miR-143在骨肉瘤细胞中的作用及机制研究

发布时间：2018-03-18 03:18

本文选题：骨肉瘤　切入点：miR-143　出处：《山东大学》2017年博士论文　论文类型：学位论文

【摘要】：研究背景骨肉瘤是最常见的起源于骨骼系统的原发性恶性骨肿瘤,特点是高发生率,高度恶性,高转移率,因此预后较差,肿瘤的发生给患者家庭和社会带来了很大的花费和负担。近20年以来,尽管国际诸多研究机构进行了大量的努力,但骨肉瘤患者生存率依然停滞不前,仿佛进入了骨肉瘤治疗的瓶颈期。同时化疗耐药的出现也让化疗方案的推广举步维艰,激进的化疗方案的副作用让很多骨肉瘤患者难以坚持完成治疗。为此,对骨肉瘤的深入研究迫在眉睫。近年来有关微小RNA(microRNA,miRNA)的研究成为肿瘤领域研究的热点。在人类多种癌细胞中都发现了miRNA的表达异常,miRNA既可以促进肿瘤生长而起到癌基因的作用,也可抑制肿瘤生长而起到抑癌基因的作用。最近的研究提示miR-143参与了肿瘤细胞的发生、增殖、分化、迁移侵袭、凋亡等生理过程,但总体而言miR-143的作用主要起到类似抑癌基因的功能。然而,针对miR-143的研究大多集中于上皮组织来源的癌细胞,对于间叶源性的恶性肿瘤细胞尤其是骨肉瘤中miR-143的作用报道较少。我们在本研究通过检测骨肉瘤组织和邻近正常组织中miR-143的表达,并通过生物信息学技术预测靶基因后检测靶基因的表达情况。通过细胞转染上调miR-143及特异性抑制序列来敲除miR-143的作用,观察体外培养的骨肉瘤细胞系中靶基因的表达情况,并检测miR-143对骨肉瘤细胞凋亡的影响,以期阐明miR-143在骨肉瘤中的作用,为靶向治疗骨肉瘤提供可能的思路。研究目的1.观察miR-143在骨肉瘤组织中的表达水平。2.观察miR-143对骨肉瘤细胞的影响。3.寻找miR-143的靶基因,阐明miR-143对靶基因的调节作用。研究方法1.收集骨肉瘤病人的标本,应用RT-PCR技术检测骨肉瘤标本及周围正常组织中miR-143的表达水平,比较miR-143在骨肉瘤和正常组织中的表达差异。2.通过生物信息学方法及软件预测miR-143的靶基因为Bcl-2,RT-PCR技术检测骨肉瘤标本及周围正常组织中Bcl-2的mRNA表达水平,应用免疫组织化学技术和Western Blot方法检测骨肉瘤标本及周围正常组织中Bcl-2的蛋白表达水平。3.利用脂质体2000将miR-143模拟物及阴性对照序列瞬时转染MG63骨肉瘤细胞系,建立过表达miR-143的骨肉瘤细胞系。4.利用CCK-8检测不同时间段过表达miR-143的骨肉瘤细胞系及阴性对照组细胞的细胞活性,观察miR-143对骨肉瘤细胞增殖能力的影响。5.利用Transwell方法检测不同时间段过表达miR-143的骨肉瘤细胞系及阴性对照组细胞的细胞穿膜数量,观察miR-143对骨肉瘤细胞迁移能力的影响。6.预测miR-143的靶基因,并构建相应靶基因的野生型和突变型荧光素酶报告基因质粒,与miR-143模拟物及阴性对照共转染MG63骨肉瘤细胞,检测荧光素酶活性,判断miR-143的靶基因。7.转染miR-143模拟物及抑制物后通过RT-PCR和Western Blot技术检测靶基因的mRNA水平和蛋白表达水平的变化,分析miR-143对靶基因的调节作用。8.将miR-143模拟物及阴性对照序列转染MG63骨肉瘤细胞系后,应用Annexin V/PI双染色后通过流式细胞术检测细胞凋亡比率的变化,来观察miR-143对骨肉瘤细胞凋亡的影响。实验结果1.12例骨肉瘤标本中的miR-143表达水平同邻近正常组织中相比明显下降,只有邻近正常组织的56%。2.通过TargetScan软件预测miR-143的靶基因可能是Bcl-2。免疫组织化学方法发现骨肉瘤标本中的Bcl-2表达水平较正常组织相比明显升高。RT-PCR检测发现Bcl-2的mRNA表达水平较正常组织明显升高,Western Blot结果显示骨肉瘤组织中Bcl-2的蛋白表达水平明显增加,约为邻近正常组织的1.9倍。3.通过细胞转染的方法成功建立miR-143高表达的骨肉瘤细胞系。4.转染miR-143 48h及72h时,骨肉瘤细胞活性明显下降,分别为对照组的78.6%和65.4%。miR-143能够抑制骨肉瘤细胞的增殖。5.转染miR-143 72h时,骨肉瘤细胞的穿出Transwell小室的细胞数量明显减少。miR-143能够抑制骨肉瘤细胞的迁移能力。6.成功构建候选靶基因Bcl-2的荧光素酶报告基因质粒,共转染miR-143和Bcl-2报告基因后荧光素酶失活,证实Bcl-2是miR-143的靶基因。7.过表达miR-143的骨肉瘤细胞中,Bcl-2的mRNA明显下调,蛋白表达水平明显降低,而应用miR-143抑制物可上调Bcl-2的蛋白表达。miR-143通过抑制Bcl-2表达发挥作用。8.过表达miR-143明显增加骨肉瘤细胞凋亡比率,其机制可能与miR-143介导的Bcl-2下调有关。结论1.骨肉瘤组织中miR-143的表达降低,而Bcl-2的表达水平增高。2.过表达miR-143能够抑制骨肉瘤细胞的增殖与迁移。3.Bcl-2可能是miR-143的靶基因之一,miR-143可介导Bcl-2的表达水平。4.miR-143可诱导骨肉瘤细胞凋亡,其机制可能与miR-143介导的Bcl-2下调有关。
[Abstract]:Background osteosarcoma is the most common origin in the skeletal system of primary malignant bone tumor, characterized by high incidence, high malignancy, high transfer rate, so the prognosis is poor, cancer has brought great cost burden for patients and families and the society. Over the past 20 years, as many international research institutions a great deal of effort, but patients with osteosarcomasurvival remains stagnant, as if into the bottleneck period of treatment of osteosarcoma. At the same time the chemotherapy resistant also make chemotherapy promotion difficult, side effects of chemotherapy of radical so many patients with osteosarcoma is difficult to adhere to the