miR-105-1对肝癌细胞NCOA1表达的影响及其预后的研究

发布时间：2018-06-06 02:25

本文选题：肝细胞肝癌 + miR-105-1　；参考：《安徽医科大学》2017年硕士论文

【摘要】：目的:越来越多的证据表明micro RNA通过调节各自的靶基因在癌症的发生发展过程中起到重要作用。本研究通过筛选出肝癌可能相关的miR-105-1及其靶基因,随后验证在肝癌和正常肝组织中进行验证miR-105-1及其靶基因NCOA1的表达量及两者的关系,并分析患者的预后,研究miR-105-1与肝癌的发生发展的可能机制。方法:我们通过使用基因微阵列和分层聚类分析,筛选肝癌可能相关的miR-105-1,收集34对人肝癌组织及对应的癌旁组织和另外120个肝癌组织,通过RT-q PCR的方法检测miR-105-1在肝癌及癌旁正常组织中的表达量差异加以验证,并进一步分析miR-105-1和患者预后的关系。通过数据库寻找miR-105-1可能的靶基因NCOA1,并通过RT-q PCR、Western-Blot及肝癌组织的免疫组化来验证miR-105-1和NCOA1的靶向关系,并联合分析miR-105-1和NCOA1与患者预后的关系。结果:我们从GEO和TCGA数据库芯片数据分析得出miR-105-1可能为我们新发现的差异基因,我们检测了miR-105-1在肝癌组织及癌旁正常组织中均有表达,miR-105-1在34个配对的HCC与癌旁正常组织中下调0.59倍(P=0.041),与34个癌旁正常组织相比,miR-105-1在154个HCC中下调0.51倍,(P=0.032)。miR-105-1在154个HCC中Kaplan-Meier生存分析OS的Log-Rank P=0.031,表明miR-105-1表达越低的HCC病人生存时间越短。PFS的Log-Rank P=0.038,表明miR-105-1表达越低的HCC病人疾病进展时间越短。我们通过GEO数据库及三个靶基因预测网站预测出NCOA1可能为miR-105-1的靶基因,Hela细胞Realtime RT-PCR检测miR-105-1 mimics(过表达)和inhibitor(knock-down)对NCOA1 m RNA含量的影响。在验证试验中,Hela细胞RT-PCR检测中,与对照组相比,miR-105-1过表达组,NCOA1表达量低(P0.001),而miR-105-1敲除组,NCOA1表达量高(P0.001)。WB检测miR-105-1过表达和敲除对NCOA1及已知的下游分子CSF1蛋白水平的影响,与对照组相比,miR-105-1过表达NCOA1及已知的下游分子CSF1蛋白表达低,相反,miR-105-1敲除后,NCOA1及已知的下游分子CSF1蛋白表达高。异位表达的miR-105-1降低NCOA1的3'-UTR的荧光素酶活性;然而,miR-105-1突变体含有三个突变的核苷没有对荧光素酶活性的抑制作用。在癌组织及癌旁组织石蜡切片的组化中,我们发现NCOA1在正常肝组织中低表达,在肝癌组织中高表达。NCOA1在154个HCC中Kaplan-Meier生存分析OS的Log-Rank p=0.011,NCOA1表达越高的HCC病人生存时间越短。PFS的Log-Rank p=0.033,NCOA1表达越高的HCC病人疾病无进展时间越短。联合分析miR-105-1和NCOA1在154个HCC中Kaplan-Meier生存分析OS的Log-Rank p0.001,miR-105-1表达越低同时NCOA1表达越高的HCC病人生存时间越短。PFS的Log-Rank p=0.002,miR-105-1越低同时NCOA1表达越高的HCC病人疾病无进展时间越短。结论1、miR-105-1在肝癌中的表达低于正常肝组织。2、miR-105-1可能通过调控靶基因NCOA1来调节肝癌的发生发展。3、miR-105-1高表达的肝癌患者预后较好,相反,低表达的患者预后不良。4、NCOA1高表达的肝癌患者预后不良,相反,低表达的患者预后较好。5、miR-105-1及NCOA1可能成为判断肝癌预后的重要的生物学指标。
[Abstract]:Objective: more and more evidence shows that micro RNA plays an important role in the development of cancer by regulating their target genes. This study screened the possible miR-105-1 and target genes of liver cancer, and then verified the expression of miR-105-1 and its target gene NCOA1 in liver cancer and normal liver tissues and both of them and both. The relationship, and the analysis of the prognosis of patients, the possible mechanism of the development of miR-105-1 and liver cancer. Methods: we use gene microarray and stratified cluster analysis to screen the possible miR-105-1 related to liver cancer, collect 34 human liver cancer tissues and corresponding para cancer tissues and other 120 liver cancer tissues, and detect miR through the RT-q PCR method. The expression of -105-1 in the normal tissues of liver cancer and para cancer was verified, and the relationship between miR-105-1 and patient's prognosis was further analyzed. The possible target gene NCOA1 of miR-105-1 was searched through database, and the targeting relationship between miR-105-1 and NCOA1 was verified by RT-q PCR, Western-Blot and liver cancer tissue, and miR-10 was analyzed jointly. The relationship between 5-1 and NCOA1 and