miR-1249-5p对前列腺癌PC-3细胞增殖、转移和细胞周期的影响

Effects of miR-1249-5p on proliferation,metastasis and cell cycle of prostate cancer PC-3 cell

目的探讨miR-1249-5p对前列腺癌PC-3细胞增殖、转移和细胞周期的影响。方法采用OncoMir肿瘤数据库(OMCD)分析miR-1249-5p表达水平与前列腺癌患者总生存期的关系。将人前列腺癌细胞株PC-3分为两组:miR-1249-5p组和阴性对照组。以Lipofectamine 2000为介导,将miR-1249-5p拟似物脂质体复合物或阴性miRNA脂质体复合物转染入对数生长期的PC-3细胞。采用实时荧光定量聚合酶链反应(RT-qPCR)检测两组PC-3细胞miR-1249-5p的表达水平,采用集落形成实验检测两组PC-3细胞增殖能力的变化,采用Transwell实验检测两组PC-3细胞侵袭变化,采用流式细胞仪检测两组PC-3细胞周期变化。通过miRNA预测软件miRGator预测miR-1249-5p的靶基因。RT-qPCR和Western blotting法分别检测miR-1249-5p的靶基因表达。计量资料以均数±标准差(x±s)表示,两组间比较采用t检验。结果与miR-1249-5p低表达的前列腺癌患者相比,miR-1249-5p表达水平较高的前列腺癌患者总生存期较长,差异具有统计学意义(P<0.01)。miR-1249-5p组miR-1249-5p表达水平(10.74±1.19)明显高于阴性对照组(1.56±0.27),差异具有统计学意义(P<0.01)。miR-1249-5p组集落形成数[(35.86±6.94)个/孔]明显少于阴性对照组[(88.94±11.66)个/孔],差异具有统计学意义(P<0.01)。miR-1249-5p组穿膜细胞数[(25.01±6.83)个/高倍视野]明显少于阴性对照组[(82.76±8.35)个/高倍视野],差异具有统计学意义(P<0.01)。miR-1249-5p组处于G_(0)~G_(1)期的细胞比例[(50.79±6.61)%]明显高于阴性对照组[(27.09±2.30)%],差异具有统计学意义(P<0.01),PC-3细胞被抑制在G_(0)~G_(1)期。神经前体细胞表达发育调控蛋白9(NEDD9)可能是miR-1249-5p的靶基因。与阴性对照组比较,miR-1249-5p组NEDD9基因表达水平显著低于阴性对照组,差异具有统计学意义(P<0.01)。结论miR-1249-5p可以抑制前列腺癌PC-3细胞增殖、转移和细胞周期,其可能通过负调控原癌基因NEDD9表达实现。

Objective To explore the effect of miR-1249-5p on the proliferation,metastasis and cell cycle of PC-3 cell in prostate cancer.Methods The relationship between the expression level of miR-1249-5p and the overall survival of prostate cancer patients was analyzed using OncoMir Cancer Database(OMCD).The human prostate cancer cell line PC-3 was divided into two groups:miR-1249-5p group and negative control group.Mediated by Lipofectamine 2000,miR-1249-5p mimics liposome complex or negative miRNA liposome complex were transfected into PC-3 cell at logarithmic growth stage.Real-time quantitative polymerase chain reaction(RT-qPCR)was used to detect the expression of miR-1249-5p in PC-3 cell of two groups.Colony formation assay was used to detect the changes of the proliferation ability of PC-3 cell in the two groups.Transwell experiment was used to detect the changes of PC-3 cell invasion in the two groups,and the cell cycle changes of the two groups of PC-3 were detected by flow cytometry.The miRNA prediction software miRGator was used to predict the target gene of miR-1249-5p.RT-qPCR and Western blotting were used to detect the target gene expression of miR-1249-5p.Measurement data were expressed as mean±standard deviation(x±s),and t-test was used for comparison between two groups.Results Compared with prostate cancer patients with low miR-1249-5p expression,prostate cancer patients with higher miR-1249-5p expression had longer overall survival,and the difference was statistically significant(P<0.01).The expression level of miR-1249-5p in the miR-1249-5p group(10.74±1.19)was significantly higher than that of the negative control group(1.56±0.27),the difference was statistically significant(P<0.01).The number of colonies formed in the miR-1249-5p group(35.86±6.94)was significantly less than that in the negative control group(88.94±11.66),and the difference was statistically significant(P<0.01).The number of transmembrane cells[(25.01±6.83)/high power field of view]in the miR-1249-5p group was significantly less than that of the negative control group[(82.76±8.35)/high power field of view],and the difference was statistically significant(P<0.01).The proportion of cells in the G_(0)-G_(1) phase in the miR-1249-5p group[(50.79±6.61)%]was significantly higher than that in the negative control group[(27.09±2.30)%],the difference was statistically significant(P<0.01),and PC-3 cell were inhibited in the G_(0)-G_(1) phase.Neural precursor cell expressed developmentally down-regulated 9(NEDD9)may be the target gene of miR-1249-5p.Compared with the negative control group,the NEDD9 gene expression in the miR-1249-5p group was significantly lower than that of the negative control group,the difference was statistically significant(P<0.01).Conclusion miR-1249-5p can inhibit the proliferation,metastasis and cell cycle of PC-3 cell in prostate cancer,which may be achieved by negatively regulating the expression of proto-oncogene NEDD9.