肾癌786-O细胞B4GALNT1基因RNA干扰模型构建与鉴定

Construction and identification of RNA interference targetingβ-1,4-N-acetyl-galactosaminyltransferase 1 on renal cell carcinoma cell line 786-O

目的构建靶向抑制β-1,4-N-乙酰氨基半乳糖转移酶1(B4GALNT1)基因表达的shRNA慢病毒载体,稳定转染至人肾癌细胞株786-O中,观察转染后抑制效率及对786-O细胞增殖活性的影响。方法构建特异性靶向抑制B4GALNT1基因表达的shRNA慢病毒载体(shB4GALNT1),通过琼脂糖凝胶电泳和阳性克隆测序鉴定载体的构建;慢病毒包装并稳定转染至肾癌786-O细胞中,通过荧光显微镜观察shRNA转染细胞;实验分为3组:空白对照(NC)组、转染空载体(VC)组和转染shB4GALNT1(KD)组,分别通过Western blot和实时荧光定量聚合酶链反应来证实转染shB4GALNT1在蛋白与mRNA表达水平的抑制效率;通过Celigo全视野细胞扫描分析抑制B4GALNT1表达对786-O细胞增殖活性的影响。结果琼脂糖凝胶电泳证实阳性克隆为341 bp,阳性克隆测序证实B4GALNT1 shRNA慢病毒表达载体构建成功;绿色荧光蛋白表达证实B4GALNT1 shRNA慢病毒表达载体转染至肾癌786-O细胞中;NC组、VC组和KD组B4GALNT1蛋白的相对表达量分别为1.013±0.057、0.916±0.055、0.340±0.030,KD组与VC组比较差异有统计学意义(P<0.01),B4GALNT1蛋白表达抑制率达63%,NC组与VC组比较差异无统计学意义(P>0.05);NC组、VC组和KD组B4GALNT1 mRNA的相对表达量分别为1.021±0.078、1.002±0.074、0.078±0.027,KD组与VC组比较差异有统计学意义(P<0.01),表达抑制率达92%,NC组与VC组比较差异无统计学意义(P>0.05);转染后第2~5天,抑制B4GALNT1表达可显著降低786-O细胞增殖活性(P<0.01)。结论本实验成功构建RNA干扰靶向抑制B4GALNT1基因的慢病毒表达载体,并稳定转染人肾癌786-O细胞,抑制B4GALNT1表达可显著降低细胞增殖活性。

Objective To construct and validate the effect of RNA interference targetingβ-1,4-N-acetyl-galactosaminyltransferase 1(B4GALNT1)by lentiviral expression vector on human renal cell carcinoma cell line 786-O in vitro,along with inhibition of cell proliferative activity.Methods Human B4GALNT1 sequence was tailored for RNA interference by lentiviral expression vector,which was verified by agarose gel electrophoresis and clone sequencing.Then B4GALNT1 lentiviral expression vector was packaged and followed by transfection into 786-O cells,which was observed by fluorescence microscope.The experiment was divided into 3 groups including negative control(NC),empty vector control(VC)and shB4GALNT1(KD).The protein level of B4GALNT1 expression was measured by western blot.The mRNA level of B4GALNT1 expression was detected by quantitative real time polymerase chain reaction.The cell proliferative activity was counted by Celigo full-field cell scanning analysis.Results Positive clone connected shRNA was 341 bp verified by agarose gel electrophoresis and lentiviral expression vector was constructed successfully validated by positive clone sequencing.Green fluorescence was observed on 786-O cells after transfection by fluorescence microscope.The relative expression level of B4GALNT1 protein in NC,VC and KD group were 1.013±0.057,0.916±0.055 and 0.340±0.030 respectively.The relative expression level of B4GALNT1 mRNA in NC,VC and KD group were 1.021±0.078,1.002±0.074 and 0.078±0.027 respectively.Both protein and mRNA of B4GALNT1 were inhibited markedly in KD group compared with VC group(P<0.01)that inhibition rate was up to 63%and 92%respectively.However,neither protein nor mRNA was changed statistically in VC group compared with NC group(P>0.05).Cell proliferative activity was decreased significantly from Day2 to Day5 after transfection in KD group compared with VC or NC group(P<0.01).Conclusion RNA interference targeting B4GALNT1 by lentiviral expression vector is constructed and identified successfully,and cell proliferative activity is inhibited after transfection with shB4GALNT1 on renal cell carcinoma cell line 786-O in vitro.