目的 观察miR-200c对人胃癌细胞增殖能力的影响,并探讨其是否通过靶向调控DNA甲基化转移酶(DNMT3B)表达发挥作用.方法 采用MTT法检测miR-200c对人胃癌MGC-803细胞生长增殖能力的影响;构建DNMT3B 3'UTR-荧光素酶报告载体,通过荧光素酶报告载体系统观察miR-200c对DNMT3B 3'UTR-荧光素酶活性的影响;将miR-200cmimics转染胃癌细胞MGC-803,采用Western blot检测DNMT3B表达水平.结果 MTT结果显示,在转染miR-200c mimics 24、48、72 h后,细胞增殖活性OD值分别为0.31±0.01、0.47±0.01、0.53 ±0.02,与对照组的0.44±0.03、0.62±0.01、0.87 ±0.01比较,P均<0.05;荧光素报告载体系统证实,DNMT3B是miR-200c直接调控的靶基因.Western blot结果显示,miR-200c可抑制DNMT3B蛋白的表达.结论 miR-200c通过靶向调控DNMT3B的表达而抑制胃癌细胞生长增殖能力.
Objective To observe effects the of miR-200c on the proliferation of gastric carcinoma cells and to investi- gate whether miR-200c suppresses cell proliferation by targeting DNA-methyhransferase (DNMT3B). Methods The cell proliferation of human gastric carcinoma cells MGC-803 treated by miR-200c was detected by MTT. DNMT3B 3'UTR-lucif- erase vector was constructed, and luciferase reporter gene assay was employed to examine the effect of miR-200c on lucifer- ase activity. MGC-803 ceils were transfected with miR-200c mimics, and Western blotting was applied to detect the expres- sion levels of DNMT3B protein. Results After transfection of miR-200c mimics for 24 h, 48 h and 72 h respectively, Mq'T assay showed the OD of cell proliferation activity was 0.31 + 0.01,0.47 _+ 0.01 and 0.53 _+ 0.02, while that in the control group was 0.44 _+ 0.03, 0.62 + 0.01 and 0.87 + 0.01 ; the differences were statistically significant ( P 〈 0.05 ). Luciferase reporter vector system confirmed that DNMT3B was a target gene of miR-200c ; Western blotting showed that the expression of DNMT3B protein was inhibited by miR-200c. Conclusion miR-200c suppresses cell proliferation by target regulating the expression of DNMT3B in gastric carcinoma.