miR-139-5p在骨肉瘤发生发展中的作用及其分子机制研究

发布时间：2018-04-23 00:26

本文选题：miR-139-5p + Cdc42　；参考：《苏州大学》2016年博士论文

【摘要】：骨肉瘤是一种起源于间叶组织的骨骼系统恶性肿瘤,其发病率和死亡率在原发性骨肿瘤中均居首位。骨肉瘤多发于15-25周岁的青少年,其发展进程快,转移及复发率高,患者生存旅较低,行单一手术切除术治疗的患者5年生存率仅为15-20%,已成为青少年健康的一大威胁。随着医疗技术的迅猛发展,新辅助放化疗联合保肢术替代截肢术成为骨肉瘤治疗的主要外科手段,虽然患者预后水平有所提高,但放化疗并不具有靶向性,并不能有效清除肿瘤细胞,患者术后复发率较高,且保肢术后还需辅助骨组织重建术,价格昂贵,给患者带来一定的经济压力。目前,骨肉瘤新型治疗手段如免疫疗法、介入治疗、中医药治疗及基因靶向治疗成为攻克骨肉瘤的突破口。其中基因靶向治疗可靶向在骨肉瘤发展进程中起重要功能的癌相关基因,从而抑制肿瘤细胞增殖、迁移和侵袭等恶性生物学特性,而成为有效治愈骨肉瘤的新希望。因此,进一步揭示骨肉瘤发生发展中的分子机制全貌,探索新的分子治疗靶点,为基因靶向治疗提供理论依据和数据支持,具有重要的临床意义和科学价值。随着第二代高通量测序技术的成熟和广泛应用,针对肿瘤组织和细胞的测序数据如外显子测序、转录组测序、miRNAs测序、lnc RNA测序和单细胞测序数据等为基因靶向治疗的研究提供了可靠的数据支持,肿瘤学研究由此迎来了“数据爆炸”时代。在当今大数据时代,一系列整合了大量肿瘤样本的基因测序数据及临床数据的数据库如TCGA、Oncomine等也应运而生,被广泛的应用到肿瘤学的研究中来。TCGA数据库是当今最大的数据库,其涵盖全球范围内29种类型的肿瘤超过1万例样本的外显子测序数据、SNP数据、CMV数据及全面的临床数据,成为肿瘤学研究的一把利剑。目前围绕TCGA数据库展开的相关研究发表在《Science》、《Nature》、《PNS》等一系列重量级科学刊物。但利用TCGA大数据进行骨肉瘤的研究则未见报道。因此,在当今大数据时代,如何运用肿瘤大数据对骨肉瘤进行深入的研究,进一步完善骨肉瘤发生发展的分子机制,成为当今肿瘤学研究的一大趋势。microRNAs是一类长度约为21-23nt的在物种间具有高度保守性的非编码RNA,之前的研究认为miRNA是一种不具备功能的基因转录的“垃圾产物”,随着深入的研究,人们逐渐发现,miRNA可以6-8个碱基不完全互补配对的方式与靶基因3’UTR区域结合,发挥对基因表达负向调控的作用。因此,miRNA在调控细胞的各项生理功能如增殖、分化、凋亡衰老方面具有重要的功能。并于2002年被《Science》杂志评为十大科技突破第一名。目前在哺乳动物中已鉴定出的miRNA多达上千条序列,并且仍不断有新的miRNAs被发现。近年来,随着对miRNA在肿瘤学研究的不断深入,人们逐渐发现肿瘤中有许多miRNA对癌相关基因具有重要的调控作用,在肿瘤的发生发展中扮演着重要的角色,被认为是治疗肿瘤的潜在的分子靶点。因此,进一步探索和研究在骨肉瘤发生发展中起关键性调控作用的miRNAs,将对于进一步明确骨肉瘤发生发展中伴随的分子机制,以及探索新的分子治疗靶点具有重要的科学意义。第一部分基于TCGA肿瘤数据分析miR-139在骨肉瘤中的表达及其表观调控机制研究在本部分的研究中,我们利用TCGA肿瘤数据库中的骨肉瘤miRNA芯片数据,筛选在骨肉瘤中发生显著差异表达的miRNAs,发现发生显著下调表达的miRNAs有70条,发生显著上调表达的miRNAs有115条。在显著上调的miRNAs中,miR-139-5p是下调最为显著且未在骨肉瘤中有报道的miRNA。我们采用q RT-PCR检测了骨肉瘤组织及细胞系中miR-139-5p的表达,结果进一步说明miR-139-5p在骨肉瘤中发生了下调表达。为了探究miR-139-5p在骨肉瘤中发生下调表达的分子机制,我们采用CHIP实验检测了miR-139启动子区组蛋白的甲基化及乙酰化修饰情况发现,miR-139在骨肉瘤中的下调表达是由miR139启动子区组蛋白H3K27、H3K9甲基化水平上调,H3K4甲基化水平下调以及H3乙酰化水平下调所介导的。此外,我们结合TCGA数据库中骨肉瘤样本的临床数据,分析了miR-139-5p表达与患者生存期的关系发现,miR-139-5p高表达患者的生存期显著高于低表达患者。通过以上结果,我们初步推断miR-139-5p在骨肉瘤发生发展中具有重要的作用。第二部分miR-139-5p对骨肉瘤恶行生物学表型的调控作用及其分子机制研究为了研究miR-139-5p在骨肉瘤发生发展中的作用,我们首先采用q RT-PCR检测了miR-139-5p mimics的转染效率。结果显示,转染miR-139-5p mimics可显著提高骨肉瘤细胞MG63及U20S中miR-139-5p水平。接下来,我们采用CCK8实验检测了miR-139-5p对骨肉瘤增殖的影响发现,miR-139-5p可显著抑制骨肉瘤的增殖。采用Transwell检测miR-139-5p对骨肉瘤细胞迁移和侵袭的影响发现,miR-139-5p可显著抑制骨肉瘤细胞的迁移和侵袭。此外,构建裸鼠皮下抑制瘤模型,体内过表达miR-139-5p后发现,miR-139-5p可显著抑制瘤体生长。因此,我们得出结论:miR-139-5p可显著抑制骨肉瘤的发生发展。为了探究miR-139-5p发挥抑瘤作用的分子机制,我们首先采用Targetscan预测了miR-139-5p的靶基因发现,miR-139-5p可靶向Cdc42 m RNA 3’UTR序列。为了进一步探究miR-139-5p对Cdc42 m RNA 3’UTR序列的靶向作用,我们构建了Cdc42m RNA 3’UTR区45-52位点的Luciferase双荧光报告载体,转染miR-139-5p后发现miR-139-5p可显著抑制荧光生成。以上结果说明miR-139-5p可靶向Cdc42 m RNA3’UTR 45-52位点。为了进一步探究miR-139-5p对Cdc42是否有负向调控作用及其作用机制,我们检测了转染miR-139-5p后,骨肉瘤细胞Cdc42 m RNA及蛋白的表达发现,miR-139-5p并不抑制Cdc42 m RNA水平,可显著抑制Cdc42蛋白的表达。通过以上结果,我们得出结论:miR-139-5p可通过抑制Cdc42 m RNA的翻译从而对Cdc42基因表达起负向调控作用。第三部分Cdc42对骨肉瘤恶行生物学表型的调控作用研究在本部分中,我们探究了Cdc42基因在骨肉瘤中的表达及其对骨肉瘤恶性生物学表型的调控作用。首先分析了TCGA数据库中骨肉瘤RNA-Seq数据发现,Cdc42m RNA并未在骨肉瘤中发生差异表达,采用q RT-PCR及Westernblot分别检测了骨肉瘤组织和细胞系中Cdc42 m RNA和蛋白表达发现,Cdc42 m RNA并未在骨肉瘤中发生差异表达,但Cdc42蛋白在骨肉瘤中发生了显著的上调表达。以上结果说明,Cdc42在骨肉瘤中发生显著的上调表达,其上调表达主要发生在转录后的调控,miR-139-5p作为Cdc42转录后的调控因子,在调控Cdc42表达上具有重要的作用。接下来我们探究了Cdc42对骨肉瘤细胞增殖和迁移的影响。CCK8结果显示,沉默Cdc42表达可显著抑制骨肉瘤细胞的增殖;划痕实验结果显示,Cdc42沉默后,骨肉瘤细胞的迁移能力降低。通过以上结果,我们得出Cdc42可促进骨肉瘤细胞的增殖和分化。综上所述,miR-139-5p在骨肉瘤发生发展中发挥着重要的作用,具体表现为通过负向调控Cdc42基因的表达,从而抑制骨肉瘤的发生发展,可以作为治疗骨肉瘤潜在的分子靶点。