completion of treatment. Therefore, in-depth study of osteosarcoma imminent. In recent years on the micro RNA (microRNA, miRNA) research has become a hot research field of tumor. In many human cancer cells are found in the abnormal expression of miRNA, miRNA can promote tumor The growth and play the role of oncogenes can also inhibit tumor growth and play the role of tumor suppressor gene. Recent studies suggest that miR-143 is involved in tumor cell proliferation, differentiation, migration, invasion, apoptosis and other physiological processes, but the overall effect of miR-143 mainly plays like the function of tumor suppressor gene. However, Research on miR-143 mostly concentrated in epithelial cancer cells for malignant mesenchymal tumor cells especially miR-143 in osteosarcoma effect reported less. In this study we through the detection of the expression of miR-143 in osteosarcoma tissues and adjacent normal tissues, and through bioinformatics prediction of target gene to detect the expression of target gene. The cells transfected with the up regulation of miR-143 and sequence specific inhibition to knock out the role of miR-143, the target gene of human osteosarcoma cell line in vitro expression of the situation, and To study the effect of miR-143 on apoptosis of osteosarcoma cells, to elucidate the role of miR-143 in osteosarcoma, as the target may provide ideas to the treatment of osteosarcoma. The purpose of the study is to observe 1. miR-143 in osteosarcoma and the expression level of.2. effect of miR-143 on osteosarcoma cell.3. targeting gene for miR-143, regulation clarify miR-143 on target gene. The method of collecting 1. patients with osteosarcoma specimens, the expression level of miR-143 RT-PCR was used to detect osteosarcoma specimens and adjacent normal tissues, miR-143 in osteosarcoma and normal tissues in the expression of the target base difference of.2. by bioinformatics methods and software for predicting miR-143 because of Bcl-2, Bcl-2 RT-PCR detection of osteosarcoma specimens and surrounding normal tissues, the expression level of mRNA, immunohistochemistry and Western Blot method for detection of osteosarcoma specimens and normal group The expression of Bcl-2 in protein level of.3. fabric by Lipofectamine 2000 analogue of miR-143 and negative control sequence transient transfection of human osteosarcoma MG63 cells, the establishment of the over expression of miR-143 osteosarcoma cell line CCK-8 was detected by.4. in different time after expression of cell activity in osteosarcoma cell line miR-143 and negative control group cells, observe the effect of miR-143 on osteosarcoma cell proliferation of.5. was detected by Transwell in different time after expression of osteosarcoma cell line miR-143 and negative control group cells penetrating quantity, the effect of miR-143 on osteosarcoma cell migration effect of.6. can force prediction of miR-143 target gene, and construct the corresponding wild-type