miR-183靶向调控MTA1基因对骨肉瘤细胞增殖及迁移的影响

发布时间：2018-06-09 02:10

本文选题：MTA1 + miR-183　；参考：《郑州大学》2017年硕士论文

【摘要】：研究背景骨肉瘤(osteosarcoma)是一种侵袭性的恶性肿瘤,从间质细胞系发展而来,具有成骨细胞分化的特点,且极易发生肺转移,同时它也是儿童癌症中生存率较低的肿瘤之一,患者的五年生存率仅为65%-70%,严重威胁着青少年的健康。近年来在基因分子的水平对疾病进行研究越来越受关注,因此从基因分子水平上研究骨肉瘤的发病机制及防治,对其未来的诊断和治疗有更积极的作用。肿瘤转移相关基因(metastasis associated gene 1,MTA1)是核小体重塑和组蛋白去乙酰化酶复合物(Nucleosome remodeling and histone deacetylase,NuRD)的重要组成部分。大量的研究发现MTA1在多种肿瘤中高表达,并促进肿瘤的恶化,参与肿瘤的增殖、侵袭、迁移、凋亡和细胞周期等过程。有研究报道miR-183在骨肉瘤细胞系MG63、U2-OS、Saos-2和HOS中均异常低表达,并能靶向埃兹基因(Ezrin,其蛋白又称细胞骨架结合蛋白)抑制MG63细胞的侵袭和迁移,多种生物信息学软件预测显示miR-183可能与MTA1存在靶向结合区。因此,探讨miR-183靶向调控MTA1基因,对骨肉瘤增殖、侵袭、迁移及凋亡的影响,将为骨肉瘤发病机制研究及临床防治提供新的思路和方向。miR-183靶向调控MTA1基因对骨肉瘤细胞的影响及其分子作用机制的研究尚未见报道。研究目的本研究将通过实验验证MTA1是miR-183的直接靶基因,转染miR-183mimic至骨肉瘤细胞系MG63,观察miR-183对MTA1的靶向调控作用,及对MG63细胞增殖、侵袭、迁移与凋亡的影响,由此寻找骨肉瘤研究及防治新的靶点和策略。研究方法1.收集标本:自2014年9月至2016年6月从郑州大学第一附属医院收集了25例骨肉瘤患者的癌组织及与其配对的癌旁组织,并置于-80℃冰箱中长期保存。采用实时荧光定量PCR技术检测已收集标本中miR-183及MTA1基因的mRNA表达水平,并通过免疫印迹Western blot检测MTA1蛋白的表达并分析结果。2.采用TargetScan和miRanda等生物信息学软件预测靶向MTA1的microRNA。3.构建含有MTA1 3’端非翻译区域(3’UTR)的野生型和突变型载体,运用双荧光素酶报告基因实验验证MTA1与miR-183的靶向作用。4.RT-qPCR法检测正常成骨细胞株hFOB1.19与人骨肉瘤细胞系MG63中miR-183与MTA1基因的mRNA表达水平。5.体外合成miR-183 mimic和miR-183 negative control,采用脂质体将其分别转染到各组人骨肉瘤细胞系MG63中。实验分为3组,miR-183 mimic组:转染miR-183 mimic;NC组:转染miR-183 negative control;Control组:仅加脂质体。6.CCK-8实验检测骨肉瘤细胞系MG63的增殖情况。7.细胞划痕实验检测骨肉瘤细胞系MG63迁移能力改变情况。8.Transwell实验检测骨肉瘤细胞系MG63侵袭能力改变的情况。9.流式细胞术及TUNEL实验检测骨肉瘤细胞系MG63凋亡的情况。10.免疫印迹Western blot法检测转染后MTA1蛋白的表达。实验结果1.RT-qPCR检测结果显示,骨肉瘤组织中miR-183的表达量显著低于其在癌旁组织中的表达(P0.01),而MTA1基因的mRNA在骨肉瘤中的表达量明显高于其在癌旁组织中的表达(P0.01)。Western blot的结果显示,与癌旁组织比较,MTA1蛋白在癌组织中呈现高表达。2.双荧光素酶报告基因实验证明miR-183与MTA1基因的3'UTR区域结合,与生物信息学软件预测结果一致,表明MTA1是miR-183的靶基因。3.RT-qPCR结果显示,在骨肉瘤细胞系MG63中miR-183的表达显著低于成骨细胞株hFOB1.19中的表达(P0.01),而MTA1基因的mRNA表达则明显高于其在成骨细胞株hFOB1.19中的表达(P0.01)。4.转染后,miR-183 mimic组的miR-183表达量明显高于NC组与Control组,差异具有统计学意义(P0.01)。表明miR-183 mimic转染骨肉瘤细胞成功。5.CCK-8结果显示,与NC组和Control组比较,转染miR-183 mimic组的骨肉瘤细胞在相同时间点的OD490吸光值有所下降,差异具有统计学意义(P0.05),而NC组与Control组比较则无显著差异(P0.05)。6.划痕实验结果显示,miR-183组细胞的迁移能力较NC组与Control组低,而NC组与Control组比较则迁移能力无明显差异。7.Transwell实验结果表明,miR-183组的骨肉瘤MG63细胞穿过基底膜的细胞数明显低于NC组与Control组,差异具有统计学意义(P0.01),但NC组与Control组穿过基底膜的细胞数量无明显差异(P0.05)。8.流式细胞术与TUNEL实验结果显示,与NC组和Control组比较,miR-183组的骨肉瘤MG63细胞的凋亡率明显升高,差异具有统计学意义(P0.05),而NC组与Control组比较,MG63细胞的凋亡率无显著差异(P0.05)。9.免疫印迹Western blot检测结果显示,转染miR-183 mimic后,与NC组和Control组比较,MTA1蛋白表达下降,而NC组与Control组比较,MTA1表达无显著差异。结论1.骨肉瘤组织与细胞系MG63中miR-183低表达,MTA1高表达。2.miR-183可以通过靶向作用MTA1基因的3’UTR区域降低其转录表达,并发挥调控作用。3.过表达miR-183可能通过降低靶基因MTA1表达而有效抑制骨肉瘤细胞系MG63的增殖、侵袭、迁移并促进其细胞凋亡。
[Abstract]:Background osteosarcoma (osteosarcoma) is an invasive malignant tumor, developed from interstitial cell lines, characterized by osteoblast differentiation, and highly susceptible to lung metastasis. At the same time, it is also one of the low survival rates in children's cancer. The five year survival rate of patients is only 65%-70%, which threatens the health of young people in recent years. It is becoming more and more concerned about the study of disease at the level of gene molecules, so it is more active to study the pathogenesis and prevention of osteosarcoma from the gene molecular level. The tumor metastasis