[Abstract]:Osteosarcoma is a malignant tumor of the skeletal system derived from interleaf tissue. The incidence and mortality of osteosarcoma are the first in primary bone tumors. Osteosarcoma often occurs in adolescents aged 15-25 years, with rapid development, high metastasis and recurrence rate and low survival. The 5 year survival rate of patients treated with single hand surgery is only 15-20%, With the rapid development of medical technology, with the rapid development of medical technology, the combination of neoadjuvant radiotherapy and chemotherapy combined with limb salvage has become the main surgical method for osteosarcoma. Although the prognosis of the patients is improved, the radiotherapy and chemotherapy is not targeted, and the tumor cells can not be effectively removed, and the recurrence rate of the patients is higher. It is also necessary to assist the reconstruction of bone tissue after limb salvage, which is expensive and brings some economic pressure to the patients. At present, the new treatment of osteosarcoma, such as immunotherapy, interventional therapy, Chinese medicine treatment and gene targeting therapy, has become the breakthrough of osteosarcoma. Gene targeting therapy can be used to play an important role in the process of osteosarcoma development. The function of cancer related genes, thus inhibiting tumor cell proliferation, migration and invasion and other malignant biological characteristics, and become a new hope to effectively cure osteosarcoma. Therefore, further reveal the molecular mechanism of osteosarcoma development, explore new molecular therapeutic targets, provide theoretical basis and data support for gene targeting therapy. Important clinical significance and scientific value. With the mature and extensive application of the second generation high-throughput sequencing technology, the research on tumor tissue and cell sequencing data, such as exon sequencing, transcriptional sequencing, miRNAs sequencing, LNC RNA sequencing and single cell sequencing data, provides reliable data support for gene targeting therapy, oncology In today's big data age, a series of data databases such as TCGA and Oncomine, which integrate a large number of tumor samples, such as TCGA, and clinical data, are also widely used in oncology research to be the largest database today, covering the global scope. Exon sequencing data, SNP data, CMV data, and comprehensive clinical data of 29 types of internal tumor types have become a sharp sword in oncology research. A series of scientific publications on , , , etc. are published around the TCGA database. However, osteosarcoma with large TCGA data is used for osteosarcoma. Therefore, in today's big data age, how to use tumor large data to study osteosarcoma, further improve the molecular mechanism of osteosarcoma development, and become a major trend in oncology research today.MicroRNAs is a class of highly conserved non coded R with a length of about 21-23nt. NA, previous studies suggest that miRNA is a "junk product" that does not have functional gene transcription. With further research, people have gradually found that miRNA can combine the 6-8 base pairs with the target gene 3 'UTR region to play the role in the negative regulation of gene