NIN1结合蛋白1激活人Src原癌基因调控前列腺癌细胞生物学特性的研究

NIN1/RPN12 binding protein 1 homolog regulates biological characteristics of prostate cancer cells by activating Src proto-oncogene

目的探讨NIN1结合蛋白1(NOB1)对前列腺癌细胞生物学特性的影响及其作用机制。方法采用实时定量PCR和Western印迹法检测常见前列腺癌细胞22RV1、PC-3、DU145、LNCap中NOB1的表达量;构建NOB1干扰慢病毒,转染前列腺癌细胞PC-3、DU145,并检测干扰效率。采用MTT法检测敲低NOB1后前列腺癌细胞PC-3和DU145增殖活性,应用流式细胞技术检测敲低NOB1后前列腺癌细胞PC-3和DU145周期,采用Transwell实验检测转染NOB1干扰慢病毒后前列腺癌细胞PC-3和DU145的侵袭能力。检测敲低NOB1后的人Src原癌基因(c-Src)磷酸化水平。结果恶性程度较高的前列腺癌细胞22RV1、PC-3、DU145的NOB1 mRNA相对表达量均显著高于恶性程度较低的前列腺癌细胞LNCaP(P值均<0.05);前列腺癌细胞22RV1、PC-3、DU145的NOB1蛋白质相对表达量均显著低于前列腺癌细胞LNCaP(P值均<0.05)。在NOB1表达量较高的前列腺癌细胞PC-3和DU145中转染NOB1干扰慢病毒(Lv-shNOB1,敲低组)和空白对照慢病毒(Lv-shCtrl,阴性对照组)以构建NOB1敲低模型,PC-3、DU145细胞的敲低组NOB1 mRNA相对表达量均显著低于空白对照组(不转染病毒)和阴性对照组(P值均<0.05),表明构建慢病毒介导NOB1稳定敲低前列腺癌细胞PC-3和DU145。PC-3、DU145细胞的敲低组NOB1蛋白质相对表达量均显著低于空白对照组和阴性对照组(P值均<0.05)。实验第3、4、5天,PC-3、DU-145细胞敲低组A595值均显著低于阴性对照组同时间(P值均<0.05)。PC-3和DU145细胞阴性对照组的G0/G1期细胞百分比均显著低于敲低组(P值均<0.05),S期细胞百分比均显著高于敲低组(P值均<0.05)。PC-3、DU145细胞敲低组的迁移细胞数均显著低于阴性对照组和空白对照组(P值均<0.01)。PC-3、DU145细胞敲低组c-Src(tyr416位点)的磷酸化半定量均显著低于阴性对照组(P值均<0.01)。结论敲低NOB1可能通过抑制c-Src的磷酸化来降低前列腺癌细胞的增殖和侵袭能力。

Objective To investigate the effect of NIN1 binding protein 1(NOB1) on the biological characteristics of prostate cancer cells and its mechanism. Methods Quantitative real-time polymerase chain reaction(PCR) and Western blot were used to detect the expression of NOB1 in prostate cancer cells(22 RV1, PC-3, DU145 and LNCap). NOB1 knockdown lentivirus was constructed and transfected into PC-3 and DU145. The interference efficiency was detected. After knockdown of NOB1 in PC-3 and DU145, their proliferative activity was detected by methyl thiazolyl tetrazolium(MTT) assay, cell cycles were measured by flow cytometry and invasion ability was detected by Transwell. The phosphorylation level of human Src proto-oncogene(c-Src) after knockdown of NOB1 was detected. Results The relative expression of NOB1 mRNA in prostate cancer cells with high malignancy(22 RV1, PC-3 and DU145) was significantly higher than that in LNCaP with low malignancy(all P<0.05). The relative NOB1 protein level in 22 RV1, PC-3 and DU145 was significantly lower than that in LNCaP(all P<0.05). NOB1 interference lentivirus(Lv-shNOB1) and blank control lentivirus(Lv-shCtrl) were transfected into PC-3 and DU145 with high NOB1 expression to construct NOB1 knockdown model. The relative expressions of NOB1 mRNA and protein in PC-3 and DU145 knockdown groups were significantly lower than those in the blank control group(no transfection) and negative control group(Lv-shCtrl transfected)(all P<0.05), indicating that the construction of lentivirus-mediated NOB1 stably knocked down PC-3 And DU145. On days 3, 4 and 5 of the experiment, the A595 values of PC-3 and DU-145 in knockdown groups were significantly lower than those in the negative control group(all P<0.05). The percentage of G0/G1 phase cells in the PC-3 and DU145 cells in the negative control group were significantly lower than those in the knockdown group(all P<0.05), while the percentage of S phase cells was significantly higher than that in the knockdown group(all P<0.05). The number of migrated cells in the PC-3 and DU145 cells in knockdown group were significantly less than that in the negative control group and blank control group(all P<0.01). The semi-quantitative phosphorylation of c-Src(site tyr416) in PC-3 and DU145 cells in knockdown group was significantly lower than that in the negative control group(all P<0.01). Conclusion The knockdown of NOB1 may reduce the proliferation and invasion of prostate cancer cells by inhibiting the phosphorylation of c-Src.