长链非编码RNA SNHG16调控乳腺癌细胞迁移的分子机制研究

发布时间：2018-01-05 02:37

本文关键词：长链非编码RNA SNHG16调控乳腺癌细胞迁移的分子机制研究　出处：《山东大学》2017年博士论文　论文类型：学位论文

【摘要】：在中国,乳腺癌已经成为发病率最高的女性恶性肿瘤。根据国家癌症中心2015年的统计数据显示,乳腺癌不仅占据了女性新发肿瘤总数的15%,也是45岁以下女性因恶性肿瘤死亡的首要病因。更为严峻的是,当前我国乳腺癌的发病率和死亡率都呈上升趋势,严重危害着广大女性的健康和生命。实体肿瘤患者的发病和死亡一般由扩散的肿瘤细胞对机体正常功能的破坏导致,因此肿瘤细胞迁移已成为癌症转移潜在机制研究的一大热点。然而,介导乳腺癌细胞迁移的分子机制并不十分明确,可预测其进展转移的分子标志物仍然有限。发现乳腺癌进展转移过程中的关键分子及调节通路是乳腺癌研究中的重点,有助于指导乳腺癌早期诊疗从而降低病死率改善预后。长链非编码RNA(long noncoding RNA,lncRNA)是近年来发现的一类不编码蛋白质的,长度超过200bp的RNA分子,许多文献报道lncRNA在肿瘤进展转移的过程中发挥重要作用。现有研究表明,lncRNA通过多种机制发挥抑癌或促癌功能,其中lncRNA与miRNA之间的相互作用机制主要有:miRNA结合并降解 lncRNA,lncRNA 作为 miRNA 海绵(miRNA sponge)结合吸附 miRNA,以及lncRNA截短生成miRNA等。长链非编码 RNA SNHG16 全称为 small nucleolar RNA host gene 16,即核内小 RNA 宿主基因 16,又叫 non-coding RNA expressed in aggressive neuroblastoma(ncRAN),是一种最早在神经母细胞瘤中发现的lncRNA,并且被证明在结直肠癌和膀胱癌中有促癌作用。然而,SNHG16是否影响乳腺癌的进展转移及其作用机制尚无报道。因此,本课题旨在研究SNHG16在乳腺癌进展转移中的作用及其涉及的分子生物学机制。研究内容主要分为以下四个部分:一.SNHG16在乳腺癌中的异常表达及其对乳腺癌细胞增殖和迁移能力的影响;二.SNHG16在乳腺癌中发挥作用的关键下游miRNA和竞争性内源RNA的预测及验证;三.SNHG16的下游分子miR-98和E2F5在乳腺癌细胞迁移中的功能及二者之间的靶向作用验证;四.SNHG16对E2F5的调节作用及对乳腺癌细胞迁移功能的影响依赖于miR-98。第一部分SNHG16在乳腺癌中的异常表达及其对乳腺癌细胞增殖和迁移能力的影响目的:以往研究表明SNHG16在神经母细胞瘤、膀胱癌以及结直肠癌中异常高表达,并发挥促癌的作用。但是SNHG16在乳腺癌中的功能尚不清楚。本研究首先确定乳腺癌组织标本中SNHG16的表达水平,再通过调控乳腺癌细胞系中SNHG16的表达来进行体外实验以确定其对乳腺癌细胞增殖迁移能力的影响。方法:收集乳腺癌肿瘤组织及癌旁非肿瘤组织共30对,提取总RNA并反转录,通过相对实时定量PCR(qRT-PCR)检测乳腺癌肿瘤组织及癌旁非肿瘤组织中SNHG16的表达差异。同时检测常用乳腺癌细胞系中SNHG16的表达水平,选取表达量较高的细胞系进行干扰实验,表达量较低的细胞系进行过表达实验。采用MTT实验测定SNHG16对乳腺癌细胞增殖能力的影响。通过Transwell细胞迁移实验中穿过小室滤膜的细胞数目来检测调控SNHG16表达后细胞迁移能力的改变。结果:QRT-PCR结果表明,30对乳腺组织中有21对中的SNHG16在乳腺癌组织比正常组织表达升高。SNHG16在MDA-MB-231和MCF-7细胞系中表达量较高,在MDA-MB-468和SK-BR-3细胞系中表达较低。MTT实验证明SNHG16对乳腺癌细胞增殖无明显作用。Transwell实验结果表明在MDA-MB-231和MCF-7细胞系中干扰SNHG16后,细胞迁移能力下降;在MDA-MB-468和SK-BR-3细胞系中过表达SNHG16后,细胞迁移能力增强。结论:SNHG16在乳腺癌中具有促癌作用,能够增强乳腺癌细胞的迁移能力,对乳腺癌细胞增殖无明显作用。第二部分SNHG16在乳腺癌中发挥作用的关键下游miRNA和竞争性内源RNA的预测及验证目的:近年来的研究表明,长链非编码RNA可能参与竞争性内源RNA(competitive endogenous RNA,ceRNA)调控网络从而发挥功能。为了进一步探究SNHG16在乳腺癌中发挥作用的分子生物学机制,本研究首先应用生物信息学方法预测SNHG16可能相互调控的ceRNA及共同作用的miRNA,并通过一系列分子生物学实验验证SNHG16与下游miRNA的直接结合,最后在乳腺癌组织中进一步验证SNHG16与下游miRNA的相关性。方法:通过数据库预测SNHG16可能相互调控的ceRNA及其共同作用的miRNA,干扰或过表达SNHG16后,采用qRT-PCR实验检测候选ceRNA及miRNA的表达量改变,从而进一步鉴定SNHG16的关键下游分子。通过DIANA TOOLS预测二者与其共同作用的miRNA相互结合的位点,构建相应的工具载体及其结合位点突变载体,进行RNA免疫共沉淀(RIP)实验和荧光素酶报告基因实验(luciferase reporter assay)验证SNHG16与下游miRNA的直接结合和具体结合位点。同时qRT-PCR检测过表达候选miRNA后SNHG16的表达水平,进一步验证二者之间的相互作用。最后在乳腺癌组织中检测SNHG16与候选下游miRNA的表达水平并进行相关性分析。结果:从starBase数据库预测SNHG16可能相互调控的ceRNA包括Rap2B,HMGA2,AMOT,SMAD2,E2F5,ZEB1等。按照竞争性内源RNA调控理论,干扰SNHG16后,候选ceRNA的表达会由于二者之间共同的miRNA增多而减少;而过表达SNHG16后,候选ceRNA的表达会由于二者之间共同的miRNA减少而增多。因此,分别过表达和干扰SNHG16后进行qRT-PCR实验验证SNHG16与候选ceRNA的调控关系,结果表明干扰或过表达SNHG16后,E2F5表达量出现了相应下降或增多,而其他候选ceRNA的表达量未出现明显一致的变化,进一步提示E2F5很有可能为SNHG16的ceRNA。随后通过DIANA TOOLS预测二者与其共同作用的miRNA及相互结合的位点,并通过qRT-PCR实验验证SNHG16可能对miR-98具有海绵吸附作用(miRNA sponge)。构建野生型和突变型载体,进行RNA免疫共沉淀实验和荧光素酶报告基因实验进一步验证SNHG16与下游miR-98的直接结合和具体结合位点。过表达miR-98后SNHG16表达降低,进一步证明SNHG16与miR-98之间的相互作用。QRT-PCR结果统计分析表明,在乳腺癌组织中SNHG16与miR-98的表达水平呈显著负相关。结论:SNHG16与miR-98存在直接相互结合作用,可能作为ceRNA与E2F5竞争性结合miR-98从而发挥促癌功能。