miR-26a/b调控p53/MDM2通路对胃癌细胞凋亡的影响

目的 探讨miR-26a/b调控p53/MDM2通路对胃癌细胞凋亡的影响。方法 实时荧光定量PCR检测胃癌细胞系MGC803、MKN-45和MKN-28中miR-26a/b的表达水平，荧光素酶报告系统分析miR-26a/b与MDM2 3’非翻译区（3’UTR）的结合情况；将化学合成miR-26a/b前体分子mimics和无关序列转染胃癌细胞MKN-45，化学合成miR-26a/b inhibitor转染胃上皮细胞GES-1后，Western blotting检测MDM2、p53及其下游分子p21和Bcl-2的表达，MTT法检测转染24、48、72 和96 h的细胞增殖情况；通过Annexin Ⅴ/PI双染检测miR-26a/b对MKN-45细胞凋亡情况。结果 与永生化胃上皮细胞GES-1相比，miR-26a/b在肿瘤细胞系MGC803、MKN-45和MKN-28中的表达均下调，在MKN-45中表达水平最低；荧光素酶活性检测显示，miR-26a/b过表达抑制MDM2 3’UTR报告载体（Wild）的荧光素酶活性，而对3’UTR突变型报告载体（Mutation）荧光素酶活性无明显影响。miR-26a/b抑制MKN-45细胞中MDM2表达并增强p53及下游分子表达，而在GES-1细胞中抑制miR-26a/b可以增强MDM2表达，降低p53及下游分子表达。MTT结果显示，miR-26a/b抑制MKN-45细胞的增殖，miR-26a/b inhibitor则明显促进GES-1细胞的增殖。通过AnnexinⅤ/PI检测细胞凋亡发现，miR-26a/b过表达的MKN-45细胞的凋亡率高于对照细胞（P＜0.01）。结论 miR-26a/b 能与MDM2 3’UTR 特异结合，调控p53/MDM2通路影响胃癌细胞的增殖及凋亡。

The apoptosis effect of miR-26a/b by regulating p53/MDM2 pathway in gastric cancer cells

Objective To investigate the apoptosis effect of miR-26a/b by regulating p53/MDM2 pathway in gastric cancer cells. Methods The expression of miR-26a/b in gastric cell lines （MGC803， MKN-45 and MKN-28） was detected by real-time PCR. The luciferase activity was analyzed to determine the binding of miR-26a/b to MDM2 3’ untranslated region （3’ UTR）. The miR-26a/b precursor molecule mimics and a scramble sequence were transfected into MKN-45 cells， and miR-26a/b inhibitors were transfected into GES-1 cells， respectively. The expression of MDM2， p53 and its downstream genes p21 and Bcl2 were detected by Western blotting. The proliferation rates of these cells were detected by MTT assay after 24， 48， 72 and 96 h. The apoptosis of cells were detected by AnnexinⅤ/PI. Results Compared with GES-1 cells， miR-26a/b was downregulated in gastric cancer cell lines， especially in MKN-45 cells. Luciferase activity analysis showed that the overexpression of miR-26a/b suppressed MDM2 3’UTR reporter vector （Wild） luciferase activity， whereas luciferase activity had no significant change in the 3’UTR mutant reporter vectors. The miR-26a/b inhibited the expression of MDM2 and enhanced the expression of p53 and downstream genes in MKN-45 cells. Inhibition of miR-26a/b enhanced the expression of MDM2， and decreased the expression of p53 and downstream genes in GES-1 cells. The Results of MTT assay showed that miR-26a/b overexpression significantly inhibited the proliferation of MKN-45 cells， while miR-26a/b inhibitor significantly promoted the cell proliferation of GES-1 cells. The AnnexinⅤ/PI assay found that the apoptosis rates of MKN-45 which overexpressed miR-26a/b were significantly higher than control （P＜0.01）. Conclusion miR-26a/b can specifically bind the 3’UTR of MDM2 and disrupt proliferation and apoptosis of gastric cancer cells by regulating p53/MDM2 pathway.