miR-495靶向SATB1基因对肾细胞癌调控作用的研究

发布时间：2018-02-25 19:19

本文关键词： 肾细胞癌 microRNA miR-495 SATB1　出处：《武汉大学》2015年博士论文　论文类型：学位论文

【摘要】：背景：肾细胞癌(RCC)是成人肾脏最常见的肿瘤类型,约占所有原发性肾肿瘤的85%,在成人所有肿瘤中的发病率约占3%。对于局限性早期肾癌可采取手术治疗,然而,仍有许多患者会面临肿瘤的复发和转移风险,因此预后多较差,伴有转移的RCC患者,其5年生存率仅9%。为了治疗这些晚期RCC患者,迫切需要发现新的、可作为治疗靶点的生物标志物。miRNAs是一类长约19-24个核苷酸,高度保守的调节分子,通过与靶基因mRNA的3‘非编码区(3'UTR)的碱基序列互补配对后降解mRNA或抑制其翻译,从而调节靶基因的表达水平。多项研究报道miRNAs在发育调节；细胞增殖、分化、侵袭和凋亡中发挥关键作用。其中miR-495在多种肿瘤中均见有异常表达,如非小细胞肺癌、乳腺癌、恶性胶质瘤、胃癌和白血病等,可能作为抑癌基因或原癌基因在肿瘤的发生发展中扮演重要作用。但目前miR-495在肾细胞癌中的表达及其功能尚属未知。目的：研究miR-495在人RCC中的表达情况,探讨miR-495的表达水平对体外RCC细胞增殖、迁徙等活性的影响。通过预测miR-495的靶基因,验证体外RCC细胞中miR-495异常表达对靶基因的调控作用,进一步探讨miR-495调控RCC的可能分子机制。方法：选择四种人RCC细胞系和一种正常人肾细胞系,qRT-PCR检测各细胞系中miR-495的表达水平,另收集临床肾癌和癌旁正常组织标本,qRT-PCR检测各组织标本中miR-495的表达差异。通过瞬时转染技术建立稳定过表达miR-495的人肾癌786-0细胞系,利用qRT-PCR验证稳定转染细胞系中miR-495的表达变化。进一步通过CCK8实验检测过表达miR-495后786-0细胞增殖活性的变化；流式细胞仪检测过表达miR-495对786-o细胞周期的影响；划痕实验验证过表达miR-495后786-0细胞迁徙能力的改变。通过检索TargetScan等生物信息学数据库预测了miR-495的靶基因SATB1,利用荧光素酶报告基因分析验证miR-495是否与靶基因SATB1mRNA的3'UTR互补结合,并通过qRT-PCR和WB方法检测人肾癌786-0细胞中miR-495对靶基因SATB1表达的抑制作用。为了进一步证实miR-495对人RCC细胞生物学活性的影响是通过其抑制靶基因SATB1表达实现的,通过SATB1表达载体的转染使稳定表达miR-495的786-0细胞中SATB1基因拯救性过表达,并利用WB验证表达载体转染后SATB1蛋白的表达水平,进一步利用CCK8、流式细胞和划痕实验检测拯救性过表达SATB1基因对于稳定表达miR-495的786-0细胞增殖活性、细胞周期以及迁徙能力的影响。结果：qRT-PCR检测结果显示四种人RCC细胞系(769-P、786-o、A498和SN12-PM6)中miR-495的表达量均较人正常肾细胞系HK-2中降低,而四种RCC细胞之间,786-0细胞内的miR-495表达水平相对最低。与癌旁正常组织相比,miR-495在临床RCC组织标本中的表达量也显著降低。通过转染人工合成的miR-495 mimics成功获得稳定表达miR-495的786-0细胞系,并利用qRT-PCR验证了转染后miR-495的表达水平明显升高。细胞周期检测显示稳定表达miR-495的786-0细胞多数细胞周期停滞在G0/G1期；CCK8检测显示稳定表达miR-495可抑制786-0细胞的增殖活性；划痕实验显示稳定表达miR-495的786-0细胞迁徙活性也明显受到抑制。通过检索TargetScan6.2等相关数据库均显示,SATB1是miR-495可能的靶基因。荧光素酶报告基因检测显示在包含p-MIR-SATB1 (SATB1-3'UTR-WT)的786-0细胞中过表达miR-495可使荧光素酶报告基因的荧光值降低约67%,而转染序列突变的p-MIR-SATB1 (SATB1-3'UTR-MUT)以及miR scramble对照组荧光素酶报告基因的荧光值没有变化。证实miR-495与SATB1 mRNA的3'UTR可互补结合。而且,利用qRT-PCR和WB检测证实了稳定表达的miR-495可抑制786-0细胞中SATB1 mRNA和蛋白水平的表达。为了进一步证实miR-495对人RCC细胞生物学活性的影响是通过靶向SATB1基因实现的,利用SATB1表达载体转染使稳定表达miR-495的786-0细胞中SATB1基因拯救性过表达,通过WB验证了载体转染后SATB1蛋白的表达明显升高。CCK8检测显示过表达SATB1可回复由于稳定表达miR-495对786-0细胞增殖活性的抑制作用；流式细胞检测也显示稳定表达miR-495的786-0细胞在过表达SATB1后细胞周期明显由G0/G1期进入到S期；划痕实验显示过表达SATB1可回复因稳定表达miR-495对786-0细胞迁徙能力的抑制作用。结论：miR-495可通过抑制SATB1表达在RCC中扮演抑癌基因的作用。miR-495对SATB1的直接作用可能提供了一个SATB1转录后调控的机制理论。miR-495有望作为将来RCC治疗的作用靶点之一。
[Abstract]:Background: renal cell carcinoma (RCC) is the most common adult kidney tumor types, accounting for all primary renal tumors in 85% of all adult cancers in the incidence rate of about 3%. can be taken for surgical treatment, limitation of early renal cell carcinoma however, there are still many patients will face tumor recurrence and metastasis risk therefore, the prognosis is poor with metastasis in patients with RCC, the 5 year survival rate of 9%. for the treatment of these patients with advanced RCC, the urgent need to find new therapeutic targets, can be used as biomarkers of.MiRNAs is 19-24 nucleotides, highly conserved regulatory molecules by mRNA and target gene 3 "non encoding region (3'UTR) of the base sequence complementary after mRNA degradation or inhibit translation, thereby regulating the expression level of target genes. Many studies reported that miRNAs in the regulation of development; cell proliferation, differentiation, invasion and apoptosis play a key role in the miR. -495 in a variety of tumors were found to have abnormal expression, such as non-small cell lung cancer, breast cancer, malignant glioma, gastric cancer and leukemia, may act as a tumor suppressor genes or oncogenes play an important role in cancer development. But at present the expression of miR-495 in renal cell carcinoma and its function is unknown Objective: To investigate the expression of miR-495 in human RCC, to investigate the expression level of miR-495 on the proliferation of RCC cells in vitro, influence the migration activity. The target gene prediction of miR-495, verification of RCC cells in vitro in miR-495 abnormal expression regulation of the target genes for further study the possible molecular mechanism of miR-495 regulation of RCC.: four human RCC cell lines and a normal renal cell line, the expression level of miR-495 qRT-PCR was detected by cell lines, another collection of clinical renal cell carcinoma and adjacent