KAI1基因外源性过表达对胆管癌细胞层黏素受体的影响

Influence of exogenous overexpression of KAI1 on the expression of laminin receptor in human cholangiocarcinoma cell line

目的研究KAI1基因外源性过表达对胆管癌细胞层黏素受体(LNR)表达的影响。方法体外培养人胆管癌QBC939细胞,分为两组:转染空载体pIRES2-EGFP对照组和转染重组真核载体pIRES2-EGFP-KAI1实验组。采用脂质体转染法分别将空载体和重组真核载体转染到QBC939细胞,经G418筛选,得到阳性克隆细胞株。通过RT-PCR、Western blotting和细胞免疫组化法,分别检测KAI1、LNR的mRNA及蛋白表达水平。结果实验组的KAI1 mRNA、蛋白表达量(分别为0.49±0.071、.06±0.05)均显著高于对照组(分别为0.22±0.02、0.59±0.02,P<0.01);实验组的LNR mRNA、蛋白表达量(分别为0.38±0.04、0.29±0.03)均显著低于对照组(分别为0.73±0.05、0.68±0.02,P<0.05)。与对照组比较,实验组KAI1蛋白在胞质的着色明显增强、LNR着色减弱。结论体外胆管癌QBC939细胞过表达KAI1可导致LNR mRNA和蛋白表达下调。

Objective To investigate the effects of exogenous overexpression of KAI1 on laminin receptor（LNR） in human cholangiocarcinoma QBC939 cell line.Methods Human cholangiocarcinoma QBC939 cells were cultured in vitro and divided into two groups：control group（transfecting empty vector,pIRES2-EGFP） and experimental group（transfecting recombinant eukaryotic vector,pIRES2-EGFP-KAI1）.The pIRES2-EGFP and pIRES2-EGFP-KAI1 were transfected into QBC939 cells line with Lipofectamine 2000.After being screened with G418,the mRNA was then extracted and detected by reverse transcription-polymerase chain reaction（RT-PCR） analysis using KAI1 and LNR-specific primers,and an aliquot of protein from each sample was subjected to Western blotting analysis using KAI1 and LNR antibodies.The protein expressions of KAI1 and LNR in two groups were also measured by immunocytochemistry.Results The mRNA and protein levels of KAI1 were significantly higher in experimental group（0.49±0.07 and 1.06±0.05） than in control group（0.22±0.02 and 0.59±0.02,P〈0.01）.On the contrary,The mRNA and protein levels of LNR were significantly lower in experimental group（0.38±0.04 and 0.29±0.03） than in control group（0.73±0.05 and 0.68±0.02,P〈0.05）.As Compared with the control group,the staining in cytoplasm of KAI1 was intensified and LNR diminished after pIRES2-EGFP-KAI1 plasmid was transfected.Conclusion Exogenous overexpression of KAI1 may down-regulate the LNR mRNA and protein expression in QBC939 cells in vitro.