第三部分SNHG16的下游分子miR-98和E2F5在乳腺癌细胞迁移中的功能及二者之间的靶向作用验证目的:为了探究SNHG16是否通过miR-98和E2F5这一调控轴线在乳腺癌中发挥作用,对于下游分子miR-98和E2F5在乳腺癌中的功能及二者之间的靶向作用需要进一步验证。方法:首先在乳腺癌细胞中过表达miR-98后进行Transwell细胞迁移实验,并进行统计分析验证miR-98对乳腺癌细胞迁移的影响。通过荧光素酶报告基因实验、qRT-PCR及Western Blot(蛋白免疫印记)实验验证miR-98与E2F5的直接靶向作用。通过干扰E2F5后的Transwell实验验证E2F5在乳腺癌细胞迁移中的作用。最后检测乳腺癌肿瘤组织及癌旁非肿瘤组织中E2F5的表达差异并进行统计分析证明E2F5在乳腺癌中的功能。结果:Transwell细胞迁移实验证明过表达miR-98或干扰E2F5后能够显著抑制乳腺癌细胞迁移。荧光素酶报告基因实验、qRT-PCR及Western Blot实验证明miR-98能够直接靶向E2F5并调控其表达。30对乳腺组织中有23对中的E2F5在乳腺癌组织比正常乳腺组织表达升高。结论:miR-98能够显著抑制乳腺癌细胞迁移,E2F5作为miR-98的直接靶基因,干扰其表达后也能发挥类似功能。第四部分SNHG16对E2F5的调节作用及对乳腺癌细胞迁移功能的影响依赖于miR-98目的:为了证明SNHG16是通过与E2F5竞争性结合miR-98发挥促进乳腺癌细胞迁移的功能,我们进一步探究SNHG16对E2F5的调节作用并验证这种调节作用和SNHG16对乳腺癌细胞迁移功能的影响是否依赖于miR-98。方法:在MDA-MB-231和MCF-7细胞系中干扰SNHG16或在MDA-MB-468和SK-BR-3细胞系中过表达SNHG16后,通过qRT-PCR及Western Blot实验检测E2F5的表达变化。在乳腺癌细胞系和乳腺癌组织中检测SNHG16和E2F5的表达水平并进行相关性分析。进行"挽救"(rescue)实验即共转染SNHG16和miR-98观察miR-98是否可以逆转SNHG16对乳腺癌细胞迁移能力的增强作用以及对E2F5的调控作用。结果:QRT-PCR及Western Blot实验结果表明干扰SNHG16后,E2F5表达量降低;过表达SNHG16后,E2F5表达量升高。在乳腺癌细胞系和乳腺癌组织中SNHG16和E2F5的表达水平呈显著正相关,也从侧面验证了 SNHG16对E2F5的正调控作用。Rescue实验结果表明,与SNHG16共转染入miR-98后,miR-98能够部分消除SNHG16介导的细胞迁移增强以及SNHG16对E2F5的正调控作用。结论:SNHG16对E2F5的表达具有正调控作用,并且SNHG16对E2F5的调节作用及对乳腺癌细胞迁移功能的影响依赖于miR-98。本论文重点阐述了 SNHG16在乳腺癌进展转移中的功能及其具体作用的分子机制。创新之处主要表现为以下几点:1.首次对SNHG16在乳腺癌细胞迁移中的作用进行探究并证明SNHG16发挥作用依赖于miR-98;2.首次提出SNHG16作为竞争性内源RNA与E2F5竞争性结合miR-98从而发挥作用;3.通过RNA免疫共沉淀和荧光素酶报告基因实验验证了 SNHG16与miR-98的直接结合,并设计突变载体证实了数据库预测的结合位点;4.揭示了 SNHG16在乳腺癌中对E2F5的正向调控作用。本论文尚存在一些不足,也是我们未来的研究方向,主要存在以下几点:1.论文主要对SNHG16在乳腺癌细胞增殖和迁移方面的功能进行了探究,尚未在乳腺癌细胞侵袭、凋亡、化疗耐药等方面发现明显调控功能,需要进一步研究。2.课题主要利用乳腺癌细胞系进行了体外实验研究SNHG16对乳腺癌转移的影响,尚需包括裸鼠成瘤及肺转移模型等体内实验进一步完善SNHG16在乳腺癌中作用功能的探究。3.本研究在临床标本中仅对SNHG16和下游分子在乳腺癌组织及癌旁非肿瘤组织中进行了表达差异及相关性分析,尚未与目前现有的乳腺癌分型指标进行对比研究,需要进一步扩大标本量进行明确的病理分型分析。
[Abstract]:In China, breast cancer has become the highest incidence of malignant tumor in women. According to the statistics in 2015 the National Cancer Center showed that breast cancer not only occupy 15% of the total number of female primary tumor, but also women under the age of 45 because the primary cause of death of malignant tumors. The more serious is that the incidence of breast cancer in China and death rates are on the rise, serious harm to women's health and life. Lead to destruction of morbidity and mortality in patients with solid tumors by diffusion of tumor cells to normal physiological function, so the migration of tumor cells has become a hot research topic in the mechanisms underlying metastasis of cancer. However, mediated molecular mechanism of breast cancer cells the migration is not very clear, molecular markers can predict the progression of metastasis is still limited. To find the key molecule in the process of metastasis of breast cancer progression and regulation pathway is breast cancer The focus of research, is helpful for the early diagnosis and treatment of breast cancer so as to reduce the mortality and improve the prognosis. Long chain encoding RNA (long noncoding non RNA, lncRNA) is a newly discovered class of encoding