miR-451对结肠癌细胞系SW620生物学行为的影响

Effects of miR-451 on the biological behavior of colorectal carcinoma cell line SW620

目的探讨miR-451对结直肠癌细胞SW620增殖、凋亡及侵袭能力的影响。方法将SW620细胞分为4组:miR-451模拟物(miR-451 mi mics)转染组(细胞转染miR-451 mi mics,终浓度100nmol/L),NC组(细胞转染miRNA阴性对照,终浓度100nmol/L),空白转染组(加入与前2组等量的Lipofectamine 2000,无miRNA片段),对照组(细胞常规培养,不加入Lipofectamine 2000和miR-NA片段)。应用荧光定量RT-PCR检测转染24h后miR-451表达量的改变,采用CCK-8试剂盒检测转染24、48、72h后细胞的增殖能力,流式细胞仪检测转染48h后细胞凋亡情况,Transwell侵袭实验检测转染24h后细胞侵袭能力的改变,细胞免疫荧光和Westernblotting检测转染48h后巨噬细胞迁移抑制因子(MIF)的表达情况。结果转染后24h,荧光定量RT-PCR检测结果显示,miR-451mi mics转染组的miR-451表达量(67.96±13.33)较NC组(以其miR-451表达量作为1)显著上调(P<0.01)。与另外3组比较,miR-451 mi mics转染组细胞增殖能力在转染后48、72h明显受抑(P<0.01),但转染后48h其细胞凋亡率无明显改变(P>0.05)。转染后48h,miR-451 mi mics转染组、NC组、空白转染组、对照组中MIF蛋白表达阳性细胞的比例分别为23.9%±14.9%、53.5%±14.1%、45.2%±19.8%、59.3%±22.2%,统计学分析显示miR-451 mi mics转染组与其他3组比较差异显著(F=7.356,P<0.01)。Transwell侵袭实验显示,miR-451 mi mics转染组侵袭细胞数(37.4±6.1个/视野)明显低于对照组(61.6±8.6个/视野)、NC组(55.6±3.6个/视野)、空白转染组(60.8±7.4个/视野,P均<0.01)。结论上调miR-451表达可抑制结直肠癌细胞的生物活性,该作用可能与抑制MIF表达有关。

Objective To explore the effects of human miR-451 on the proliferation,apoptosis and invasive ability of colon cancer cell line SW620.Methods Four groups were designed in the present experiment,i.e.,cells were transfected with 100nmol/L of miR-451 mimics in miR-451 mimics group,with 100nmol/L of negative control in negative control（NC） group,with Lipo 2000 alone in blank control group,and without transfection of either Lipo2000 or miRNA in control group.The expression of miR-451 was measured by real-time RT-PCR 24h after transfection,and proliferation of cells was examined by using the Cell Counting Kit-8 at respective time of 24,48,72 hours after transfction.The apoptotic rate was assessed by flow cytometry（FCM） and invasive ability was determined by Transwell assay.The effect of miR-451 mimics on the expression of macrophage migration inhibitory factor（MIF） protein was measured by immunofluorescent and Western blot assay.Results Twenty-four hours after transfection,an increase in expression of miR-451 was noted in miR-451 mimics group（67.96±13.33） when compared with NC group,in which the expression of miR-451 was defined as 1（P〈0.01）.In comparison with the other groups,miR-451 mimics group showed a significant inhibition of proliferation at 48 and 72 hours after transfection（P〈0.01）,but no significant change in apoptotic rate was found at 48 hour after transfection（P〉0.05）.Immunofluorescent assay showed that MIF positive rate of miR-451 mimics group（23.9%±14.9%） was decreased when compared with that of NC group（53.5%±14.1%）,blank transfection group（45.2%±19.8%） and blank control group（59.3%±22.2%）（F=7.356,P〈0.01）.Transwell assay illustrated that the invasive ability in miR-451 mimics group（37.4±6.1 invasive cells per field） was lower than that in negative control group（55.6±3.6 invasive cells per field）,blank transfection group（60.8±7.4 invasive cells per field） and blank control group（61.6±8.6 invasive cells per field,P〈0.01）.Conclusion Up-regulation of miR-451 expression may inhibit the bioactivity of colon cancer cells,and it may be attributed to the inhibition of MIF expression.