长链非编码RNA-H19促进乳腺癌细胞“干性”的维持

发布时间：2018-01-03 13:22

本文关键词：长链非编码RNA-H19促进乳腺癌细胞“干性”的维持　出处：《大连医科大学》2017年硕士论文　论文类型：学位论文

更多相关文章： H19 LIN28 let-7 乳腺癌干细胞

【摘要】：长链非编码RNA-H19(long non-coding RNA-H19,H19)是由H19基因编码,长度为2.3kb,不能进行编码蛋白的RNA,H19由RNA聚合酶II转录,是一个印记癌基因。其次H19母系等位基因表达,是最早被发现的非编码RNA。近期研究发现,H19可在多种原位或转移的肿瘤中高表达,且与患者的不良预后有很大的相关性。由此证明,H19在肿瘤的发生发展中发挥了重要的作用。研究报道,许多患乳腺癌的病人经过常规的放化疗治疗后,常会伴随肿瘤细胞的复发与转移,且造成的危害更加严重。鉴于此,科学家们对肿瘤的复发及转移等机制进行了进一步的研究,提出肿瘤干细胞(cancer stem cells,CSCs)是造成癌症复发和转移的根本原因。虽然经过了数十年不断的研究,至今还没有找到可以靶向治疗CSCs的治疗方法。此外,一些列研究表明在乳腺癌中存在一小群乳腺癌肿瘤干细胞(breast cancer stem cells,BCSCs),是造成乳腺癌无法治愈的主要原因。迄今为止,对于H19如何调控肿瘤发生发展的研究已有很多,也得到了许多令人振奋的结果。但H19在肿瘤干细胞中的调控机制还未能阐明。本课题通过对H19的深入研究,表明了H19对BCSCs干性的维持有不可或缺的作用。研究证实,在乳腺癌中H19可以作为竞争性内源RNA(competing endogenous RNA,ceRNA)的功能作用于microRNA let-7(let-7),一个抑癌microRNA。所以在乳腺癌中H19可以通过吸附let-7来促进肿瘤的进程并调控乳腺癌细胞的干性能力。此外,实验数据表明,H19可正向调控LIN28的表达,LIN28的3’UTR区域存在可以和let-7结合的序列。我们得出结论,在乳腺癌中H19可通过调控microRNAs及肿瘤多潜能因子影响乳腺癌的发生发展进程。我们的主要研究流程及结果如下所示:1.我们在乳腺癌病人组织中验证了H19的表达水平明显升高,随后应用流式细胞分选实验技术分选出BCSCs并验证H19的表达水平。表明了H19在乳腺癌病人组织及乳腺癌干细胞样细胞中显著高表达。2.随后对乳腺癌细胞中的H19进行异常表达处理(高表达或者敲降),通过细胞微球形成实验发现在乳腺癌细胞中异常高表达H19可明显促进肿瘤细胞干性能力的增强;与之相反,当在细胞中敲降H19后可抑制BCSCs的干性特征。3.此后,我们发现在乳腺癌细胞中,H19可调控CSCs标记物LIN28的表达水平。并且LIN28表达水平的升高可以恢复由于H19降低而抑制的BCSCs的干性能力。相应的,通过对乳腺癌病人组织分析的数据表明H19和LIN28在原发性乳腺癌中的表达水平有很大的相关性。4.在机制上,我们从以前的研究中发现H19可调控let-7的表达,而LIN28可调控let-7的生成。我们在乳腺癌细胞中研究发现,let-7的表达水平没有受H19表达的变化而变化,因此H19只影响let-7的生物利用度而不影响其表达水平,由此H19可作为ceRNA的功能吸附let-7,进而使其不能抑制其下游靶向基因的表达。而功能受到抑制的let-7,不能再抑制乳腺癌细胞中干细胞多潜能因子LIN28的表达,因此LIN28的表达水平会随之升高,进而促进BCSCs干性能力的增强。结论:在此课题中,我们的研究结果揭示了在乳腺癌细胞中lncRNA-H19、microRNA let-7和BCSCs标记物LIN28可形成一个调节通路,用于调控BCSCs的干性特征。因此,在乳腺癌的治疗中,靶向此通路为我们提供了一种新的乳腺癌的治疗策略。
[Abstract]:Long chain non RNA-H19 (long non-coding RNA-H19 encoding, H19 encoding by H19 gene) is not, the length of 2.3kb, encoding protein RNA, H19 transcription by RNA polymerase II, is a hallmark of cancer genes. Secondly H19 maternal allele expression, RNA. encoding is the first discovery of a recent study found that H19 can be expressed in a variety of in situ or metastatic tumors, and have great correlation with poor prognosis. It is proved that H19 in tumor development has played an important role. The research reports, many breast cancer patients after radiotherapy and chemotherapy in the treatment of conventional, often accompanied with the recurrence and metastasis of tumor cells the harm and cause more serious. In view of this, the scientists such as recurrence and metastasis of tumor mechanism was further studied, the cancer stem cells (cancer stem cells, CSCs) is caused by primary cancer recurrence and metastasis Because. Despite the continuous research for decades, has yet to find targeted therapy in the treatment of CSCs. In addition, some studies show that in breast cancer there is a small group of breast cancer stem cells (breast cancer stem cells, BCSCs), is the main cause of breast cancer cannot be cured. So far. H19 for the study of how to control the occurrence and development of tumor has many, also had many exciting results. But H19 in cancer stem cells in the