protein, RNA molecular length of more than 200bp, many reported that lncRNA play an important role in tumor progression and metastasis in the current study shows that lncRNA, through a variety of mechanisms play a tumor suppressor or cancer promoting function, the interaction mechanism between lncRNA and miRNA are: miRNA binding and degradation of lncRNA, lncRNA as miRNA (miRNA sponge) combined with sponge adsorption miRNA, and generate miRNA. LncRNA truncated long chain non encoding RNA SNHG16 small nucleolar RNA host gene 16, the small nuclear RNA gene 16, also called non-coding RNA expressed in aggressive neuroblastoma (ncRAN), is one of the earliest in neuroblastoma cells in tumor Now lncRNA, and proved in the colorectal cancer and bladder cancer in the cancer promoting effects. However, the influence of SNHG16 on metastasis and mechanism of breast cancer has not been reported. Therefore, the purpose of this paper is to study the role of SNHG16 in progression of metastasis in breast cancer and its mechanism of molecular biology. The research contents are mainly divided into the following four parts: the abnormal expression of.SNHG16 in breast cancer and its effect on the proliferation and migration of breast cancer cells; the prediction and verification of two.SNHG16 play a role in breast cancer miRNA key downstream and competitive endogenous RNA; between three.SNHG16 downstream molecules miR-98 and E2F5 in breast cancer cell migration in the function and the two target verification; four the regulative effect of.SNHG16 on E2F5 and the influence on the function of breast cancer cell migration depends on the first part of the miR-98. abnormal expression of SNHG16 in breast carcinoma The influence on the proliferation and migration of breast cancer cells Objective: Previous studies showed that SNHG16 in neuroblastoma, bladder cancer and colorectal cancer in abnormal high expression, and play the role of promoting cancer. But the SNHG16 function in breast cancer is unclear. In this study, to determine the expression level of SNHG16 mammary gland carcinoma specimens the in vitro experiment to the regulation of SNHG16 expression in breast cancer cell lines to determine their impact on proliferation and migration of breast cancer cells. Methods: We collected and cancer of breast tumor adjacent non tumor tissue of a total of 30, total RNA extraction and reverse transcription by relative quantitative real-time PCR (qRT-PCR) detection breast cancer tumor tissues and non differential expression of SNHG16 in tumor tissue. At the same time to detect the expression of SNHG16 in breast cancer cell lines, select the interference experiment high expression cell lines, expression Low cell lines were overexpression experiments. Determination of effects of SNHG16 on cell proliferation of human breast cancer by MTT assay. Transwell cell migration by cell number through the experiment to detect the cell membrane regulates the expression of SNHG16 after the changes of cell migration. Results: