上／下调miR-21对结肠癌细胞的生物学作用及对西妥昔单抗药物敏感性的影响

Up /down-regulation of miR-21 changes biological function of colon cancer cells and sensitivity to cetuximab

目的:探讨miR-21在结肠癌细胞中的生物学功能及对EGFR单抗西妥昔敏感性的影响。方法:通过慢病毒载体的构建及包装生成上调/下调miR-21的慢病毒LV-miR-21和LV-anti-miR-21并感染人结肠癌RKO细胞,采用qRT-PCR、MTT、非锚定依赖性细胞生长、流式细胞术、CCK-8等技术检测上调/下调miR-21后细胞的miR-21表达水平、细胞增殖、克隆形成能力、细胞凋亡能力及对西妥昔单抗药物敏感性的变化。结果:LV-miR-21与LV-anti-miR-21慢病毒的滴度分别为3.0×1012TU/L和2.0×1012TU/L,将病毒颗粒分别感染人结肠癌RKO细胞,观察绿色荧光,感染效率在80%以上。LV-miR-21组的miR-21表达水平、细胞增殖能力和克隆形成能力均高于LV-anti-miR-21组,差异有统计学意义(P<0.05)。而LV-anti-miR-21组的细胞早期凋亡率及西妥昔单抗对细胞的抑制率均高于LV-miR-21组(P<0.05)。结论:miR-21可促进结肠肿瘤细胞的增殖。下调miR-21可以增加结肠癌细胞对靶向治疗药物西妥昔单抗的敏感性。

AIM： To explore the effects of miR-21 on biological behavior of colon cancer cells and their sensi- tivity to epidermal growth factor receptor monoclonal antibody cetuximab. METHODS： Lentiviral vectors were constructed to generate up- and down-regulations of miR-21 lentiviruses （LV-miR-21 and LV-anti-miR-21, respectively）, and the cor- responding negative control viruses （LV-miR-21 NC and LV-anti-miR-21 NC, respectively） were also constructed. The vi- ruses were used to infect human colon cancer RKO cells. The changes of the miR-21 expression level, the cell prolifera- tion, the colony-forming ability, the cell apoptosis and the sensitivity of the cells to cetuximab were detected by real-time PCR, MT＇I＂ assay, soft agar colony assay, flow cytometry and CCK-8 assay. RESULTS： The lentivirus titers of LV-miR- 21, LV-miR-2 NC, LV-anti-miR-21 and LV-anti-miR-21 NC were 3.0 × 1012 TU/L, 6.0× 1011 TU/L, 2.0 × 1012 TU/L and 8.0 × 10H TU/L, respectively. The infection efficiency was over 80% by the observation of green fluorescence. The miR-21 expression level, the cell proliferation, and the colony-forming ability in LV-miR-21 group were significantly higher than those in LV-anti-miR-21 group. The early apoptotic rate and the inhibitory rate of cetuximab for the cells in LV-anti- miR-21 group were higher than those in LV-miR-21 group. CONCLUSION： miR-21 promotes the proliferation of colon cancer cells. Down-regulation of miR-21 enhances the sensitivity of the colon cancer cells to the targeted therapy drug cetuximab.