related gene (metastasis associated gene 1, MTA1) is the recombination of nucleosome remodeling and histone deacetylase. An important part of Nucleosome remodeling and histone deacetylase (NuRD). A large number of studies have found that MTA1 is highly expressed in a variety of tumors and promotes the deterioration of the tumor and participates in the process of tumor proliferation, invasion, migration, apoptosis and cell cycle. It is reported that miR-183 is different in the osteosarcoma cell lines MG63, U2-OS, Saos-2, and HOS. It is often low expressed and can target Ezi gene (Ezrin, its protein also called cytoskeleton binding protein) to inhibit the invasion and migration of MG63 cells. A variety of bioinformatics software predicts that miR-183 may have a target binding zone with MTA1. Therefore, the effect of miR-183 targeting MTA1 gene on proliferation, invasion, migration and apoptosis of osteosarcoma will be discussed. The study of the pathogenesis and clinical prevention of osteosarcoma provides new ideas and directions for the study of the effect of.MiR-183 targeting MTA1 gene on osteosarcoma cells and its molecular mechanism. The purpose of this study is to verify that MTA1 is a direct target gene of miR-183 and transfected from miR-183mimic to osteosarcoma cell line MG63. The effects of miR-183 on the targeting of MTA1, and the effects on proliferation, invasion, migration and apoptosis of MG63 cells were observed, and the new targets and strategies for the study and prevention of osteosarcoma were sought. Methods 1. collection of specimens from the First Affiliated Hospital of Zhengzhou University from September 2014 to June 2016 were collected from the cancer tissues of 25 patients with osteosarcoma and their pairing. The para cancerous tissue was preserved in the -80 C refrigerator for a long time. The mRNA expression level of the miR-183 and MTA1 genes in the collected specimens was detected by real-time fluorescence quantitative PCR, and the expression of MTA1 protein was detected by immunoblotting Western blot and the results of.2. were predicted by the bioinformatics software such as TargetScan and miRanda. ORNA.3. constructs a wild type and mutant vector containing MTA1 3 'untranslated region (3' UTR), and uses double luciferase reporter gene test to verify the target action of MTA1 and miR-183 to detect the mRNA expression level.5. in normal osteoblast cell line hFOB1.19 and human osteosarcoma cell line MG63, miR-183 and MTA1 genes in vitro synthesis 3 mimic and miR-183 negative control were transfected into the human osteosarcoma cell line MG63 respectively with liposomes. The experiment was divided into 3 groups, and the miR-183 mimic group was transfected with miR-183 mimic; NC group: transfected to miR-183 negative. Changes in the migration ability of osteosarcoma cell line MG63.8.Transwell test detection of invasion ability of osteosarcoma cell line MG63.9. flow cytometry and TUNEL test detection of apoptosis in osteosarcoma cell line MG63.10. immunoblotting Western blot method to detect the expression of MTA1 protein after transfection. Experimental results 1.RT-qPCR detection junction The results showed that