重要的促肿瘤蛋白。我们第三部分实验结果显示,miR-195可作用于CCND2及MYB基因的3’-UTR而直接抑制蛋白质表达,该结果初步阐明了miR-195抑制宫颈癌HeLa细胞增殖、侵袭及迁移的分子机制。本论文的基本框架和主要研究内容如图1所示,各部分的详细研究思路和技术路线将在论文中作进一步的阐述。第一部分：miR-195在子宫颈癌组织中的表达研究目的：检测子宫颈癌组织及癌旁组织中miR-195的表达情况,并结合临床病理资料分析miR-195与子宫颈癌发生、转移的关系。方法：在本部分研究中,我们首先收集69对子宫颈癌的临床样本,并提取子宫颈癌组织和对应癌旁组织的RNA,然后利用实时荧光定量PCR技术对miR-195表达水平进行检测,明确miR-195在子宫颈癌组织及邻近癌旁组织中的表达情况。最后,分析miR-195表达与子宫颈癌发病的相关性；进一步结合临床病理资料分析miR-195表达与子宫颈癌转移的相关性。结果：1.相对于癌旁组织,miR-195在子宫颈癌组织中的表达明显下调,统计分析结果显示差异具有显著性。2.与没有发生转移的子宫颈癌样本(38例)相比,miR-195在发生转移的子宫颈癌样本(31例)中表达显著下调(降为0.43倍)。3.在没有发生淋巴结转移的子宫颈癌病例中,18例(47%)肿瘤组织中miR-195表达水平较癌旁组织降低,8例(21%)肿瘤组织中miR-195表达水平较癌旁组织未发生改变,12例(32%)肿瘤组织中miR-195表达水平较癌旁组织升高。在发生淋巴结转移的病例中,20例(65%)肿瘤组织中miR-195表达水平较癌旁组织降低,6例(19%)肿瘤组织,中miR-195表达水平较癌旁组织未发生改变,5例(16%)肿瘤组织中miR-195表达水平较癌旁组织升高。结论：1.相对于癌旁组织,miR-195在子宫颈癌组织中的表达明显下调,说明miR-195在子宫颈癌中可能作为抑癌基因发挥作用。2.相对于未发生盆腔淋巴结转移的子宫颈癌样本,miR-195在发生转移的子宫颈癌样本中表达显著下调,说明miR-195在子宫颈癌的发展过程中可能发挥了重要的作用。第二部分：miR-195对子宫颈癌细胞(HeLa)增殖、迁移及侵袭能力的影响目的：体外细胞模型研究miR-195对子宫颈癌细胞(HeLa)增殖、迁移、侵袭能力的影响。方法：在本部分研究中,我们在HeLa细胞中高表达miR-195,通过对细胞进行直接计数,从而明确miR-195对HeLa细胞增殖的影响。利用流式细胞术检测HeLa细胞中表达miR-195后细胞周期的改变,从而明确miR-195对HeLa细胞周期的影响。利用伤口愈合模型来研究miR-195对HeLa细胞迁移能力的影响。利用基于Matrigel的Transwell法研究miR-195对HeLa细胞侵袭能力的影响。结果：1.miR-195对HeLa细胞增殖的影响第5天假转染组(Mock)细胞数为(14.5±2.1)×105；对照组细胞数为(14.6±0.8)×105；而表达miR-195组细胞数为(10.4±1.0)×105,与对照组比较差异有显著性(P0.05)。CCK-8检测结果显示,与Mock组相比,转染对照RNA对活力细胞数无影响(0.97±0.11倍),而高表达miR-195组细胞生长较对照组比较显著降低(0.69±0.08倍),两者比较差异有显著性(P0.05)。2.miR-195对HeLa细胞周期的影响Mock组中处于G0/G1期的细胞占总细胞数的50%,S期细胞约占39%；G2/M期细胞约占11%；对照组中处于G0/G1期的细胞占总细胞数的49%,S期细胞约占41%；G2/M期细胞约占10%；高表达miR-195后,处于G0/G1期的细胞比例增加(60%)；处于S期的细胞比例减少(30%)。三次实验结果统计显示,表达miR-195组G0/G1期细胞比例较对照组显著增加(P0.05),而S期细胞比例较对照组显著减少(P0.05)。3.miR-195对子宫颈癌HeLa细胞伤口愈合能力的影响与对照组相比,高表达miR-195的HeLa细胞伤口愈合能力显著下降。4.miR-195对子宫颈癌HeLa细胞侵袭能力的影响细胞接种24小时后,miR-195高表达组迁移穿过Matrigel的细胞数明显少于对照组；结晶紫染色结果显示,miR-195高表达组迁移穿过Matrigel的细胞染色吸光值为对照组的0.69±0.06倍(P0.05)。结论：1.miR-195在子宫颈癌中可能作为抑癌基因发挥作用,可抑制HeLa细胞生长、增殖、迁移和侵袭。2.miR-195可作为子宫颈癌治疗的潜在靶点,为子宫颈癌的治疗提供新的思路。第三部分：miR-195功能靶基因CCND2和MYB的鉴定目的：预测并验证miR-195在HeLa细胞中的靶向基因,从而明确miR-195调控子宫颈癌细胞行为的作用机制。方法：本部分研究中,我们选择较为权威的生物信息学工具TargetScan、DIANA microT和PicTar对miR-195的靶基因进行预测,得到一批共同预测到的靶基因。经过功能注释以及文献检索,最终将目标锁定在CCND2和MYB两个基因上。进一步利用定量PCR、蛋白质免疫印迹等方法来验证miR-195对CCND2口MYB表达的调控作用；并将包含有miR-195结合区的CCND2和MYB基因的3’-UTR序列构建到荧光素酶报告质粒中,然后将该质粒与miR-195共同转染到HeLa细胞中进行双荧光素酶活性检测。最后,在高表达miR-195的细胞中回补CCND2和MYB,检测细胞增殖、迁移及侵袭能力。结果：1.经过生物信息学预测及功能注释和文献检索,最终将目标锁定在CCND2和MYB两个基因上。其中CCND2与细胞周期密切相关,而MYB与肿瘤细胞转移、侵袭有密切相关性。2.定量PCR实验结果显示,在HeLa细胞中高表达miR-195后,CCND2基因的mRNA水平下降至对照组的0.85±0.07倍(P0.05),MYB基因的mRNA水平下降至对照组的0.79±0.06倍(P0.05)。蛋白质免疫印迹结果显示,高表达miR-195后CCND2和MYB的蛋白水平也显著降低。以上结果表明,miR-195在HeLa细胞中抑制CCND2和MYB基因的mRNA及蛋白质表达。荧光素酶检测结果显示,含CCND2基因3’-UTR的报告系统中,高表达miR-195组荧光素酶活性降低为对照组的0.65±0.07倍(P0.05)；含MYB基因3’-UTR的报告系统中,高表达miR-195组荧光素酶活性降低为对照组的0.53±0.08倍(P0.05)。3.在高表达miR-195的细胞中回补CCND2可以使细胞增殖能力回复到对照组的0.85±0.07倍,与转染miR-195组(为对照组的0.65±0.07倍)相比差异有显著性,回补CCND2对miR-195抑制的细胞迁移、侵袭能力无影响。4.在高表达miR-195的细胞中回补MYB可以使细胞增殖能力回复到对照组的0.77±0.05倍,与转染miR-195组(为对照组的0.65±0.07倍)相比差异有显著性；回补MYB使miR-195抑制的细胞迁移得以部分回复；回补MYB可以将被miR-195抑制的细胞侵袭能力回复到对照组的0.86±0.07倍,与转染miR-195组(为对照组的0.69±0.05倍)相比差异有显著性。结论：1.在子宫颈癌细胞中,miR-195可以直接与CCND2及MYB基因的3’-UTR区结合,从而抑制二者的表达。2.miR-195可以直接抑制CCND2和MYB基因的表达,从而发挥其抑制子宫颈癌细胞增殖、迁移和侵袭的作用。