QRT-PCR results showed that 30 of the breast tissues of 21 SNHG16 in breast cancer tissue than normal tissue expression increased.SNHG16 expression was higher in MDA-MB-231 and MCF-7 cell lines, expression in MDA-MB-468 and SK-BR-3 cell lines with low.MTT experiments show that SNHG16 on the proliferation of breast cancer cells had no obvious effect of.Transwell experimental results show that the interference of SNHG16 in MDA-MB-231 and MCF-7 cell lines, cell migration decreased; overexpression of SNHG16 in MDA-MB-468 and SK-BR-3 cell lines, cell migration ability. Conclusion: SNHG16 has a tumor promoting role in breast cancer, can increase The migration ability of breast cancer cells, has no obvious effect on the proliferation of breast cancer cells. The prediction and verification of purpose of the second part of the role of SNHG16 in breast cancer in key downstream miRNA and competitive endogenous RNA: recent studies show that long chain non encoding RNA may participate in the competition of endogenous RNA (competitive endogenous RNA, ceRNA) to play regulatory network function. In order to further explore the molecular mechanism of SNHG16 play a role in breast cancer, in this study we used bioinformatics methods to predict SNHG16 may regulate each other jointly by ceRNA and miRNA, and by directly binding to a series of molecular biology experiments of SNHG16 and miRNA downstream, finally verify the correlation SNHG16 and downstream of miRNA in breast cancer. Methods: the database may predict SNHG16 mutual regulation ceRNA and their interaction with miRNA, Interference or overexpression of SNHG16, the expression of qRT-PCR and miRNA ceRNA experiment to detect candidate key changes, thus further downstream molecular identification of SNHG16 DIANA TOOLS. Through the combination of the two forecast together with the miRNA site, and the corresponding construction tool carrier binding site mutant vector RNA immunoprecipitation (RIP) experiment and luciferase reporter assay (luciferase reporter assay) with direct verification of SNHG16 and downstream miRNA and specific binding sites. At the same time, qRT-PCR detected the expression level of SNHG16 expression of candidate miRNA, further verify the interaction between the two. The detection of SNHG16 and miRNA and the expression level of candidate downstream in breast cancer correlation analysis. Results: the prediction of SNHG16 may regulate each other ceRNA including Rap2B, HMGA2, AMOT, SMAD2, E2F5, ZEB1 from starBase database. According to the According to the competitive regulation of endogenous RNA interference theory, SNHG16, expression of candidate ceRNA decreases due to the increase in miRNA in common between the two; and the expression of SNHG16, expression of candidate ceRNA that increases due to the decrease of miRNA in common between the two. Therefore, the regulation relationship between expression and interference after the SNHG16 qRT-PCR experiment verify the SNHG16 and candidate ceRNA, results show that