miRNA-93对结肠癌HT-29细胞株化疗敏感性的影响

目的 探讨miR-93与结肠癌HT-29细胞株对氟尿嘧啶（5-FU）敏感性的影响及可能机制。方法 miR-93抑制物转染HT-29细胞，同时设置空白对照组和阴性对照组（NC），采用MTT法检测细胞增殖活性。流式细胞术、Transwell细胞侵袭实验和划痕实验分别检测miR-93抑制物组、空白对照组和NC组HT-29细胞凋亡、侵袭和迁移。双荧光素酶报告实验验证PTEN与miR-93的关系。采用实时荧光定量PCR（QPCR）检测3组细胞miR-93表达，以及对p-VEGFR2、p-Akt、PTEN mRNA表达的影响。结果 miR-93抑制物组中miR-93的相对表达量为0.40±0.05，显著低于空白对照组和NC组的1.13±0.08、1.12±0.09，差异有统计学意义（P＜0.05）。经1、4、16、64、256 μg／ml的5-FU处理后，miR-93抑制物组HT-29细胞的增殖抑制率高于同浓度下NC组和空白对照组，差异有统计学意义（P＜0.05）。经16 μg／ml 5-FU处理后，miR-93抑制物组HT-29细胞凋亡率、侵袭率和愈合率分别为（52.75±7.44）％、（45.76±15.85）％、（12.64±3.29）％，与空白对照组和NC组比较，差异有统计学意义（P＜0.05）。双荧光素酶报告实验分析PTEN可能是miR-93的下游靶基因。miR-93抑制物组p-VEGFR2、p-Akt和PTEN表达水平分别为1.28±0.09、0.85±0.08、1.22±0.09，与空白对照组和NC组比较，VEGFR2和Akt磷酸化水平下调，PTEN表达上调。结论 下调miR-93可以增加PTEN的表达，抑制VEGFR-2和Akt磷酸化水平，从而提高结肠癌细胞对5-FU的敏感性。

Effect of miRNA-93 on chemosensitivity of colon cancer cell line HT-29

Objective To investigate the effect of miR-93 and the sensitivity of colon cancer HT-29 cells on 5-FU and its possible mechanism. Methods miR-93 inhibitor was transfected into HT-29 cells， and blank control group and negative control group （NC） were also set up. Cell proliferation was detected by MTT assay. The apoptosis， invasion and migration activities of HT-29 cells in the miR-93 inhibitor group， blank control group and NC group were detected by flow cytometry， Transwell cell invasion test and scratch test respectively. Double luciferase reporter gene method was used to verify the relationship between PTEN and miR-93. Real-time fluorescence quantitative PCR （QPCR） was used to detect the expression of miR-93 and the effect on the expression of p-VEGFR2， p-Akt and PTEN mRNA in the three groups. Results The relative expression of miR-93 in the miR-93 inhibitor group was 0.40±0.05， significantly lower than that in the blank control group and NC group （1.13±0.08，1.12±0.09）， and the difference was statistically significant （P＜0.05）. The proliferation inhibition rate of HT-29 cells treated with 1， 4， 16， 64， 256 μg／ml 5-FU in miR-93 inhibitor group was higher than that in the NC group and the blank control group （P＜0.05）. The apoptosis rate， invasion rate and healing rate of HT-29 cells treated with 16 μg／ml 5-FU in the inhibitor group were （52.75 ±7.44）％， （45.76 ±15.85）％， （12.64 ±3.29）％ respectively， which were significantly different from those in the control group and NC group （P＜0.05）. PTEN might be a target protein of miR-93 which was verified by the dual luciferase reporter gene system. The expression levels of p-VEGF R2， p-Akt and PTEN in HT-29 cells treated with inhibitor of miR-93 were 1.28±0.09，0.85±0.08 and 1.22±0.09， respectively. Compared with control group and NC group， the phosphorylation levels of VEGFR2 and Akt were down-regulated and the expression of PTEN was up-regulated. Conclusion Down-regulation of miR-93 can increase the expression of PTEN， inhibit the phosphorylation of VEGFR-2 and Akt， and thus enhance the sensitivity of colon cancer cells to 5-FU.