and mutant luciferase gene reporter plasmid were co transfected into MG63 osteosarcoma cells with miR-143 mimics and negative, detection of luciferase activity,.7. gene was transfected into miR miR-143 The level of mRNA and expression of target gene RT-PCR and Western Blot technology, the expression of -143 mimics and inhibitors, miR-143 analysis on target gene regulation of.8. analogue of miR-143 and negative control sequence were transfected into MG63 osteosarcoma cell lines, to detect the changes of cell apoptosis rate by flow cytometry by Annexin V/ PI staining to observe the effect of miR-143 on apoptosis of osteosarcoma cells. Results expression in 1.12 cases of osteosarcoma specimens miR-143 levels in adjacent normal tissues compared with adjacent normal tissue decreased significantly, only 56%.2. by TargetScan software to predict the target gene miR-143 may be Bcl-2. immunohistochemistry found in osteosarcoma specimens the expression level of Bcl-2 was significantly higher than normal tissue.RT-PCR detected Bcl-2 mRNA expression level increased significantly compared with the normal tissue, Western Bl Ot results showed that the expression level of Bcl-2 protein in osteosarcoma was significantly increased, about 1.9 times the.3. of the adjacent normal tissue by transfected cells successfully established the high expression of miR-143 in osteosarcoma cell line.4. transfected with miR-143 48h and 72h, osteosarcoma cell activity decreased significantly, the control group respectively 78.6% and 65.4%.miR-143 can inhibit the growth of osteosarcoma cells.5. transfected with miR-143 luciferase reporter gene plasmid 72h, the number of cells through the Transwell cell of osteosarcoma cells significantly reduced.MiR-143 can inhibit osteosarcoma cell migration.6. successfully constructed candidate target genes of Bcl-2, miR-143 and Bcl-2 were co transfected with reporter gene luciferase inactivation, confirmed that Bcl-2 is the target gene of.7. miR-143 the expression of miR-143 in osteosarcoma cells, Bcl-2 mRNA down-regulation of protein expression level decreased significantly, and the inhibition effect of miR-143. Can up regulate the protein expression of Bcl-2.MiR-143 through inhibiting the expression of Bcl-2 play a role in.8. over expression of miR-143 significantly increased the apoptosis rate of osteosarcoma cells, and its mechanism may be mediated by down-regulation of miR-143 Bcl-2. Conclusion the expression of miR-143 decreased 1. in osteosarcoma, and the expression level of Bcl-2 increased in.2. overexpression of miR-143 could inhibit the proliferation and migration of.3.Bcl-2 osteosarcoma cells may be one of the target genes of miR-143, miR-143 expression level of.4.miR-143 mediated Bcl-2 can induce apoptosis of osteosarcoma cells, and its mechanism may be mediated by down-regulation of miR-143 Bcl-2.

【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】：R738.1

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