the interference or overexpression of SNHG16, E2F5 expression appeared decreased or increased, whereas the expression of other candidate ceRNA showed no obvious consistent changes, further suggesting that E2F5 is likely to SNHG16 ceRNA. followed by DIANA TOOLS two and predict the joint effect of miRNA and the interaction site of qRT-PCR, and through experimental verification of SNHG16 may have a sponge adsorption effect on miR-98 (miRNA sponge). Wild type and mutant vector construction, RNA co immunoprecipitation experiments And the luciferase reporter assay further verified with SNHG16 and downstream miR-98 and specific binding sites. Overexpression of SNHG16 decreased the expression of miR-98, further proof that.QRT-PCR result analysis of interaction between SNHG16 and miR-98, in breast cancer tissues, the expression level of SNHG16 and miR-98 showed a significant negative correlation. Conclusion: SNHG16 and miR-98 the direct interaction between ceRNA and E2F5, may serve as a competitive binding of miR-98 to promote tumor function. Between the third part of the SNHG16 downstream molecules miR-98 and E2F5 in breast cancer cell migration function and two targeting Test Objective: To explore whether SNHG16 through miR-98 and E2F5 control of this axis in breast cancer play a role for downstream molecules miR-98 and E2F5 function in breast cancer and two targeting methods need to be further verified: The expression of miR-98 in breast cancer cells after Transwell cell migration experiments and statistical analysis to verify the effect of miR-98 on the migration of breast cancer cells. The luciferase reporter assay, qRT-PCR and Western Blot (Western blot) directly target experimental verification of miR-98 and E2F5 to Transwell. Through experimental verification of E2F5 interference E2F5 the role in breast cancer cell migration. Finally, the detection of breast cancer tumor tissues and non differential expression of E2F5 in tumor tissue and statistical analysis showed that the E2F5 function in breast cancer. Results: Transwell cell migration experiments showed that overexpression of miR-98 or E2F5 interference can inhibit breast cancer cell migration luciferase reporter. The qRT-PCR and Western gene, Blot miR-98 can be proved directly targeting E2F5 and its regulation on the expression of.30 in breast tissue of 23 in E 2F5 in breast cancer tissue than in normal breast tissue expression. Conclusion: miR-98 can significantly inhibit the migration of breast cancer cells, E2F5 as a direct target gene of miR-98 interference expression can also play a similar role function. The fourth part of the SNHG16 of E2F5 and the influence on the function of breast cancer cell migration depends on the objective: to miR-98 SNHG16 was proved by miR-98 combined with E2F5 competition to promote breast cancer cell migration function, we further explore the regulatory role of SNHG16 on E2F5 and verify the regulation effect and SNHG16 on the function of breast cancer cell migration is dependent