expression. Therefore, miRNA regulates every cell in the cell. Physical function, such as proliferation, differentiation and apoptosis, has important functions. In 2002, it was first named ten major scientific and technological breakthroughs in magazine. At present, up to thousands of miRNA sequences have been identified in mammals, and new miRNAs has been found. In recent years, with the deepening of oncology research on miRNA, people We have gradually found that many miRNA plays an important role in the regulation of cancer related genes and plays an important role in the development of cancer. It is considered as a potential molecular target for cancer treatment. Therefore, further exploration and study of the regulatory role of the key role of miRNAs in the development of osteosarcoma will be further clear. The molecular mechanism associated with the development of the osteosarcoma and the exploration of new molecular therapeutic targets are of great scientific significance. Part 1 based on the analysis of TCGA tumor data, the expression of miR-139 in osteosarcoma and its apparent regulation mechanism are studied in this part, and we use the number of miRNA chips in the osteosarcoma in the TCGA tumor database. According to the screening of significant differential expression of miRNAs in osteosarcoma, there were 70 significant down-regulation miRNAs and 115 significant up-regulated miRNAs. In a significantly up-regulated miRNAs, miR-139-5p was the most significant down-regulation and was not reported in osteosarcoma. We used Q RT-PCR to detect osteosarcoma tissues and fines. The expression of miR-139-5p in the cell lines further indicated that miR-139-5p was downregulated in osteosarcoma. In order to explore the molecular mechanism of the down-regulation of miR-139-5p in osteosarcoma, we detected the methylation and acetylation of the histone in the miR-139 promoter region by CHIP test, and miR-139 was found in the osteosarcoma. The expression is mediated by the miR139 promoter histone H3K27, the up regulation of H3K9 methylation level, the downregulation of H3K4 methylation level and the downregulation of H3 acetylation. In addition, we combine the clinical data of the osteosarcoma samples in the TCGA database to analyze the relationship between the expression of miR-139-5p and the survival period of the patients, and the birth of the patients with high expression of miR-139-5p. The survival time is significantly higher than that of the low expression patients. Through the above results, we preliminarily deduce that miR-139-5p plays an important role in the development of osteosarcoma. Second the role of miR-139-5p in the regulation of the biological phenotype of osteosarcoma and its molecular mechanism are studied to study the role of miR-139-5p in the development of osteosarcoma. Q RT-PCR was used to detect the transfection efficiency of miR-139-5p mimics. The results showed that the transfection of miR-139-5p mimics could significantly increase the miR-139-5p level in osteosarcoma cells MG63 and U20S. Next, we detected miR-139-5p on the proliferation of osteosarcoma by CCK8 experiment, and miR-139-5p could significantly inhibit the proliferation of osteosarcoma. The effect of miR-139-5p on the migration and invasion of