[Abstract]:Background: cervical cancer is one of the most common female malignant tumors in the world. The incidence of the disease is the second place next to breast cancer, and the mortality rate is fifth. It is a serious threat to human life and health. According to statistics, there are about 50 new cases of cervical cancer in the world, accounting for 5% of all new cases of cancer in the world and nearly 270 thousand. The death of the patients is 13-15 million new cases in China each year, accounting for about 1/3 of the global total. The death toll of cervical cancer in China is about 50 thousand each year. The main pathological types of cervical cancer are squamous and adenocarcinoma. The progress is a very complicated process, which is related to the abnormal regulation of multi gene and multiple stages. Our understanding of the mechanism of the development of cervical cancer is still not comprehensive. Research has proved that persistent infection of high risk human papillomavirus (HPV) is a clear cause of cervical cancer. However, women infected with HPV do not necessarily develop cervical cancer; in addition, not all patients in the cervical epithelial cancer can eventually develop. This suggests that the development of cervical cancer is a complex process, other than human HPV virus, and other factors play an important role in the development of cervical cancer. Therefore, the molecular mechanism of the pathogenesis and progression of cervical cancer still needs to be studied in depth (.MicroRNAs (miRNAs) is composed of 20-25 nucleotides. " RNA, by combining with the 3 'untranslated region of the target gene (3' -UTR) to promote the degradation of mRNA or inhibit the translation of mRNA. By regulating gene expression at the post transcriptional level, miRNAs plays its important functions in many life activities, including cell proliferation, differentiation, withering, and development. Many diseases such as diabetes, cardiovascular disease, and rheumatoid arthritis are closely related. In recent years, studies have shown that miRNAs plays an important role in many human cancers, and participates in many processes, such as the occurrence, development and metastasis of cancer. Some studies have found that some of the miRNAs play the role of oncogene, and some other miRNAs hair. The role of the oncogene.MiRNAs and its regulatory target gene regulation network together regulate the occurrence and development of the tumor. The study also confirmed that the abnormal expression of miRNAs in the serum and cancer tissues of cancer patients is closely related to the incidence, progression, metastasis, invasion and resistance of cancer. Therefore, miRNAs may be a good target for the diagnosis of cancer. Sub.MicroRNA-195 (miR-195) is an important tumor suppressor miRNA, which plays the role of tumor suppressor gene in all kinds of tumor, such as lung cancer and breast cancer. However, the role of miR-195 in cervical cancer is not completely clear. In order to clarify the role of miR-195 in the development of cervical cancer, the first part of this thesis first started from clinical samples to detect M The relationship between the expression of iR-195 in cervical cancer tissue and para cancerous tissue and the correlation between its expression and the occurrence, development and metastasis of cervical cancer. The results suggest that miR-195 may play the role of tumor suppressor gene in cervical cancer. The proliferation and metastasis of tumor cells are the key link in the development of cervical cancer. In order to further clarify the inhibitory effect of miR-195 on the pathogenesis of cervical cancer, the second part of this dissertation