miR-221通过调节PTEN影响肺癌细胞对吉非替尼的耐药性

miR-221 changes resistance of lung cancer cells to gefitinib by regulating PTEN

目的:探讨微小RNA-221(miR-221)在人肺癌细胞对吉非替尼耐药中的作用及其相关机制。方法:RT-qPCR检测人肺腺癌吉非替尼敏感细胞PC9和吉非替尼耐药细胞PC9/GR中miR-221的表达;通过脂质体试剂Lipofectamine 2000把miR-221 inhibitor转染入肺癌PC9/GR细胞内,CCK-8法检测细胞对吉非替尼敏感度的变化,Western blot检测第10号染色体缺失的磷酸酶及张力蛋白同源物(PTEN)的蛋白表达。构建含PTEN 3’-UTR的萤光素酶报告载体,验证miR-221对PTEN的靶向调控作用。结果:PC9/GR细胞的miR-221表达水平明显高于PC9细胞(P<0.05)。PC9/GR细胞中PTEN表达水平低于PC9细胞(P<0.05)。转染miR-221 inhibitor后,吉非替尼对PC9/GR细胞的IC_(50)明显降低,PTEN的蛋白表达增加(P<0.05)。萤光素酶活性检测实验显示抑制miR-221表达能增强PTEN的萤光素酶活性(P<0.05)。结论:miR-221可能通过抑制PTEN的表达,增强肺癌细胞对吉非替尼的耐药性。

AIM: To explore the effect of microRNA-221(miR-221) on resistance of lung cancer cells to gefitinib, and to investigate its related mechanism. METHODS: RT-qPCR was used to detect the levels of miR-221 expression between gefitinib-sensitive cell line PC9 and gefitinib-resistant cell line PC9/GR. The PC9/GR cells were transfected with miR-221 inhibitor by Lipofectamine 2000. The drug sensitivity of these cells to gefitinib was determined by CCK-8 assay. The protein expression level of phosphatase and tensin homologue deleted on chromosome ten(PTEN) was determined by Western blot. The 3’-UTR of PTEN was cloned into luciferase reporter vector and its luciferase activity was detected to verify whether miR-221 targets PTEN. RESULTS: The expression level of miR-221 in the PC9/GR cells was significantly higher than that in the PC9 cells(P<0.05). The protein expression level of PTEN in the PC9/GR cells was lower than that in the PC9 cells(P<0.05). The IC50 of gefitinib was significantly reduced in the PC9/GR cells after transfection with miR-221 inhibitor(P<0.05). The protein expression level of PTEN in the cells transfected with miR-221 inhibitor was increased as compared with control group and blank group(P<0.05). Inhibition of miR-221 expression enhanced the enzymatic activity of luciferase reporter vector of PTEN. CONCLUSION: miR-221 enhances the resistance of lung cancer cells to gefitinib by down-regulating the protein expression of PTEN.