miR-145-5p通过靶向PAK4促进非小细胞肺癌对吉非替尼的敏感性

miR-145-5p promotes sensitivity to gefitinib in non-small cell lung cancer by targeting PAK4

目的研究miR-145-5p通过靶向p21活化激酶(PAK)4对非小细胞肺癌对吉非替尼的敏感性的作用及机制。方法RT-qPCR检测miR-145-5p mRNA和PAK4 mRNA的表达,利用Lipofectamine2000将miR-NC(miR-NC组)、miR-145-5p mimic(miR-145-5p mimic组)、miR-145-5p inhibitor(miR-145-5p inhibitor组)、pc-PAK4质粒分别或联合转染进入A549/GR细胞,未转染的A549/GR细胞为对照组。噻唑蓝(MTT)法检测细胞活性,流式细胞法检测细胞凋亡,双荧光素酶报告检测靶向关系,Western印迹检测PAK4蛋白相对表达水平。在裸鼠左腋下皮下注射转染过的A549/GR细胞,分为空白组、miR-145-5p mimic组、pc-PAK4组、miR-145-5p mimic+pc-PAK4组。每隔一天给每只小鼠口服吉非替尼(100 mg/kg)。第24天颈椎脱位法处死裸鼠,完整取出皮下肿瘤,电子天平称重,RT-PCR检测小RNA和靶基因表达,TUNEL染色检测凋亡。结果与正常肺细胞BEAS-2B相比,A549和A549/GR细胞中miR-145-5p明显低表达(P<0.01)。与对照组相比,miR-145-5p inhibitor组A549/GR细胞活性从24 h开始明显升高,细胞凋亡率明显降低(P<0.01,P<0.05);miR-145-5p mimic组A549/GR细胞活性从36 h开始明显降低,细胞凋亡率明显升高(P<0.01,P<0.05)。miR-145-5p对野生型PAK4荧光素酶活性有明显下调作用(P<0.01),对突变型PAK4荧光素酶活性没有明显作用(P>0.05)。共转染pc-PAK4明显逆转miR-145-5p高表达对A549/GR细胞作用(P<0.01)。miR-145-5p高表达明显减小A549/GR移植瘤体积,减轻A549/GR移植瘤重量,明显上调miR-145-5p mRNA表达,明显下调PAK4 mRNA表达,明显上升细胞凋亡率;共转染pc-PAK4明显逆转miR-145-5p高表达对A549/GR移植瘤作用(P<0.01)。结论miR-145-5p通过靶向PAK4促进非小细胞肺癌对吉非替尼的敏感性。

Objective To investigate the effect and mechanism of miR-145-5p on the sensitivity of non-small cell lung cancer to gefitinib by targeting p21 activated kinase(PAK)4.Methods The expressions of miR-145-5p mRNA and PAK4 mRNA were detected by RT-qPCR.The miR-NC(miR-NC group),miR-145-5p mimic(miR-145-5p mimic group),miR-145-5p inhibitor(miR-145-5p inhibitor group),and pc-PAK4 plasmids were transfected into A549/GR cells separately or in combination by Lipofectamine2000,untransfected A549/GR cells were used as the control group.Cell activity was detected by thiazole blue(MTT)method,apoptosis was detected by flow cytometry,targeting relationship was detected by dual luciferase report,the relative expression level of PAK4 protein was detected by Western blotting.Nude mice were subcutaneously injected with each transfected A549/GR cell under the left armpit,and divided into blank group,miR145-5p mimic group,pc-PAK4 group,and miR-145-5p mimic+pc-PAK4 group.Gefitinib(100 mg/kg)was administered orally to each mouse every other day.On day 24,nude mice were sacrificed by cervical dislocation,and the subcutaneous tumors were completely removed and weighed.The expression of small RNA and target genes was detected by RT-PCR,and apoptosis was detected by TUNEL staining.Results Compared with normal lung cells BEAS-2B,miR-145-5p expression was lower in A549 and A549/GR cells(P<0.01).Compared with the control group,the activity of A549/GR cells in the miR-145-5p inhibitor group was significantly increased from 24 h,and the apoptosis rate was significantly decreased(P<0.01,P<0.05);the activity of A549/GR cells in the miR-145-5p mimic group was significantly decreased from 36 h,and the apoptosis rate was significantly increased(P<0.01,P<0.05).miR-145-5p significantly down-regulated the activity of wild-type PAK4 luciferase(P<0.01),and had no significant effect on the activity of mutant PAK4 luciferase(P>0.05).Co-transfection of pc-PAK4 significantly reversed the effect of miR-145-5p overexpression on A549/GR cells(P<0.01).The high expression of miR-145-5p significantly reduced the volume of A549/GR transplanted tumors,reduced the weight of A549/GR transplanted tumors,significantly up-regulated miR-145-5p mRNA expression,significantly down-regulated PAK4 mRNA expression,and significantly increased the rate of apoptosis;co-transfected pc-PAK4 significantly reversed the effect of miR-145-5p overexpression on A549/GR xenografts(P<0.01).Conclusions miR-145-5p could promote the sensitivity of non-small cell lung cancer to gefitinib by targeting PAK4.