Hsa-miR-19b-1-5p在前列腺癌中的作用及其机制的研究

发布时间：2018-04-14 11:07

本文选题：前列腺肿瘤 + miR-19b-1-5p　；参考：《青岛大学》2017年硕士论文

【摘要】：研究目的:前列腺癌(prostate cancer,PCa)主要发生于老年人群。数年前,我国前列腺癌的发病率还处在一个很低的程度,但随着人们生活水准的逐渐西式化以及诊断程度的不断上升,我国前列腺癌的检出率和发病率开始连年增高,已然成为危及中老年男性生命的重要疾病。本研究基于微小RNA(miRNA,miR)对前列腺癌存在抑制或促进的作用而产生,旨在探讨miR-19b-1-5p对人前列腺癌细胞生物学行为的影响,阐明miR-19b-1-5p在前列腺癌中的作用以及发生发展的主要机制。研究方法:收集临床标本,划分前列腺癌组和良性前列腺组织组,各组标本分别为77例和68例。实时荧光定量聚合酶链反应技术(Real-time PCR)检测各组织中miR-19b-1-5p的表达情况,分析mi R-19b-1-5p的相对表达量与前列腺癌Gleason评分和临床病理特征之间的关系。Real-time PCR检测不同前列腺癌细胞系和正常前列腺上皮细胞中miR-19b-1-5p的表达情况。将miR-19b-1-5p的模拟物(mimic)、抑制物(inhibitor)经化学转染试剂分别转染进人前列腺癌细胞系中。细胞计数(Cell Counting Kit-8,CCK-8)试剂盒检测细胞的增殖能力变化,Transwell小室检测细胞的侵袭和转移能力,划痕实验检测细胞迁移能力,流式细胞仪分析细胞凋亡能力,以此探究miR-19b-1-5p对前列腺癌细胞生物学行为的影响及其作用。应用靶基因预测软件miRanda、TargetScan、PicTar等预测miR-19b-1-5p的潜在靶基因。Real-time PCR检测miR-19b-1-5p与靶基因mRNA的相对表达量,分析二者之间的相互关系,Western blot验证靶基因的表达变化。结果:mi R-19b-1-5p在良性前列腺组织内高表达,而在前列腺癌组织中表达量较低,两组比较差异有显著性(t=24.87,P0.05),miR-19b-1-5p的相对表达量与肿瘤的Gleason评分、临床分期间有关。前列腺癌细胞系PC-3、DU145、LNCap、C4-2同正常前列腺上皮细胞RWPE-1比较发现,miR-19b-1-5p在前列腺癌细胞系中表达量较低,RWPE-1中表达量较高,而在DU145、PC-3中表达量最低。CCK-8实验表明转染miR-19b-1-5p-mimic组的细胞增殖率显著低于miR-19b-1-5p-inhibitor组。Transwell实验中,转染mi R-19b-1-5p-mimic组的细胞通过含基质胶的Transwell小室数量明显少于miR-19b-1-5p-inhibitor组。划痕实验中,转染miR-19b-1-5p-mimic组的细胞闭合率明显小于miR-19b-1-5p-inhibitor组。流式分析实验显示转染miR-19b-1-5p-mimic组的细胞凋亡率增加。靶基因软件预测CXCR6为mi R-19b-1-5p的下游靶基因,Real-time PCR结果显示miR-19b-1-5p与CXCR6存在负相关,Western blot实验也证实转染miR-19b-1-5p-mimic后CXCR6的表达水平受到抑制。结论及意义:miR-19b-1-5p在前列腺癌组织内低表达,起抑癌基因的作用。随着Gleason评分的增高、临床分期级别增高而表达量减少。上调miR-19b-1-5p的表达可明显抑制前列腺癌细胞株的增殖、侵袭和转移能力,促进其凋亡,可能是通过抑制下游靶基因CXCR6的表达实现。
[Abstract]:Objective: prostate cancer (PCA) mainly occurs in the elderly.A few years ago, the incidence of prostate cancer in China was still at a very low level. However, with the gradual westernization of people's standard of living and the rising diagnostic level, the detection rate and incidence of prostate cancer in China began to increase year after year.It has become an important disease that endangers the life of middle-aged and aged men.The aim of this study was to investigate the effect of miR-19b-1-5p on the biological behavior of human prostate cancer cells and to elucidate the role of miR-19b-1-5p in prostate cancer and the main mechanism of its development.Methods: clinical specimens were collected and divided into prostate cancer group (77 cases) and benign prostate tissue group (68 cases).Real-time PCR was used to detect the expression of miR-19b-1-5p in tissues.To analyze the relationship between the relative expression of mi R-19b-1-5p and Gleason score and clinicopathological features of prostate cancer. Real-time PCR was used to detect the expression of miR-19b-1-5p in different prostate cancer cell lines and normal prostate epithelial cells.The mimic of miR-19b-1-5p was transfected into human prostate cancer cell line by chemical transfection reagent.Cell Counting Kit-8 CCK-8 kit was used to detect cell proliferation ability. Cell invasion and metastasis were detected by Transwell chamber, migration ability was detected by scratch assay, and apoptosis was analyzed by flow cytometry.To investigate the effect of miR-19b-1-5p on the biological behavior of prostate cancer cells.The relative expression of miR-19b-1-5p and target gene mRNA was detected by using the target gene prediction software miRanda Target Scann PicTar to predict the potential target gene. Real-time PCR was used to detect the relative expression of miR-19b-1-5p and target gene mRNA, and the correlation between them was analyzed. Western blot was used to verify the change of target gene expression.Results there was a significant difference between the two groups in the high expression of R-19b-1-5p in the benign prostate tissue and the low expression in the prostate cancer tissue. The relative expression of the two groups was correlated with the Gleason score of the tumor and the clinical stage.Compared with normal prostatic epithelial cells (RWPE-1), prostate cancer cell line PC-3DU145LNCapC4-2 was found to have a higher expression level of miR-19b-1-5p in prostate cancer cell line than that in prostate cancer cell line RWPE-1.CCK-8 assay showed that the cell proliferation rate of miR-19b-1-5p-mimic group was significantly lower than that of miR-19b-1-5p-inhibitor group. The number of Transwell cells transfected with mi R-19b-1-5p-mimic group was significantly lower than that of miR-19b-1-5p-inhibitor group.In scratch test, the cell closure rate of miR-19b-1-5p-mimic transfected group was significantly lower than that of miR-19b-1-5p-inhibitor group.Flow cytometry showed that the apoptosis rate of miR-19b-1-5p-mimic transfected group was increased.Real-time PCR of CXCR6 as a downstream target gene of mi R-19b-1-5p was predicted by target gene software. The results showed that there was a negative correlation between miR-19b-1-5p and CXCR6. Western blot experiment also confirmed that the expression of CXCR6 was inhibited after transfection of miR-19b-1-5p-mimic.Conclusion the low expression of 1: miR-19b-1-5p in prostate cancer may play an important role in tumor suppressor gene.With the increase of Gleason score, the clinical stage grade increased and the expression decreased.Upregulating the expression of miR-19b-1-5p can obviously inhibit the ability of proliferation, invasion and metastasis of prostate cancer cell line and promote its apoptosis, which may be realized by inhibiting the expression of downstream target gene CXCR6.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.25

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