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目的转录因子Kaiso是BTB/POZ蛋白家族的重要成员,能够调控多种重要的侵袭、增殖相关基因的表达。本研究拟通过shRNA(smallhairpinRNA)技术干扰Kaiso蛋白的表达,观察Kaiso对肺癌细胞系增殖和侵袭等生物学行为的影响。方法运用shRNA重组质粒转染肺癌细胞系LTE和SPC,特异性下调Kaiso的表达,免疫荧光观察核内Kaiso的表达情况,四甲基偶氮唑盐(MTT)和体外基质胶侵袭(Transwell)实验分别检测肺癌细胞系的增殖和侵袭能力,RT-PCR方法检测Kaiso下游靶基因Matrilysin的转录水平。结果转染shRNA质粒特异性地下调了Kaiso的mRNA和蛋白表达(P<0.05),LTE和SPC细胞核内Kaiso的荧光信号显著减弱甚至消失,肺癌细胞的增殖活性和侵袭能力明显增强,Matrilysin基因的转录水平显著上调。结论细胞核内Kaiso蛋白的表达下调,通过调控重要靶基因的表达,影响肺癌细胞系的增殖和侵袭能力。
The target transcription factor Kaiso is an important member of BTB / POZ protein family and regulates a variety of important invasion and proliferation related gene expression. This study intends to interfere with the expression of Kaiso protein by shRNA (smallhairpinRNA) technology to observe the biological effects of Kaiso on the proliferation and invasion of lung cancer cell lines. Methods shRNA recombinant plasmids were transfected into lung cancer cell lines (LTE and SPC) to down-regulate the expression of Kaiso. The expression of Kaiso in the nucleus was observed by immunofluorescence. MTT assay and Transwell assay The proliferation and invasion ability of lung cancer cell lines were detected respectively. The transcription level of Matrilysin, a target gene of Kaiso, was detected by RT-PCR. Results The transfected shRNA plasmid specifically down-regulated the mRNA and protein expression of Kaiso (P <0.05). Fluorescence signals of Kaiso in LTE and SPC cells significantly decreased or even disappeared, and the proliferative activity and invasiveness of lung cancer cells were significantly enhanced. The expression of Matrilysin gene Transcriptional levels were significantly up-regulated. Conclusions The expression of Kaiso protein in the nucleus is down-regulated and the proliferation and invasion ability of lung cancer cell lines are affected by regulating the expression of important target genes.