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目的:研究前列腺特异转录因子NKX3.1对Dicer1基因表达的影响。方法:转染pcDNA3.1-NKX3.1至前列腺癌PC3细胞,通过G418筛选,有限稀释法克隆培养获得NKX3.1稳定转染的PC3细胞-PC3(+);采用基因芯片检测NKX3.1对前列腺癌PC3细胞基因组表达谱的影响。进一步应用RT-PCR、Western blot-ting进行验证。为进一步检测Dicer1表达增高、促进microRNA的成熟作用,构建了microRNA let-7a-1靶序列-报道基因融合质粒,转染PC3细胞和PC3(+)细胞,通过报道基因检测成熟microRNA let-7a-1对其靶序列的作用。结果:基因组芯片显示,NKX3.1稳定表达的PC3(+)细胞中,Dicer1表达明显高于PC3细胞。RT-PCR、Western blotting结果验证PC3(+)细胞中Dicer1mRNA和蛋白质的表达水平均高于PC3细胞。microRNA let-7a-1靶序列-报道基因融合质粒,转染PC3细胞和PC3(+)细胞后,报告基因检测,PC3(+)细胞中荧光素酶活性明显低于PC3细胞。结论:NKX3.1可上调前列腺癌PC3细胞中Dicer1的表达,促进microRNA的成熟及作用。
Objective: To study the effect of prostate specific transcription factor NKX3.1 on Dicer1 gene expression. Methods: pcDNA3.1-NKX3.1 was transfected into prostate cancer PC3 cells and cloned by G418 and limited dilution method to obtain PC3 cells stably transfected with NKX3.1-PC3 (+). The gene chip was used to detect NKX3.1 Effect of prostate cancer PC3 cell genome expression profile. Further use of RT-PCR, Western blot-ting validation. To further detect the increased expression of Dicer1 and enhance the maturation of microRNAs, a microRNA let-7a-1 target sequence-reporter fusion plasmid was constructed and transfected into PC3 cells and PC3 (+) cells. The expression of mature microRNA let-7a- 1 on its target sequence. Results: Genomic chip showed that the expression of Dicer1 was significantly higher in PC3 (+) cells stably expressing NKX3.1 than in PC3 cells. The results of RT-PCR and Western blotting showed that the expression of Dicer1 mRNA and protein in PC3 (+) cells was higher than that in PC3 cells. After transfected with PC3 cells and PC3 (+) cells, the reporter gene was detected and the luciferase activity in PC3 (+) cells was significantly lower than that in PC3 cells by microRNA let-7a-1 target sequence-reporter fusion plasmid. Conclusion: NKX3.1 up-regulates the expression of Dicer1 in PC3 cells and promotes the maturation of microRNAs.