慢病毒靶向干扰Cx26抑制人高转移性肝癌HCCLM3细胞增殖及迁移

摘要

目的：探讨缝隙连接蛋白26（connexin 26，Cx26）对人高转移性肝癌 HCCLM3细胞增殖、凋亡、迁移能力的影响。方法构建Cx26干扰慢病毒载体感染HCCLM3细胞，用嘌呤霉素筛选稳定干扰 Cx26表达的靶细胞，通过 Real-time PCR和Western blot鉴定Cx26的mRNA和蛋白表达情况；用“parachute”荧光示踪法检测细胞缝隙连接（gap junc-tion，GJ）功能；用MTT法、流式细胞术、Transwell迁移实验分别检测干扰Cx26对HCCLM3细胞增殖、凋亡、迁移能力的影响。结果 LV-Cx26组的Cx26 mRNA和蛋白表达明显低于LV-NC组和野生组细胞（P＜0.01），并且LV-Cx26组的GJ功能也较LV-NC组和野生组明显减弱（P＜0.01）。干扰Cx26可明显抑制HCCLM3细胞增殖（P＜0.01），促进HC-CLM3细胞凋亡（P ＜0.01），降低其体外迁移能力（P ＜0.01）。结论慢病毒靶向干扰Cx26表达可明显减弱其GJ功能，并抑制高转移性肝癌HCCLM3细胞增殖、迁移能力，促进凋亡，降低其恶性生物学特性。%Aim To investigate effects of Cx26 on pro-liferation,apoptosis,migration of human highly meta-static hepatocellular carcinoma HCCLM3 cells.Meth-ods The HCCLM3 cells were infected with lentiviral vector targeting interference of Cx26 and the stable transfectants were selected by puromycin.The interfer-ence efficiency of Cx26 was detected by Real-time PCR and Western blot.Gap junction function was assessed by “parachute”dye-coupling assay. The effects of Cx26 interference on proliferation,apoptosis,migra-tion of HCCLM3 cells were determined by MTT assay, flow cytometry,transwell migration assay,respective-ly.Results The mRNA and protein expression of Cx26 in LV-Cx26 group was significantly lower than the LV-NC group and wide group (P<0.01),and GJ function in LV-Cx26 group also significantly reduced compared with LV-NC group and wild group (P <0.01 ).The Cx26 interference significantly inhibited the proliferation (P<0.01)),promoted the apoptosis (P <0.01 ),and decreased migration of HCCLM3 cells in vitro (P <0.01 ).Conclusion Lentiviral vector targeting interference Cx26 expression of HC-CLM3 cells can significantly reduce its GJ function,in-hibit the proliferation and migration,promote apopto-sis,and reduce its malignant properties.