regulation also failed to clarify this topic. Through in-depth research on H19, H19 on BCSCs show that the dry maintenance has an integral role. The research shows that, in breast cancer H19 can be used as a competitive endogenous RNA (competing endogenous RNA, ceRNA) function in microRNA let-7 (let-7), a tumor suppressor in breast cancer microRNA. so that H19 can promote tumor by adsorption let-7 The process and regulation of breast cancer cell dry ability. In addition, the experimental data show that H19 can positively regulate LIN28 expression, sequence binds to the let-7 and 3 'UTR region of LIN28. We conclude that in breast cancer H19 by regulating microRNAs and tumor pluripotent factor influence the progression of breast cancer the main research results and process. We are as follows: 1. in patients with breast cancer tissues we verified the expression level of H19 was significantly increased, the expression level of flow cytometry technique were selected BCSCs and validation of the H19 application. Then it H19 in patients with breast cancer and breast cancer stem cell like cells the increased expression of.2. after treatment of abnormal expression of H19 in breast cancer cells (high expression or knockdown), through cell microsphere formation experiment found in breast cancer cells with high expression of H19 can be obviously abnormal To promote and enhance tumor cell dry ability; on the contrary, when the drying characteristics of.3. knockdown of H19 in cells after inhibition of BCSCs since we found in breast cancer cells, H19 expression regulation of CSCs marker LIN28. And the increase of expression of LIN28 can be recovered by H19 and reduce the inhibition of BCSCs the drying ability. Accordingly, through the analysis of the patients with breast cancer data showed that the expression level of H19 and LIN28 in primary breast cancer.4. have great correlation in the mechanism, the H19 can regulate the expression of let-7 we found from previous studies, while LIN28 can be generated. We study the regulation of let-7 found in breast cancer cells, the expression level of let-7 was not affected by the expression of H19 and H19 changes, therefore only affect the bioavailability of let-7 without affecting the expression level of H19, which can be used as a function of adsorption of let-7 ceRNA, and then So that it can not inhibit the expression of target gene expression. And the function was inhibited by let-7, can inhibit the expression of pluripotent stem cell factor LIN28 in breast cancer cells, the expression level of LIN28 will increase, and then promote the enhancement of BCSCs dry ability. Conclusion: in this paper, our results reveal lncRNA-H19 in breast cancer cells, microRNA let-7 and BCSCs marker LIN28 can form a regulatory pathway for dry characteristic regulation of BCSCs. Therefore, in the treatment of breast cancer, targeting this pathway provides us a new therapeutic strategy for breast cancer.

【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.9

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