论文部分内容阅读
目的:通过研究VTCN1在鼻咽癌细胞中的表达情况,采用慢病毒载体靶向沉默VTCN1基因体外对人鼻咽癌细胞的抑制作用。方法:通过RT-PCR技术和蛋白质免疫印迹法,检测人鼻咽癌细胞株CNE1,HONE-1,NP65和HNE2的VTCN1表达情况,筛选出VTCN1表达量最多的细胞株,进行后续的实验。用脂质体lipofectamine2000将沉默VTCN1的慢病毒载体转染高表达VTCN1的HNE2细胞,筛选出稳定细胞株;采用RT-PCR技术和蛋白质免疫印迹法检测VTCN1ShRNA对VTCN1基因的靶向沉默效率;ckk-8、基质胶侵袭实验检测VTCN1沉默后HNE2细胞增殖和侵袭的影响;蛋白质免疫印迹实验检测VTCN1沉默后,JAK/STAT通路主要蛋白的磷酸化水平的变化。结果:VTCN1靶向沉默的细胞VTCN1表达受到显著抑制;RT-PCR和蛋白质免疫印迹法检测发现稳定转染ShRNA后,VTCN1的mRNA表达水平和蛋白表达水平均明显下降(P<0.05);转染VTCN1ShRNA后HNE2细胞的增殖和侵袭能力均明显降低(P<0.05);VTCN1沉默后,JAK、STAT磷酸化水平明显下降(P<0.05)。结论:VTCN1ShRNA能有效沉默VTCN1的表达,显著抑制HNE2细胞的增殖和侵袭,推测其与下调JAK/STAT信号通路蛋白的活化相关。
OBJECTIVE: To study the inhibitory effect of lentiviral vector targeting VTCN1 gene on human nasopharyngeal carcinoma cells in vitro by studying the expression of VTCN1 in nasopharyngeal carcinoma cells. Methods: The expression of VTCN1 in human nasopharyngeal carcinoma cell lines CNE1, HONE-1, NP65 and HNE2 was detected by RT-PCR and Western blotting, and the cell lines with the highest expression of VTCN1 were screened out for further experiments. The lentiviral vector with VTCN1 silencing was transfected into VTCN1-expressing HNE2 cells by Lipofectamine 2000 to screen stable cell lines. The targeting silencing efficiency of VTCN1ShRNA to VTCN1 gene was detected by RT-PCR and Western blotting. The expression of ckk- Matrigel invasion assay was used to detect the effect of VTCN1 silencing on the proliferation and invasion of HNE2 cells. Western blotting was used to detect the changes of the phosphorylation level of major proteins in JAK / STAT pathway after VTCN1 silencing. Results: The expression of VTCN1 in VTCN1 silencing cells was significantly inhibited. RT-PCR and Western blotting showed that VTCN1 mRNA and protein expression were significantly decreased (P <0.05) VTCN1ShRNA HNE2 cells proliferation and invasion were significantly reduced (P <0.05); VTCN1 silence, JAK, STAT phosphorylation levels were significantly decreased (P <0.05). Conclusion: VTCN1ShRNA can effectively silence the expression of VTCN1 and significantly inhibit the proliferation and invasion of HNE2 cells, suggesting that VTCN1ShRNA is associated with the down-regulation of JAK / STAT signaling pathway.