on the miR-98. method in MDA-MB-231 and MCF-7 cell lines in SNHG16 or MDA-MB-468 interference and SK-BR-3 cells in the over expression of SNHG16, expression of qRT-PCR and Western by Blot assay. E2F5 in breast cancer cells and breast cancer tissues. Correlation analysis was performed to detect the SNHG16 and E2F5 expression level. The "save" (rescue) is the experimental co transfection of SNHG16 and miR-98 on miR-98 can enhance the reversal effect of SNHG16 on the migration of breast cancer cells and the effect on the regulation of E2F5. Results: QRT-PCR and Western Blot showed that the interference SNHG16, the expression of E2F5 decreased; after overexpression of SNHG16, E2F5 expression increased. There was a positive correlation between the level of SNHG16 in breast cancer cells and breast cancer tissues and E2F5, also from the side to verify the SNHG16 of E2F5 positive role in the regulation of experimental results show that the.Rescue and SNHG16 were co transfected into miR-98, miR-98 can eliminate the SNHG16 cell migration regulation effect of SNHG16 on enhancement and E2F5. Conclusion: the expression of SNHG16 has a positive regulatory effect on E2F5, and the effect of SNHG16 on E2F5 and on the migration of breast cancer cells The effect depends on the miR-98. this paper focuses on the molecular mechanism of SNHG16 in the progression and metastasis of breast cancer. The function and specific role innovation mainly as follows: 1. for the first time on the role of SNHG16 in breast cancer cell migration to explore and prove the role of SNHG16 depends on the miR-98 of SNHG16 is proposed as 2.; the competition of endogenous RNA and E2F5 competitive binding miR-98 can play a role; 3. RNA by immunoprecipitation and luciferase assay verified the direct combination of SNHG16 and miR-98, and the design of the mutant vector confirmed the binding site prediction database; 4. reveals the positive regulation effect of SNHG16 on E2F5 in breast cancer. This thesis is there are some shortcomings, but also our future research direction, mainly has the following several points: 1. the main thesis of SNHG16 in breast cancer cell proliferation and migration of the The function of the inquiry, yet in the invasion, apoptosis of breast cancer cells, chemotherapy resistance etc. obvious regulation function, the need for further research project mainly uses the.2. breast cancer cell lines by in vitro experiment to study the effect of SNHG16 on breast cancer metastasis, still need to include the tumorigenicity and lung metastasis model in vivo to further improve in SNHG16 breast cancer in this study and Research on.3. function in clinical specimens only on SNHG16 and downstream molecules in breast cancer tissues and adjacent non tumor tissues were analyzed and the correlation between the expression of differences, not with the existing classification of breast cancer index

【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R737.9

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