the expression of miR-183 in osteosarcoma was significantly lower than that in paracancerous tissue (P0.01), while the expression of mRNA in osteosarcoma was significantly higher than that of its expression in the paracancerous tissues (P0.01).Western blot, which showed that MTA1 protein expressed a high expression of.2. double fluorescein in the tissue of the cancer tissue compared with that of the para cancerous tissue. The enzyme reporter gene experiment showed that miR-183 was associated with the 3'UTR region of MTA1 gene, which was consistent with the prediction results of bioinformatics software, indicating that MTA1 was the target gene of miR-183 and that the expression of miR-183 in the osteosarcoma cell line MG63 was significantly lower than the expression of the osteoblast strain hFOB1.19 (P0.01), while mRNA expression of the MTA1 gene was clear. The expression of miR-183 in the miR-183 mimic group was significantly higher than that in the osteoblast cell line hFOB1.19 (P0.01).4.. The difference was statistically significant (P0.01). The results showed that the miR-183 mimic transfected osteosarcoma cells were successfully transfected with the osteosarcoma cells. The OD490 absorption value of the tumor cells decreased at the same time point, the difference was statistically significant (P0.05), but there was no significant difference between the NC group and the Control group (P0.05).6. scratch test results showed that the migration ability of the miR-183 group was lower than that of the NC group and the Control group, while the NC and Control group had no significant difference in the migration capacity. The experimental results showed that the number of osteosarcoma MG63 cells passing through the basement membrane in the miR-183 group was significantly lower than that in the NC group and the Control group. The difference was statistically significant (P0.01), but there was no significant difference in the number of cells passing through the basement membrane in the NC group and Control group (P0.05).8. flow cytometry and TUNEL experimental results, compared with NC and Control groups. The apoptosis rate of osteosarcoma MG63 cells in the group was significantly higher, the difference was statistically significant (P0.05), but there was no significant difference in the apoptosis rate of MG63 cells in the group NC and the Control group (P0.05).9. immunoblotting Western blot detection results showed that after the miR-183 mimic, the expression of the protein decreased with the NC group and the group. Conclusion there is no significant difference in MTA1 expression. Conclusion 1. osteosarcoma tissue and cell line MG63 are low expression of miR-183, MTA1 high expression.2.miR-183 can reduce its transcriptional expression through the 3 'UTR region targeting the target action of MTA1 gene, and play the regulatory role of.3. overexpression miR-183 may effectively inhibit the osteosarcoma cell line MG by reducing the target gene MTA1 expression. 63 of the proliferation, invasion, migration, and promotion of cell apoptosis.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R738.1

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