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背景与目的:mi R-17-92基因簇与多种疾病的发生密切相关,其在肺癌、肝癌、胃癌和前列腺癌等多种肿瘤细胞中均高表达。本研究利用慢病毒包装系统建立稳定高表达mi R-17-92基因簇的DU145细胞株,探讨mi R-17-92基因簇对前列腺癌DU145细胞的迁移、侵袭能力及对顺铂耐药性的影响。方法:构建高表达mi R-17-92基因簇的表达载体,转染DU145细胞株,同时转染空载体作为对照,并用实时荧光定量聚合酶链反应(real-time fluorescent quantitative polymerase chain reaction,RTFQ-PCR)进行鉴定。用x CELLigence系统监测细胞的迁移、侵袭能力及顺铂处理后的生长情况;通过划痕实验观察细胞的迁移情况;采用蛋白[质]印迹法(Western blot)、凝胶酶谱实验和RTFQ-PCR检测相关蛋白质和基因的表达以探讨mi R-17-92增强DU145细胞的迁移、侵袭能力及对顺铂耐药性的相关机制。结果:DU145-mi R-17-92细胞迁移速率和侵袭能力高于DU145-control细胞(P<0.01)。DU145-mi R-17-92细胞中整合素β1的蛋白质表达水平和基质金属蛋白酶-9(matrix metalloprotein-9,MMP-9)的活性显著高于DU145-control细胞。顺铂处理后,DU145-mi R-17-92细胞的生长速度自12 h起快于DU145-control细胞并呈顺铂耐药性(P<0.01)。细胞外调节蛋白激酶1/2(extracellular regulated protein kinases,ERK1/2)在DU145-mi R-17-92细胞中呈现持续高水平磷酸化,顺铂处理后,其磷酸化水平无明显变化。DU145-mi R-17-92细胞中切除修复互补交叉基因1(excision repair cross complementing1,ERCC1)的m RNA和蛋白质表达水平显著高于DU145-control细胞。结论:高表达mi R-17-92增强了DU145细胞的迁移、侵袭能力,其机制与整合素β1的表达上调及MMP-9活性增强有关。此外,高表达mi R-17-92增强了DU145细胞对顺铂的耐药性,该过程与ERK1/2的磷酸化水平增加和ERCC1的表达水平上调相关。
BACKGROUND & OBJECTIVE: mi R-17-92 gene cluster is closely related to the occurrence of various diseases. It is highly expressed in many tumor cells such as lung cancer, liver cancer, gastric cancer and prostate cancer. In this study, the lentiviral packaging system was used to establish the DU145 cell line with stable and highly expressed mi R-17-92 gene cluster, and to explore the migration and invasion ability of mi R-17-92 gene cluster on the prostate cancer DU145 cell line and its resistance to cisplatin Impact. METHODS: The expression vector of mi R-17-92 gene cluster was constructed and transfected into DU145 cell line. Meanwhile, empty vector was transfected into the vector as a control. Real-time fluorescent quantitative polymerase chain reaction (RTFQ) -PCR). Cell migration, invasion and cell growth after cisplatin treatment were monitored by x CELLigence system. Cell migration was observed by scratch assay. Western blot, gel zymography and RTFQ- PCR to detect the expression of related proteins and genes to explore the mi R-17-92 enhanced DU145 cell migration, invasion and cisplatin resistance mechanisms. Results: DU145-mi R-17-92 cells had higher migration rate and invasiveness than DU145-control cells (P <0.01). The protein expression level of integrin β1 and the activity of matrix metalloprotein-9 (MMP-9) in DU145-mi R-17-92 cells were significantly higher than those in DU145-control cells. After cisplatin treatment, the growth rate of DU145-mi R-17-92 cells was faster than that of DU145-control cells at 12 h (P <0.01). The extracellular regulated protein kinase 1/2 (ERK1 / 2) showed sustained high level of phosphorylation in DU145-mi R-17-92 cells. After cisplatin treatment, its phosphorylation level did not change significantly. The m RNA and protein levels of excision repair cross complementing1 (ERCC1) in DU145-mi R-17-92 cells were significantly higher than those in DU145-control cells. Conclusion: The overexpression of mi R-17-92 enhances the migration and invasion of DU145 cells. The mechanism is related to the up-regulation of integrin β1 and the enhancement of MMP-9 activity. In addition, overexpression of mi R-17-92 enhanced DU145 cell resistance to cisplatin, which is associated with increased ERK1 / 2 phosphorylation and ERCC1 expression.