摘要
目的研究抑制丝裂原活化蛋白激酶/细胞外信号调节激酶-激酶3(MEKK3)基因表达促进TRAIL诱导人肝癌HCC-9204细胞凋亡的作用。方法构建人MEKK3基因的siRNA重组腺病毒载体,转导人肝癌HCC-9204细胞,以RTPCR和印迹法检测MEKK3 mRNA和蛋白的表达,筛选并建立稳定沉默MEKK3基因表达的人肝癌HCC-9204细胞株,以流式细胞仪检测TRAIL诱导细胞凋亡的情况。结果①成功构建并鉴定表达人MEKK3基因的siRNA重组腺病毒载体。②建立MEKK3基因有效且稳定沉默的人肝癌HCC-9204细胞株。③500 g/LTRAIL处理MEKK3基因沉默的HCC-9204细胞株,细胞凋亡率较野生HCC-9204细胞株显著提高。结论本研究成功建立有效且稳定沉默MEKK3基因表达的人肝癌HCC-9204细胞株模型,初步证实MEKK3基因表达与TRAIL诱导肝癌细胞凋亡的敏感性相关。
Objective To investigate the promoting effect of silencing MEKK3 gene on TRAIL-induced apoptosis of liver cancer HCC-9204 cells, so as to search for a novel treatment for liver cancer. Methods MEKK3 siRNA recombinant adenovirus vector was constructed and transduced into liver cancer HCC-9204 cells. RT-PCR and Western blotting was performed to analyze the suppression of MEKK3 in the transduced cells. Flow cytometry was used to measure TRAIL- induced apoptosis. Results DNA sequencing and RT-PCR confirmed that the MEKK3 siRNA expression adenovirus vectors were successfully constructed. RT-PCR and Western blotting showed that MEKK3 stably and effectively silenced HCC-9204 cells were established. The significant increasing of TRAIL（500 g/L）-induced apoptosis was observed in MEKK3 silenced HCC-9204 cells by flow cytometry analysis. Conclusion The MEKK3 stably and effectively silenced liver cancer HCC-9204 cells were successfully established, the correlation between MEKK3 expression and TRAIL- induced apoptosis was demonstrated. The MEKK3 silenced HCC-9204 cells provide a tool for further investigation of the mechanism.
出处
《世界临床药物》
CAS
2013年第12期720-726,共7页
World Clinical Drug