expresses miR-195 in the cervical cancer cell (HeLa) cultured in vitro, and detects the effect of miR-195 on cell proliferation, invasion and migration. Based on the ability of miR-195 to inhibit the proliferation, invasion and migration of HeLa cells, the third part of this thesis has studied the molecular mechanism of miR-195, and screened and identified the target gene directly regulated by cell cycle protein D2 (CyclinD2, CCND2) and transcription factor MYB. Cell proliferation is closely related to cell cycle, and CCN D2 is an important cyclin.CCND2 that combines with cyclin dependent kinase 4 (CDK4) and cyclin dependent kinase 6 (CDK6) to regulate the activity of CDK4 and CDK6, thereby promoting the transformation of cells from the G1 phase to S phase. The study shows that the abnormal expression of CCND2 is closely related to the occurrence of a variety of tumors such as glioma and gastric cancer in human brain tissue Related.MYB is a widely existing transcription factor. It can play an important role in the differentiation and proliferation of cells by regulating gene expression..MYB is highly expressed in a variety of tumor tissues such as neuroblastoma and breast cancer, and the expression level is closely related to the prognosis. In addition, the study shows that the MYB protein is also expressed. It is closely related to tumor invasion and metastasis. It is seen that CCND2 and MYB are two important tumor stimulating proteins. Our third experimental results showed that miR-195 could act on the 3 '-UTR of CCND2 and MYB gene and directly inhibit protein expression. The results preliminarily elucidate the molecular mechanism of miR-195 inhibiting the proliferation, invasion and migration of HeLa cells in cervical cancer. The basic framework and main research contents of this paper are illustrated in Figure 1. The detailed research ideas and technical routes of each part will be further elaborated in this paper. Part 1: the purpose of miR-195 expression in cervical cancer tissue: to detect the expression of miR-195 in cervical cancer tissues and adjacent tissues, and to combine with clinical pathology. Data analysis of the relationship between miR-195 and the occurrence and metastasis of cervical cancer. Methods: in this part of the study, we first collected 69 clinical samples of cervical cancer and extracted the cervical cancer tissue and the RNA corresponding to the paracancerous tissue, and then detected the level of miR-195 by real time fluorescence quantitative PCR technique, and made clear the miR-195 in the cervical cancer group. In the end, the correlation between the expression of miR-195 and the incidence of cervical cancer was analyzed. The correlation between the expression of miR-195 and the metastasis of cervical cancer was analyzed by the clinicopathological data. Results: 1. compared with the adjacent tissue, the expression of miR-195 in the cervical cancer group was obviously down down, and the statistical analysis showed that the results were obvious. Compared with non metastatic cervical cancer samples (38 cases), the expression of miR-195 was significantly reduced in 31 cases of metastatic cervical cancer (0.43 times),.3. in cervical cancer cases without lymph node metastasis, and 18 (47%) of the tumor tissues in 18 cases (47%) were lower than those in the paracancerous tissue, 8 cases (2). 