MicroRNA-29a靶向PTEN/AKT信号通路调节结直肠癌耐药敏感性

MicroRNA-29a regulate resistance to colorectal cancer via targeting on PTEN/AKT signaling pathway

目的探讨MicroRNA-29a(miR-29a)调控结直肠癌细胞HCT-8对化疗药物5-氟尿嘧啶(5-FU)敏感性,以及miR-29a对PTEN蛋白的靶向作用。方法体外培养结直肠癌细胞株HCT-8和5-FU耐药细胞株HCT-8/FU,分别加入适量5-FU,终浓度为0、12.5、25、50、100、200μg/ml的5-FU分别处理HCT-6和HCT-8/FU,培养48h后,用CCK-8法测定,计算IC50;用RT-PCR法检测两株细胞中miR-29a和PTEN的表达情况。耐药细胞株中转染miR-29a inhibitor,CCK-8检测药物敏感性。通过Targetscan网站预测miR-29a的靶基因,并用双荧光素酶报告基因实验验证。shRNA抑制细胞中PTEN的表达,CCK-8法检测不同浓度5-FU作用HCT-8/FU细胞的IC50;Western blot检测敲低miR-29a时,PTEN、AKT及p-AKT蛋白表达变化。结果HCT-8/FU的IC50显著高于HCT-8细胞,miR-29a在HCT-8/FU细胞中表达上调,但PTEN在HCT-8/FU细胞表达下调。miR-29a inhibitor转染HCT-8/FU细胞后,HCT-8/FU细胞的IC50降低。PTEN是miR-29a的靶基因,PTEN表达下调可升高HCT-8/FU细胞IC50,miR-29a抑制物可促进PTEN表达,并抑制p-AKT蛋白水平。结论抑制miR-29a在耐药结直肠癌细胞中的表达可增加细胞对5-FU的药物敏感性,与PTEN/AKT信号通路有关。

Objective To investigate the effect of MicroRNA-29 a(miR-29 a)on the sensitivity of colorectal cancer cell HCT-8 to chemotherapeutic drug 5-fluorouracil(5-FU)and the relationship between miR-29 a and PTEN proteins.Methods The colorectal cancer cell line HCT-8 and 5-FU resistant cell line HCT-8/FU were cultured in vitro.Then appropriate amounts of 5-FU were added to a final concentration of 0,12.5,25,50,100,200 ug/mL of 5-FU for treatment of HCT-6 and HCT-8/FU respectively,and cell activity was measured by CCK-8 after 48 hours cultivation to calculate IC50.The level of miR-29 a and PTEN were detected by RT-PCR in cell lines.Then,miR-29 a inhibitor were transfected into HCT-8/FU cell,and IC50 of HCT-8/FU with miR-29 a inhibitor was detected by CCK-8 assay.The targeting of miR-29 a was predicted through website,targetscan,and verified by double luciferase assay.shRNA was used to inhibit the expression of PTEN,and IC50 was detected.Western blot was used to detect the expression of PTEN,AKT and p-AKT.Results The IC50 of HCT-8/FU was significantly higher than that of HCT-8 cells,miR-29 a was up-regulated and PTEN was down-regulated in HCT-8/FU cells.After miR-29 a inhibitor was transfected into HCT-8/FU cells,the IC50 of HCT-8/FU cells was decreased.3’UTR of PTEN was demonstrated to bind with miR-29 a detected by luciferase assay,down-regulation of PTEN expression increased the IC50 of HCT-8/FU cells,down-regulation of miR-29 a promoted PTEN expression and inhibited the expression of p-AKT.Conclusion Inhibition of miR-29 a in drug-resistant colorectal cancer cells could increase the drug sensitivity of the cells,which is related to PTEN/AKT signaling pathway.