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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-374a and miR-548 regulating gefitinib resistance in non-small cell lung cancer. Methods The relative expression levels of miR-374a or miR-548b in HCC827-Gef and Calul cells transfected with miR-Zip-347a or p-miR-548b were detected by qRT-PCR. The expression of pcDNA-Wnt5a and pcDNA-CCNB1 And its control vector, relative expression levels of Wnt5a and CCNB1 in HCC827-Gef cells. The sensitivity to Gefitinib of knockdown of miR-374a or HCC827-Gef and Calul cells overexpressing miR-548b was detected by the 2MTS assay and the sensitivity of gentamicin to HCC827-Gef cells overexpressing the protein Wnt5a or CCNB1 was analyzed Luciferase reporter gene luciferase reporter gene co-transfected vector HEK293 cells luciferase activity. 4 Western blotting was used to analyze the expression levels of Wnt5a or CCNB1 in cells transfected with p-miR-374a, p-miR-548b and p-miR-control. Results 1 The expression of miR-374a was significantly inhibited in non-small cell lung cancer cells Calul and HCC827-Gef or the sensitivity to gefitinib was significantly increased when miR-548b was overexpressed. The expression of miR-374a and miR in 2HCC827-Gef cells -548b overexpression significantly decreased the expression of Wnt5a and CCNB1, respectively. Overexpression of Wnt5a partially reversed the resistance of gefitinib to non-small cell lung cancer cells in HCC827-Gef cells. Overexpression of HCC827-Gef Re-expression of CCNB1 in cells also demonstrates the sensitivity of the cells to gefitinib. Conclusion miR-374a and miR-548b regulate the sensitivity of non-small cell lung cancer cells to gefitinib by targeting Wnt5a and CCNB1 genes, respectively.