1%) the expression level of miR-195 was not changed in tumor tissue, and in 12 cases (32%), the expression level of miR-195 was higher than that of para cancerous tissue. In the cases of lymph node metastasis, the expression level of miR-195 in 20 cases (65%) was lower than that of para cancer tissue, 6 cases (19%), and the expression level of miR-195 was less than that of para cancerous tissue. The expression level of miR-195 in 5 cases (16%) was higher than that of para cancerous tissue. Conclusion: the expression of miR-195 in cervical cancer tissue is obviously down regulated by 1. relative to the paracancerous tissue, indicating that miR-195 may play a role in the tumor suppressor gene in cervical cancer as compared to the cervical cancer samples without pelvic lymph node metastasis, miR- 195 significantly down-regulation in the metastasis of cervical cancer samples, indicating that miR-195 may play an important role in the development of cervical cancer. Second: the effect of miR-195 on the proliferation, migration and invasion of cervical cancer cells (HeLa): in vitro cell model study on the proliferation of cervical cancer cells (HeLa) by miR-195 The effect of migration and invasion ability. Methods: in this part of the study, we expressed miR-195 in HeLa cells, and by direct counting the cells to determine the effect of miR-195 on the proliferation of HeLa cells. Flow cytometry was used to detect the changes in the cell cycle of the HeLa cells after the expression of miR-195, thus defining miR-195 to HeLa cell cycle. The effect of the wound healing model was used to study the effect of miR-195 on the migration ability of HeLa cells. The effect of miR-195 on the invasive ability of HeLa cells was studied using the Transwell method based on Matrigel. Results: the effect of 1.miR-195 on the proliferation of HeLa cells (Mock) was (14.5 + 2.1) x 105, and the number of cells in the control group was (14.6 + 0) (14.6 + 0). .8) x 105, and the number of cells expressed in the miR-195 group was (10.4 + 1) x 105, and there was a significant difference between the control group and the control group (P0.05).CCK-8 detection results showed that compared with the Mock group, the transfected control RNA had no effect on the number of active cells (0.97 + 0.11 times), while the cell length of the high expression miR-195 group was significantly lower than that of the control group (0.69 + 0.08 times), and the differences were poor. The effect of P0.05.2.miR-195 on the cell cycle of HeLa cells was 50% of the total number of cells in the G0/G1 stage, 39% in S, 11% in G2/M, 49% in the total number of cells in the control group, 41% in S, 10% in G2/M, and in G0/G1, in G0/G1, and in G0/G1. The proportion of cells in the period of S was increased (60%), and the proportion of cells in the stage of stage was decreased (30%). The three times of the experimental results showed that the proportion of G0/G1 phase cells in the miR-195 group was significantly higher than that of the control group (P0.05), while the proportion of S stage cells was significantly lower than that of the control group (P0.05) the effect of.3.miR-195 on the healing ability of cervical cancer HeLa cells was compared with the control group. The wound healing ability of HeLa cells with high expression of miR-195 significantly decreased the effect of.4.miR-195 on the invasive ability of HeLa cells in cervical cancer. After 24 hours of cell inoculation, the number of cells migrating through Matrigel