miR-301在结肠癌细胞增殖和凋亡中的作用

The role of miR-301 in colon cancer cell proliferation and apoptosis

背景与目的:miR-301在多种恶性肿瘤中表达上调,然而其在结肠癌中的表达及功能尚不清楚。本研究旨在检测微小RNA miR-301在结肠癌组织中的表达,并在体内外研究miR-301反义寡核苷酸技术(ASO)对结肠癌细胞增殖和凋亡的影响。方法:运用荧光定量PCR定量分析120例结肠癌患者癌组织及对应癌旁组织中miR-301的表达;通过miR-301ASO降低结肠癌SW620细胞中miR-301的表达,采用MTT、克隆形成实验、流式细胞技术及体内实验观察miR-301ASO对SW620细胞产生的生物学效应。结果:在120例结肠癌患者中,63.33%(76/120)的结肠癌组织miR-301表达明显高于对应癌旁组织(P=0.00);与对照组miR-301的表达量(0.50±0.07)相比,miR-301ASO组可以显著降低miR-301的表达(0.09±0.01,P=0.00);MTT实验结果显示,转染miR-301 ASO组24、48、96 h SW620细胞的存活数量均明显低于对照组(P<0.05);克隆形成实验结果显示,miR-301ASO组克隆形成率(5.33%±0.74%)较对照组(33.33%±8.38%)显著降低(P=0.00);体内研究进一步证实miR-301ASO可以抑制肿瘤细胞的增殖,从而导致肿瘤生长较对照组慢(P=0.01),肿瘤的体积较对照组明显减小(P=0.01);流式细胞仪检测显示,转染miR-301ASO组SW620细胞凋亡指数(15.68±1.46)较随机染转染ASO组(3.36±0.88)明显增高(P=0.02);另外,降低miR-301的表达发现Bcl-2的mRNA和Bcl-2蛋白均明显下降(P=0.00,P=0.00)。结论:miR-301在结肠癌组织中表达上调,降低miR-301的表达可有效抑制结肠癌细胞生长、促进细胞凋亡。miR-301有可能成为结肠癌基因表达调控的新靶点。

Background and purpose： The miR-224 in a variety of malignant tumors is overexpression, however, its expression and function in colon cancer are not clear. The aim of this study was to investigate the expression of miR-301 in colon cancer tissues and demonstrate the regulative effects of miR-301 ASO on the proliferation and apoptosis of colon cancer cell in vitro and in vitro. Methods： The expression of miR-301 in 120 colon cancer tissues and their adjacent tissues was detected by real-time quantitative PCR method. After transfection with miR-301ASO, the biological effects of miR-301 in SW620 cells were measured by MTT assay, the colony formation experiment, flow cytometry and the in vivo experiment. Results： The expression level of miR-301 was found to be overexpressed in 63.33% （76/120） of the colon cancer cases （P〈0.05）. miR-301 expression in SW620 cells （transfection with miR-301 ASO, 0.09±0.01） was significantly less than control group （0.50±0.07, P=0.00）. MTT assay results showed that SW620 cells survived rate at 24, 48 and 96 h decreased greatly after transfection with miR- 301ASO （P=-0.00）. Clone formation assay revealed that miR-301 ASO group colony formation rate （5.33%±0.74%） was significantly lower than the control group （33.33%±8.38%, P=-0.00）. In vivo study further confirmed that miR- 301ASO could inhibit the proliferation of SW620 cells （P〈0.05）, and miR-301ASO group grew substantially slow compared with the negative control group （P=0.00）. Flow cytometry indicated that the apoptotic index in miR-301 ASO group （15.68±1.46） was significantly higher than the control group （3.36±0.88, P=0.02）. In addition, the Bcl2 mRNAand protein were significantly decreased after reduce the expression of miR-301 （P=0.00, P=-0.00）. Conclusion： MiR- 301 was overexpressed in human colon cancer. Reduce the expression of miR-301 can effectively inhibit the growth of colon cancer cells and promote apoptosis. MiR-301 may become a new target for the regulation of gene expression in colon cancer.