miR-29a抑制胃癌细胞内靶基因VEGF-A的表达及意义

Suppression of target gene VEGF-A expression in gastric cancer cells by miR-29a and its significance

目的验证胃癌细胞株SGC-7901内miR-29a对靶基因VEGF-A的直接调控作用。方法采用负载miR-29a的腺病毒载体(Ad-miR29a)感染人胃癌细胞株SGC-7901,上调SGC-7901细胞内miR-29a的表达丰度后,采用RT-qPCR及Western blot法分别检测SGC-7901细胞内VEGF-A在mRNA及蛋白水平的表达;进一步采用双荧光素酶实验及突变实验,验证miR-29a是否通过结合在VEGF-A 3'UTR而直接抑制靶基因表达。结果与感染阴性对照Ad-LacZ的SGC-7901细胞相比,感染Ad-miR29a的SGC-7901细胞内VEGF-A蛋白表达下降(0.42±0.02 vs.0.91±0.03,P<0.01),且VEGF-A mRNA水平亦下调(0.75±0.21 vs.1.15±0.25,P<0.05);荧光素酶实验显示,与阴性对照相比,转染miR-29a mimic后,HEK293细胞萤火虫荧光素酶活性明显下降(0.56±0.13 vs.0.93±0.06,P<0.05);而在VEGF-A 3'UTR与miR-29a的结合位点被突变之后,HEK293细胞萤火虫荧光素酶活性得以恢复。结论 miR-29a通过结合于VEGF-A 3'UTR相应位点,直接抑制VEGF-A在mRNA及蛋白水平的表达。miR-29a可能成为胃癌基因治疗的有效靶标。

Objective To identity the direct control effect of miR-29a in gastric cancer cell strain SGC-7901 on target gene VEGF-A. Methods Adenovirus vector Ad-miR29a which was loaded with miR-29a was used to infect the human gastric cancer cell strain SGC-7901. After the up-regulation of the miR-29a expression abundance in SGC-7901 cell, RT-qPCR and Western blot assay were used to examine the expression of VEGF-A in SGC-7901 cells. Furthermore, dual luciferase experiment and mutation experiment were carried out to identify whether miR-29a inhibited directly the expression of target gene through the combination with VEGF-A 3＇ UTR. Results The protein expression level of VEGF-A decreased in the SGC-7901 cells infected with Ad-miR29a compared with the level in those with negative infection with Ad-LacZ （0.42 ± O. 02 vs. O. 91 ± 0.03, P 〈 O. 01 ）, and the mRNA level of VEGF-A also down-regulated （0.75 ± 0.21 vs. 1.15± 0.25,P 〈 0.05 ）. The luciferase experiment indicated that compared with the negative control group, the activity of firefly lueiferase in HEK293 cells decreased significantly after the transfeetion of miR-29a mimic（0.56 ± 0.13 vs. 0.93 ± 0.06, P 〈 0.05 ）. After the binding site of VEGF-A 3 UTR and miR-29a was mutated, the activity of firefly luciferase in HEK293 cell was recovered. Conclusion Through the combination to the relative sites of VEGF-A 3 ＇UTR, miR-29a can directly inhibit the expression of VEGF-A in mRNA and the protein level. MiR-29a may become an effective target in the gene therapy for gastric cancer.