目的：探讨靶向葡萄糖调节蛋白78（glucose regulated protein 78， GRP78 ）基因的miRNA真核表达质粒，对人食管癌EC109细胞增殖的影响。 方法： 设计合成4对针对 GRP78 的特异性miRNA干扰序列和1对阴性对照序列，定向克隆至pcDNATM6.2-GW/EmGFP-miR真核表达载体上，构建靶向 GRP78 的miRNA重组质粒：pcDNATM6.2-miR78-1、pcDNATM6.2-miR78-2、pcDNATM6.2-miR78-3、pcDNATM6.2-miR78-4，并通过脂质体法转染至HEK293细胞；杀稻瘟菌素筛选2周形成稳定转染细胞系；荧光显微镜检测转染效率。将筛选出的最佳干扰质粒转染至EC109细胞，采用RT-PCR法检测 GRP78 mRNA的表达，CCK8法检测其对EC109细胞增殖的影响。 结果： 测序结果表明成功构建4种靶向 GRP78 的miRNA重组质粒，经转染和筛选，所有转染后HEK293细胞均有GFP的表达；与未转染组及阴性对照组（pcDNATM6.2-Ctrl）相比，4种干扰质粒组HEK293细胞中 GRP78 mRNA的表达均下降（P<0.05）；干扰效率以转染pcDNATM6.2-miR78-1为最高。pcDNATM6.2-miR78-1转染EC109细胞，与未转染组、pcDNATM6.2-Ctrl组相比，EC109细胞中 GRP78 mRNA的表达明显下降\[（0.38±0.02） vs （1.03±004）、（1.00±0.03），均P<0.05\]。CCK8法检测结果显示，转染 pcDNATM6.2-miR78-1干扰质粒12、24、48、72 h后，EC109细胞的增殖被显著抑制\[（0.028±0.001） vs （0.086±0.010），（0.035±0.003） vs （0.155±0.011），（0.112±0.009） vs （0389±0.008）、（0.169±0.013） vs （0.433±0.009）；均P<0.05\]。 结论： 4对靶向 GRP78 的miRNA表达载体构建成功，其中pcDNATM6.2-miR78-1干扰质粒沉默效果最佳，能有效抑制EC109细胞的增殖。

Objective:To explore the effect of miRNA eukaryotic expression vector targeting glucose regulated protein 78 ( GRP78 ) gene on proliferation of human esophageal carcinoma EC109 cells. Methods: Four pairs of specific miRNA interference sequences targeting GRP78 gene and one pair of negative control sequence were designed and synthesized. The recombined plasmids of miRNA targeting GRP78 , pcDNATM6.2-miR78-1, pcDNATM6.2-miR78-2, pcDNATM6.2-miR78-3 and pcDNATM6.2-miR78-4, were constructed using pcDNATM6.2-GW/EmGFP-miR eukaryotic expression vector, and were transfected into HEK293 cells by lipofectamine. After the treatment with blasticidin for two weeks, the stably transfected HEK293 cells were obtained. The transfection efficiency was observed by fluorescence microscopy. The best interference plasmid was then selected and transfected into EC109 cells. The expression of GRP78 mRNA and the proliferation of EC109 cells were detected by RT-PCR and CCK-8 assay, respectively. Results: Sequencing results indicated that four recombined plasmids of miRNA targeting GRP78 were successfully constructed. After transfection and screening, the expression of GFP was observed in all transfected HEK293 cells. The expression of GRP78 mRNA in HEK293 cells transfected with all the four interference plasmids was decreased (P<0.05), comparing with the untransfected or negative control group (pcDNATM6.2-Ctrl), in which the highest interference efficiency was observed in pcDNATM6.2-miR78-1 group. After pcDNATM6.2-miR78-1 transfection, the expression of GRP78 mRNA in EC109 cells was significantly decreased compared with the untransfected group and the pcDNATM6.2-Ctrl group (\[0.38±0.02\] vs \[1.03±004\], \[1.00±0.03\], P<0.05), respectively. CCK8 assay showed that EC109 cell proliferation was significantly suppressed after pcDNATM62-miR78-1 transfection in 12, 24, 48, 72 h (\[0.028±0.001\] vs \[0.086±0.010\], \[0.035±0.003\] vs \[0.155±0011\], \[0.112±0.009\] vs \[0.389±0.008\], \[0.169±0.013\] vs \[0.433±0.009\]; P<005). Conclusion: Four pairs of miRNA expression vectors targeting GRP78 gene are successfully constructed, in which pcDNATM62-miR78-1 shows the best gene silencing efficiency and efficiently inhibits the proliferation of EC109 cells.

国家自然科学基金资助（No. 30972925）