小干扰RNA靶向沉默CXCR2基因抑制肝癌细胞的增殖

Targeted silencing of CXCR2 via small interference RNA for inhibition of liver carcinoma cells proliferation

目的 研究CXCR2-小干扰RNA（siRNA）基因体外抑制肝癌细胞增殖的作用.方法 将CXCR2-siRNA以脂质体转染法转染肝癌细胞hepG2,24h后荧光显微镜观察细胞荧光含量.采用RT-PCR及免疫印迹法分别检测正常肝Chang细胞、未转染肝癌hepG2细胞以及转染CXCR2-siRNA基因后hepG2细胞在转染24、48、72h后的CXCR2 mRNA和蛋白表达水平.以未转染的hepG2细胞为对照,CCK8法检测转染CXCR2-siRNA的hepG-2细胞在转染24、48、72 h后的增殖.以未转染的hepG2细胞为对照,检测转染CXCR2-siRNA 24 h后hepG2细胞与Transwell小室孵育24h时的侵袭能力.结果 CXCR2-siRNA转染hepG2 24 h后镜下可见大量绿色荧光,转染率为80％.hepG2细胞CXCR2mRNA和蛋白表达水平高于Chang细胞（mRNA：1.69±0.22比0.63±0.31,蛋白：1.93±0.25比0.84±0.29,均P＜0.05）.转染CXCR2-siRNA24和48 h后hepG2细胞CXCR2mRNA表达量低于未转染的hepG2细胞（0.75±0.24、0.83±0.21比1.78±0.25,均P＜0.05）,72 h后CXCR2-siRNA虽然仍能抑制hepG2细胞CXCR2mRNA的表达,但与未转染的hepG2细胞相比差异并无统计学意义（1.35±0.18比1.78±0.25,P＞0.05）.转染CXCR2-siRNA24和48 h后的hepG2细胞CXCR2蛋白表达低于未转染的hepG2细胞（0.91±0.25、1.16±0.23比1.98±0.31,均P＜0.05）,转染72 h后蛋白水平有所上升,与对照组相比差异无统计学意义（1.71±0.18比1.98±0.31,P＞0.05）.转染CXCR2-siRNA 24、48、72 h后hepG2细胞mRNA和蛋白表达差异均无统计学意义.与未转染的hepG2细胞相比,转染CXCR2-siRNA 24、48、72 h后hepG2细胞增殖受到抑制（均P＜0.05）.转染CXCR2-siRNA的hepG2细胞对Transwell小室的侵袭能力明显低于未转染的hepG2细胞[（36±5）个腐倍视野（＆#215;200）比（526±9）个府倍视野（＆#215;200）,P＜0.05].结论 siRNA沉默CXCR2基因体外可有效抑制肝癌细胞的增殖.

Objective To investigate the inhibitory effect of siRNA-CXCR2 on in vitro proliferation of hepatocellular carcinoma.Methods CXCR2-siRNA plasmid was transfected by using the liposome technique into hepG2,the hepatocellular carcinoma cells,for the determination of fluorescence under fluorescence microscope at hour 24.The expression of CXCR2 mRNA and protein in the Chang cells derived from normal liver tissues,untransfected hepG2 cells and the hepG2 cells transfected with siRNA-CXCR2 was detected by reverse transcriptase polymerase chain reaction and Western blotting at hours 24,48 and 72,respectively.The CCK8 technique was,by using the untransfected hepG2 cells as control,employed to examine the proliferation of hepG2 cells transfected with CXCR2-siRNA at hours 24,48 and 72.The invasive capacity of the transfected hepG2 cells at hour 24 following transfection and following incubation in a Transwell chamber was assessed respectively compared with hepG2 cells.Results Following transfection,there was a preponderance of green fluorescence （80%） in hepG2 cells at hour 24 under fluorescent microscope and the transfected rate was 80%.Compared with Chang cells,the transfected hepG2 cells yielded a higher levels of CXCR2 mRNA and protein expression （1.69±0.22 vs 0.63 ±0.31 for mRNA and 1.93±0.25 vs 0.84±0.29 for protein,both P〈0.05）.Compared with untransfected hepG2 cells,the transfected cells were associated with a reduced level of CXCR2 mRNA expression at hours 24 and 48 （0.75±0.24 and 0.83±0.21 vs 1.78±0.25,both P〈0.05）.This held true even at hour 72,however,the level of which was not statistically different from that of untransfected cells （1.35 ±0.18 vs 1.78 ±0.25,P〉0.05）.The transfected cells were,compared with untransfected cells,linked to lower level of CXCR2 protein expression at hours 24 and 48 （0.91±0.25 and 1.16±0.23 vs 1.98±0.31,P〈0.05）.Despite that there was an increase in CXCR2 protein expression at hour 72,which was not markedly different from that of untransfected cells （1.71 ±0.18 vs 1.98±0.31,P〉0.05）.Furthermore,there was no statistically significant difference in the mRNA and protein expression in the transfected hepG2 cells at hours 24,48 and 72.However,transfection with CXCR2-siRNA resulted in inhibition of hepG2 cell proliferation at hours 24,48 and 72 compared with untransfected cells （all P〈0.05）.In addition,compared with untransfected cells,the transfeeted cells yielded a markedly attenuated capacity of invasion to the Transwell chamber [（36±5）/HP（×200） vs （526±9）/HP（×200）,P〈 0.05].Conclusion Silencing of CXCR2 genes with siRNA effectively inhibits proliferation of hepatocellular carcinoma cells.