CAJ | 학술논문

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MDM23’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AnnexinⅤ／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MDM23’ 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 in?hibitor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MDM23’ UTR 特异结合，调控p53／MDM2通路影响胃癌细胞的增殖及凋亡。
목적：탐토miR?26a／b조공p53／MDM2통로대위암세포조망적영향。방법실시형광정량PCR검측위암세포계MGC803、MKN?45화MKN?28중miR?26a／b적표체수평，형광소매보고계통분석miR?26a／b여MDM23’비번역구（3’ UTR）적결합정황；장화학합성miR?26a／b전체분자mimics화무관서렬전염위암세포MKN?45，화학합성miR?26a／b inhibitor전염위상피세포GES?1후，Western blotting검측MDM2、p53급기하유분자p21화Bcl?2적표체，MTT법검측전염24、48、72화96 h적세포증식정황；통과AnnexinⅤ／PI쌍염검측miR?26a／b대MKN?45세포조망정황。결과여영생화위상피세포GES?1상비，miR?26a／b재종류세포계MGC803、MKN?45화MKN?28중적표체균하조，재MKN?45중표체수평최저；형광소매활성검측현시，miR?26a／b과표체억제MDM23’ UTR보고재체（ Wild）적형광소매활성，이대3’ UTR돌변형보고재체（ Mutation）형광소매활성무명현영향。 miR?26a／b억제MKN?45세포중MDM2표체병증강p53급하유분자표체，이재GES?1세포중억제miR?26a／b가이증강MDM2표체，강저p53급하유분자표체。 MTT결과현시，miR?26a／b억제MKN?45세포적증식，miR?26a／b in?hibitor칙명현촉진GES?1세포적증식。통과AnnexinⅤ／PI검측세포조망발현，miR?26a／b과표체적MKN?45세포적조망솔고우대조세포（ P＜0?01）。결론 miR?26a／b 능여MDM23’ UTR 특이결합，조공p53／MDM2통로영향위암세포적증식급조망。
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 blot?ting. 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 as?say showed that miR?26a/b overexpression significantly inhibited the proliferation of MKN?45 cells, while miR?26a/b inhibitor signifi?cantly promoted the cell proliferation of GES?1 cells. The AnnexinⅤ/PI assay found that the apoptosis rates of MKN?45 which overex?pressed 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.