normal tissues, qRT-PCR tissues were detected by The differential expression of miR-495. Through transient transfection technology to establish the stable over expression of miR-495 in human renal carcinoma cell line 786-0, the expression of miR-495 qRT-PCR to verify the stable transfection cell lines. Further changes in 786-0 by the CCK8 assay the cell proliferation activity of miR-495 expression after detection; overexpression of miR-495 effect on 786-o cell cycle by flow cytometry the scratch test verified; 786-0 cell migration miR-495 expression changes. By searching the TargetScan database of bioinformatics to predict the target gene SATB1 miR-495, analyzed by luciferase reporter to verify whether miR-495 and SATB1mRNA target gene 3'UTR complementary binding and through inhibition of miR-495 detection of human renal cell carcinoma qRT-PCR and WB 786-0 cells the target gene of SATB1. To further confirm the effect of miR-495 on the activity of RCC in cell biology is through The inhibition of SATB1 target gene expression, SATB1 786-0 cells by SATB1 vector transfection and expression of the stable expression of miR-495 gene in rescue over expression, and use WB to verify the expression level of SATB1 protein expression after transfection, further using CCK8, flow cytometry and scratch assay of overexpression of SATB1 gene for rescue 786-0 stable expression of miR-495 cell proliferation activity, cell cycle effects and migration ability. Results: qRT-PCR results showed that four kinds of human RCC cell lines (769-P, 786-o, A498 and SN12-PM6) expression in miR-495 were lower than that of normal human kidney cell line HK-2 decreased, and four kinds of RCC cells, 786-0 cells the expression level of miR-495 is the lowest. Compared with normal tissues, the expression of miR-495 in clinical RCC in the samples was also significantly decreased. By transfection of synthetic miR-495 mimics successfully obtained stability The expression of miR-495 786-0 cell lines, and the use of qRT-PCR to verify the expression level of miR-495 increased significantly after transfection. Cell cycle analysis showed that the expression of most G0/G1 cell cycle arrest in 786-0 stable miR-495 cells; CCK8 assay showed that the stable expression of miR-495 can inhibit the proliferation of the cells in 786-0; scratch test showed that stable expression of miR-495 gene in 786-0 cell migration the activity was also inhibited. By searching the TargetScan6.2 database show that SATB1 is the target gene of miR-495. Luciferase reporter gene assay showed that contain p-MIR-SATB1 (SATB1-3'UTR-WT) 786-0 cells that overexpressed miR-495 fluorescent luciferase reporter gene decreased about 67%, while transfection of the sequence of the p-MIR-SATB1 mutant (SATB1-3'UTR-MUT) and fluorescence the control group miR scramble luciferase reporter gene and confirmed that the miR-495 value does not change. SATB1 mRNA 3'UTR can be combined with complementary. Moreover, detected by qRT-PCR and WB confirmed the stable expression of miR-495 can inhibit the expression of SATB1 mRNA and protein levels in 786-0 cells. To further confirm the effect of miR-495 on the activity of RCC in cell biology is to achieve the target gene by SATB1 and SATB1 786-0 cells transfected to stable expression the use of miR-495 SATB1 gene in rescuing expression by WB to verify the expression of SATB1 protein increased significantly after transfection.CCK8 assay showed that the overexpression of SATB1 can reply due to stable expression of inhibitory effect of miR-495 on 786-0 cell proliferation; flow cytometry showed stable expression of miR-495 786-0 cells in the expression of cell cycle after SATB1 obviously from G0/G1 to S; the scratch test showed that overexpression of SATB1 can reply for stable expression of miR-495 in 786-0 cell migration inhibition ability Conclusion: miR-495 can inhibit the expression of SATB1 and play a role in tumor suppressor gene in RCC. The direct effect of.MiR-495 on SATB1 may provide a mechanism of SATB1 post transcriptional regulation..miR-495 is expected to be one of the targets of RCC therapy in the future.

【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R737.11

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