miR-374a和miR-548b调控非小细胞肺癌对吉非替尼耐药的研究

Research on miR-374a and miR-548b regulate gefitinib resistance in non-small cell lung cancer

目的探讨miR-374a和miR-548调控非小细胞肺癌对吉非替尼耐药的机制。方法 1qRT-PCR检测转染载体miR-Zip-347a或p-miR-548b的HCC827-Gef和Calul细胞中miR-374a或miR-548b的相对表达水平;并检测转染pcDNA-Wnt5a和pcDNA-CCNB1及其对照载体的HCC827-Gef细胞中Wnt5a和CCNB1的相对表达水平。2MTS法检测敲除miR-374a或过表达miR-548b的HCC827-Gef和Calul细胞对吉非替尼的敏感性,并分析过表达蛋白Wnt5a或CCNB1的HCC827-Gef细胞对吉非替尼的敏感性;3荧光素酶报告基因检测共转染相应载体后的HEK293细胞中荧光素酶活性。4蛋白印迹分析转染p-miR-374a、p-miR-548b和p-miR-control的细胞中Wnt5a或CCNB1的表达水平。结果 1非小细胞肺癌细胞Calul和HCC827-Gef中miR-374a表达明显受到抑制或miR-548b过表达时,其对吉非替尼的敏感性显著升高;2HCC827-Gef细胞miR-374a和miR-548b过表达分别显著降低Wnt5a和CCNB1蛋白的表达;HCC827-Gef细胞中Wnt5a过表达可部分逆转非小细胞肺癌细胞对吉非替尼的耐药;转染p-miR-548b的HCC827-Gef细胞中CCNB1的再表达也说明了细胞对吉非替尼的敏感性。结论 miR-374a和miR-548b分别通过靶向Wnt5a和CCNB1基因调控非小细胞肺癌细胞对吉非替尼的敏感性。

Objective To investigate the mechanism of miR-374 a and miR-548 in the regulation of non-small cell lung cancer to gefitinib resistance. Methods ①The expression of miR-374 a and miR-548 b in HCC827-Gef and Calul cells transfected with miR-Zip-374 a or p-miR-548 b were detected by qRT-PCR. And the expression levels of CCNB1 and Wnt5 a in HCC827-Gef cells transfected with pcDNA-Wnt5 a,pcDNA-CCNB1 and their control vectors were also detected by qRT-PCR. ②MTS assay was used to detect the sensitivity of HCC827-Gef and Calul cells knocked down of miR-374 a or overexpressed of miR-548 b to gefitinib. And the sensitivity of HCC827-Gef was also detected by MTS when protein Wnt5 a or CCNB1 were overexpressed. ③Luciferase reporter assay was used to detect the luciferase activity of HEK293 cells after co-transfected with corresponding vector. ④Western blot was used to analyze the expression levels of Wnt5 a or CCNB1 in cells transfected with p-miR-374 a,p-miR-548 b and p-miR-control. Results ①When the expression of miR-374 a was significantly inhibited or miR-548 b overexpressed in Calul and HCC827-Gef cells,their sensitivity to gefitinib was increased. ②Overexpression of miR-374 a and miR-548 b in HCC827-Gef cells separately reduced the expression of Wnt5 a and CCNB1 protein; And the overexpression of Wnt5 a in HCC827-Gef cells could partly reverse the drug resistance of non-small cell lung cancer cells to gefitinib; In the same way,the re-expression of CCNB1 in HCC827-Gef cells transfected with p-miR-548 b also showed the sensitivity of the cells to gefitinib. Conclusion MiR-374 a and miR-548 b regulate the sensitivity of non-small cell lung cancer to gefitinib respectively by targeting Wnt5 a and CCNB1 genes.