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目的前列腺癌在我国的发病率及病死率逐年上升,但其发病机制尚未明确,本研究从转录后水平初步探讨RNA结合蛋白HuR参与调控前列腺癌细胞活力的机制。方法免疫荧光检测前列腺增生细胞(BPH)及前列腺癌细胞(PC3)中HuR蛋白的定位,蛋白质印迹法检测HuR及环氧合酶-2(cyclooxygenase,COX-2)蛋白在细胞内的表达,MTT法检测细胞活力,脂质体转染法转染质粒及干扰片段,qPCR检测干扰效率,RNA-pull down实验验证HuR对COX-2的调控。结果 HuR在BPH细胞主要定位于细胞核,在PC3细胞中主要定位于胞质。PC3细胞中HuR表达水平为1.699±0.011,高于BPH细胞的0.654±0.028,差异有统计学意义,t=58.67,P<0.001。PC3细胞中过表达HuR后,转染空质粒组细胞活力为1.038±0.117,转染HuR表达质粒后细胞活力为1.838±0.057,差异有统计学意义,t=10.656,P<0.001。干扰HuR表达后,转染siNC组细胞活力为1.254±0.095,转染siHuR干扰片段组细胞活力为0.66±0.102,t=7.383,P=0.002;BPH组和PC3组COX-2表达水平分别为0.449±0.055和1.066±0.068,t=12.147,P<0.001;过表达COX-2后PC3细胞活力明显增加,转染空质粒组细胞活力为1.296±0.114,转染COX-2表达质粒后细胞活力为1.954±0.062,t=18.062,P<0.001。干扰COX-2表达后,PC3细胞活力明显降低,转染siNC组细胞活力为1.233±0.145,转染siCOX-2干扰片段后细胞活力为0.661±0.096,t=5.688,P=0.005。HuR可以结合于COX-2的3′-UTR并上调COX-2蛋白表达,转染空质粒组COX-2蛋白表达水平为0.638±0.067,转染HuR表达质粒组为1.703±0.022,t=26.26,P<0.001。结论HuR参与调控前列腺癌细胞活力可能是通过上调COX-2表达而实现。
Objective The incidence and mortality of prostate cancer in our country have been increasing year by year, but its pathogenesis has not yet been clarified. In this study, the mechanism of HuR involved in the regulation of the activity of prostate cancer cells was investigated from the post-transcriptional level. Methods Immunofluorescence was used to detect the localization of HuR protein in prostate hyperplasia (BPH) and prostate cancer (PC3) cells. Western blotting was used to detect the expression of HuR and cyclooxygenase-2 (COX-2) The cell viability was detected by Lipofectamine 2000 method. Plasmids and interference fragments were transfected by lipofectamine. The interference efficiency was detected by qPCR, and the regulation of HuX on COX-2 by RNA-pull down assay. Results HuR mainly localized in the nucleus of BPH cells and localized in the cytoplasm in PC3 cells. The expression level of HuR in PC3 cells was 1.699 ± 0.011, which was higher than that of BPH cells (0.654 ± 0.028), the difference was statistically significant, t = 58.67, P <0.001. The viability of cells transfected with empty vector was 1.038 ± 0.117 after transfection with HuR in PC3 cells. The viability of cells transfected with HuR plasmid was 1.838 ± 0.057, with a significant difference (t = 10.656, P <0.001). After interfering with HuR expression, the cell viability of transfected siNC group was 1.254 ± 0.095, and the viability of transfected siHuR interference fragment was 0.66 ± 0.102, t = 7.383, P = 0.002. The expression of COX-2 in BPH group and PC3 group were 0.449 ± 0.055 and 1.066 ± 0.068, t = 12.147, P <0.001. The viability of PC3 cells was significantly increased after COX-2 overexpression. The viability of cells transfected with empty plasmid was 1.296 ± 0.114. The viability of cells transfected with COX-2 was 1.954 ± 0.062, t = 18.062, P <0.001. The viability of PC3 cells was significantly reduced after interfering with COX-2 expression. The viability of transfected siNC cells was 1.233 ± 0.145. The viability of transfected siCOX-2 cells was 0.661 ± 0.096, t = 5.688, P = 0.005. HuR could bind to 3’-UTR of COX-2 and up-regulate the expression of COX-2 protein. The expression of COX-2 protein was 0.638 ± 0.067 in transfected empty plasmid group and 1.703 ± 0.022 in transfected HuR plasmid group, t = 26.26 , P <0.001. Conclusion HuR involved in the regulation of prostate cancer cell viability may be through the up-regulation of COX-2 expression.