dsRNA上调Numb基因表达对人前列腺癌PC-3细胞影响的研究

发布时间：2018-06-27 21:31

本文选题：小激活RNA + Numb　；参考：《福建医科大学》2015年硕士论文

【摘要】：目的基于小激活RNA(small activating RNA,sa RNA)的肿瘤治疗策略,为Numb基因筛选出具有激活功能并相对高效的sa RNA分子。继而探讨雄激素及抗雄激素药物对转染Numb-sa RNA的人前列腺癌PC-3细胞生长的影响。方法1.设计与合成针对Numb基因的3对候选小分子双链RNA(double-stranded RNA,ds RNA)分子,对照组ds RNA(ds Control)设计成与人类基因组序列非同源。将ds RNA分子转染人前列腺癌PC-3细胞。2.采用Real-time quantitative PCR(RT-q PCR)法检测转染后人前列腺癌PC-3细胞中靶基因Numb的m RNA表达水平。3.Western Blot验证转染后人前列腺癌PC-3细胞靶基因Numb的蛋白表达水平。4.将筛选出的Numb-sa RNA转染入人前列腺癌PC-3细胞,实验组分为转染组和非转染组,以未转染以及未行药物处理的细胞作为阴性对照组。分别采用不同浓度的二氢睾酮(dihydrotestosterone,DHT),氟他胺(flutamide)、二氢睾酮-氟他胺混合液(dihydrotestosterone-flutamide mixture,DHT-F)处理实验组细胞,采用MTT法在药物处理48h后分别测定细胞活性;5.根据MTT法检测结果,筛选出有效的药物浓度,分别处理转染组和非转染细胞,培养48h后,采用Annexin V-FITC/PI检测各组细胞的凋亡情况。结果1.ds Numb-870、ds Numb-948均未能上调人前列腺癌PC-3细胞中靶基因Numb m RNA水平;而ds Numb-298能上调细胞内Numb m RNA和蛋白水平。2.采用MTT法检测细胞增殖情况,DHT处理细胞后,发现转染组在10-3mol/L、10-4mol/L、10-5mol/L三个浓度组的相对存活率均明显低于阴性对照组,差异有统计学意义(P0.05);转染组在10-3mol/L和10-4mol/L浓度组的相对存活率明显低于非转染组,差异有统计学意义(P0.05)。氟他胺处理细胞后,转染组在10-3mol/L、10-4mol/L、10-5mol/L三个浓度组的相对存活率均低于阴性对照组,差异有统计学意义(P0.05);转染组在10-4mol/L浓度组的相对存活率明显低于非转染组,差异有统计学意义(P0.05)。二氢睾酮-氟他胺混合液处理后,转染组各个浓度组的相对存活率较阴性对照组均明显降低,差异有统计学意义(P0.05);且转染组在各个浓度的相对存活率均明显低于非转染组,差异均有统计学意义(P0.05)。3.采用Annexin V-FITC/PI双染法,利用流式细胞技术检测细胞凋亡情况,DHT处理后,转染组的凋亡率较阴性对照组下降,差异有统计学意义(P0.05);转染组的凋亡率较非转染组明显下降,差异有统计学意义(P0.05)。氟他胺处理后,转染组的凋亡率较阴性对照组明显上升,差异有统计学意义(P0.05);转染组较非转染组的凋亡率明显上升,差异有统计学意义(P0.05)。二氢睾酮-氟他胺混合液处理后,转染组的凋亡率较阴性对照组明显上升,差异有统计学意义(P0.05);转染组的凋亡率多于非转染组,差异有统计学意义(P0.05)。结论1.经筛选ds Numb-298具有特异性激活人前列腺癌PC-3细胞中Numb基因表达的功能。2.将Numb-sa RNA转染入人前列腺癌PC-3细胞中,能在一定程度上使人前列腺癌PC-3恢复对激素的敏感性,诱导其凋亡。
[Abstract]:Objective to screen active and relatively efficient sa RNA molecules for numb gene based on small activated (small activating RNA-sa RNA. Then we investigated the effects of androgen and androgen drugs on the growth of PC-3 cells transfected with Numb-sa RNA. Method 1. Three pairs of candidate small molecule double-stranded RNAs (double-stranded RNAs RNAs) were designed and synthesized for numb gene, while the control group DS RNA (DS control) was designed to be not homologous to the human genome sequence. The DS RNA molecule was transfected into human prostate cancer PC-3 cell line. 2. Real-time quantitative polymerase chain reaction (RT-q PCR) was used to detect the mRNA expression level of target gene numb in human prostate cancer PC-3 cells. 3. Western blot was used to verify the protein expression level of the target gene numb of human prostate cancer PC-3 cells. The selected Numb-sa RNA was transfected into human prostate cancer PC-3 cells. The experimental group was divided into transfection group and non-transfection group. Untransfected and untreated cells were used as negative control group. The experimental cells were treated with different concentrations of dihydrotestosterone (DHT) and flutamide (flutamide), mixture (dihydrotestosterone-flutamide mixture- DHT-F), respectively. The cell activity was measured by MTT assay after 48 hours of treatment. According to the results of MTT assay, the effective drug concentration was selected. The transfected cells and non-transfected cells were treated respectively. After 48 hours of culture, Annexin V-FITC / Pi was used to detect the apoptosis of the cells in each group. Results 1.ds Numb-870 DS Numb-948 could not upregulate the level of target gene numm RNA in human prostate cancer PC-3 cells, while DS Numb-298 could up-regulate the level of numb mRNA and protein in PC-3 cells. MTT assay was used to detect the proliferation of cells treated with DHT. It was found that the relative survival rate of the transfected group in 10-3 mol / L 10-4 mol / L 10-5 mol / L group was significantly lower than that in the negative control group. The relative survival rate of transfection group in 10-3 mol / L and 10-4 mol / L groups was significantly lower than that in non-transfection group (P0.05). After treated with flutamide, the relative survival rate of transfected cells in 10-3 mol / L 10-4 mol / L 10-5 mol / L group was significantly lower than that in negative control group (P0.05), and the relative survival rate in 10-4 mol / L group was significantly lower than that in non-transfection group (P0.05). The relative survival rate of each concentration group in transfection group was significantly lower than that in negative control group (P0.05), and the relative survival rate in each concentration of transfection group was significantly lower than that in non-transfection group. The difference was statistically significant (P0.05). Using Annexin V-FITC / Pi double staining method and flow cytometry, the apoptosis rate of transfected group was lower than that of negative control group (P0.05), and the apoptosis rate of transfected group was significantly lower than that of non-transfected group. The difference was statistically significant (P0.05). After flutamide treatment, the apoptosis rate of transfection group was significantly higher than that of negative control group (P0.05); the apoptosis rate of transfection group was significantly higher than that of non-transfection group (P0.05). The apoptosis rate of transfection group was significantly higher than that of negative control group (P0.05), and the apoptosis rate of transfection group was more than that of non-transfection group (P0.05). Conclusion 1. DS Numb-298 has the function of specifically activating the expression of numb gene in human prostate cancer PC-3 cells. Transfection of Numb-sa RNA into human prostate cancer PC-3 cells can restore the hormone sensitivity and induce apoptosis in human prostate cancer PC-3 cells to a certain extent.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R737.25

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