in miR-195 high expression group was significantly less than that in the control group; the result of crystal violet staining showed that the miR-195 high expression group migrated through Matrigel cells. The value of light was 0.69 + 0.06 times (P0.05) of the control group. Conclusion: 1.miR-195 may play a role in the tumor suppressor gene in cervical cancer. It can inhibit the growth, proliferation, migration and invasion of HeLa cells as potential targets for the treatment of cervical cancer, and provide new ideas for the treatment of cervical cancer. The third part: miR-195 function target gene CCN. The purpose of D2 and MYB identification: to predict and verify the targeting gene of miR-195 in HeLa cells, and to clarify the mechanism of miR-195 regulating the behavior of cervical cancer cells. Methods: in this part, we choose the more authoritative bioinformatics tool TargetScan, DIANA microT and PicTar to predict the target gene of miR-195, and obtain one of the target genes of miR-195. Through functional annotation and literature retrieval, the target was finally locked on two genes of CCND2 and MYB. Quantitative PCR, protein immunoblotting and other methods were used to verify the regulation of miR-195 on the MYB expression of CCND2 mouth, and the 3 '-UTR sequence containing the CCND2 and MYB genes in the miR-195 binding region. The plasmid was built into the luciferase reporter plasmid, and then the plasmid and miR-195 were transfected into HeLa cells for double luciferase activity detection. Finally, the CCND2 and MYB were supplemented in the cells with high expression of miR-195, and the cell proliferation, migration and invasion ability were detected. Results: 1. through bioinformatics prediction, functional annotation and literature retrieval, the results will eventually be carried out. The target is locked on two genes of CCND2 and MYB, of which CCND2 is closely related to the cell cycle, while MYB and tumor cell metastasis, and the invasion of the.2. quantitative PCR test results show that the mRNA level of the CCND2 gene drops to 0.85 + 0.07 times as much as the control group (P0.05) in the HeLa cell, and the level of the MYB gene drops to the level to the control group. 0.79 + 0.06 times (P0.05) of the control group. Protein immunoblotting showed that the protein levels of CCND2 and MYB were also significantly reduced after high expression of miR-195. The results showed that miR-195 inhibited the mRNA and protein expression of the CCND2 and MYB genes in HeLa cells. The results of luciferase detection showed that the high table in the reporting system containing the CCND2 gene 3 '-UTR. The activity of luciferase in the miR-195 group decreased to 0.65 + 0.07 times (P0.05) of the control group. In the report system containing MYB gene 3 '-UTR, the luciferase activity of the high expression miR-195 group decreased to 0.53 + 0.08 times of the control group (P0.05).3. in the cells with high expression of miR-195, which could restore the cell proliferation ability to 0.85 + 0.07 times as much as the control group. Compared with the transfected miR-195 group (0.65 + 0.07 times as the control group), there was a significant difference between the transfected group and the transfected group (CCND2). The reactivation of miR-195 inhibited cell migration and invasion.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R737.33

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