osteosarcoma cells found that miR-139-5p could significantly inhibit the migration and invasion of osteosarcoma cells. In addition, the subcutaneous tumor model of nude mice was constructed, and after the overexpression of miR-139-5p in the body, miR-139-5p could significantly inhibit the growth of the tumor. Therefore, we conclude that miR-139-5p can significantly inhibit osteosarcoma. Development. In order to explore the molecular mechanism of miR-139-5p playing a tumor suppressor, we first used Targetscan to predict the target gene discovery of miR-139-5p, and miR-139-5p can target the Cdc42 m RNA 3 'UTR sequence. In order to further explore the target of miR-139-5p to Cdc42 m RNA 3' sequence, we construct 45-52 bits. Luciferase double fluorescent report carrier, miR-139-5p can significantly inhibit fluorescence generation after transfection of miR-139-5p. The above results show that miR-139-5p can target Cdc42 m RNA3 'UTR 45-52 site. In order to further explore the negative regulation of miR-139-5p on Cdc42 and its mechanism of action, we detected osteosarcoma after miR-139-5p transfection. The expression of Cdc42 m RNA and protein found that miR-139-5p did not inhibit the Cdc42 m RNA level and could significantly inhibit the expression of Cdc42 protein. Through the above results, we concluded that miR-139-5p can be used to regulate the expression of the gene by inhibiting the translation of Cdc42 m RNA. The third part of the biological phenotype of osteosarcoma is the biological phenotype of osteosarcoma. In this part, we explored the expression of Cdc42 gene in osteosarcoma and its regulatory role in the malignant biological phenotype of osteosarcoma. First, we analyzed the RNA-Seq data of osteosarcoma in the TCGA database, and Cdc42m RNA was not expressed differently in osteosarcoma, and the bone was detected by Q RT-PCR and Westernblot, respectively. The expression of Cdc42 m RNA and protein in sarcomas and cell lines found that Cdc42 m RNA did not express differential expression in osteosarcoma, but the expression of Cdc42 protein in osteosarcoma was significantly up-regulated. The above results indicated that Cdc42 was significantly up-regulated in osteosarcoma, and its up regulation was mainly regulated after transcriptional regulation, miR-139-5p As a regulatory factor after Cdc42 transcriptional regulation, it plays an important role in regulating the expression of Cdc42. Then we explored the effect of Cdc42 on the proliferation and migration of osteosarcoma cells..CCK8 results showed that silent Cdc42 expression significantly inhibited the proliferation of osteosarcoma cells; the scratch test showed that the migration ability of osteosarcoma cells after Cdc42 was silent. Through the above results, we conclude that Cdc42 can promote the proliferation and differentiation of osteosarcoma cells. To sum up, miR-139-5p plays an important role in the development of osteosarcoma, which is manifested by negative regulation of the expression of Cdc42 gene, thus inhibiting the occurrence and development of osteosarcoma, and can be used as a potential molecular target for the treatment of osteosarcoma. Point.

【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R738.1

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