the prognosis of patients. Results: We obtained from GEO and TCGA database chip data analysis that miR-105-1 may be our new differentially identified gene. We detected the expression of miR-105-1 in liver cancer tissues and normal tissues adjacent to cancer, and miR-105-1 was reduced by 0.59 times (P=0.041) in 34 paired HCC and adjacent normal tissues (P=0.041). Compared with 34 adjacent normal tissues, miR-105-1 was down 0.51 times in 154 HCC, and (P=0.032).MiR-105-1 in 154 HCC, Kaplan-Meier survival analysis of OS Log-Rank P=0.031, indicating that the lower the miR-105-1 expression, the shorter the survival time of the HCC patient was, the shorter the disease progression time for the patients with the lower expression. The GEO database and three target gene prediction sites were used to predict that NCOA1 might be the target gene of miR-105-1. Hela cells Realtime RT-PCR detected the influence of miR-105-1 mimics (over expression) and inhibitor (knock-down) on NCOA1 m. P0.001, miR-105-1 knockout group, NCOA1 expression high (P0.001).WB detection of miR-105-1 overexpression and knockout of NCOA1 and known downstream molecules CSF1 protein level, compared with the control group, miR-105-1 overexpression NCOA1 and known downstream molecules CSF1 protein table, on the contrary, miR-105-1 knockout, and the known downstream points The expression of CSF1 protein was high. The miR-105-1 of ectopic expression reduced the luciferase activity of NCOA1 3'-UTR; however, the miR-105-1 mutant contained three mutant nucleosides that did not inhibit the activity of luciferase. In the histochemistry of paraffin sections of cancer tissue and paracancerous tissue, we found that NCOA1 was low expression in normal liver tissue and in liver cancer tissue The higher expression of.NCOA1 in 154 HCC is Log-Rank p=0.011 for the survival analysis of OS, the higher the NCOA1 expression is, the shorter the.PFS Log-Rank p=0.033 of HCC patients, the higher the NCOA1 expression, the shorter the disease progression time. The lower the expression of -105-1 and the higher the expression of NCOA1 in HCC patients, the shorter the.PFS Log-Rank p=0.002, the lower the miR-105-1 and the higher the NCOA1 expression, the shorter the progression free time of the disease. Conclusion 1, the expression of miR-105-1 in the liver cancer is lower than that of the normal liver tissue, and miR-105-1 can regulate the hair of the liver cancer by regulating the target gene. The prognosis of liver cancer patients with high expression of.3 and miR-105-1 is better. On the contrary, the prognosis of patients with low expression is poor.4, and the prognosis of the patients with high expression of NCOA1 is poor. On the contrary, the prognosis of the patients with low expression is better.5. MiR-105-1 and NCOA1 may be the important biological indicators to judge the prognosis of liver cancer.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.7

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