CAJ | 학술논문

目的构建人DNA依赖蛋白激酶催化亚基(DNA-PKcs)基因RNA干扰慢病毒载体,观察在非小细胞肺癌细胞株中沉默DNA-PKcs对C225诱导的表皮生长因子受体(EGFR)核转位及C225抑制细胞增殖的影响.方法构建慢病毒干扰载体(LV-si-DNA-PKcs)感染A549细胞.荧光定量聚合酶链反应(FQ-PCR)检测DNA-PKcs mRNA表达;Western blot检测DNA-PKcs和细胞核EGFR蛋白的表达.细胞计数试剂盒(CCK-8)法检测C225对细胞的增殖抑制作用.结果 LV-si-DNA-PKcs感染的A549细胞见绿色荧光,感染率达95％以上;LV-si-DNA-PKcs有效干扰DNA-PKcs蛋白及mRNA表达水平(P＜0.05).加C225前未见细胞核EGFR表达.C225处理1h,LV-si-DNA-PKcs细胞株见细胞核EGFR表达,持续用药4、24 h细胞核EGFR表达无明显增加;在LV-si-control细胞株及空白组,C225处理1、4、24h后细胞核EGFR表达逐渐增多.C225处理48 h后,LV-si-DNA-PKcs细胞株的细胞存活率开始低于其他2组细胞,72 h细胞存活率明显降低[(40.04±2.89)％比(55.82±7.11)％、(52.67±1.43)％,F=9.392,P＜0.01].结论抑制A549细胞中DNA-PKcs表达,在一定程度上抑制了C225诱导的EGFR细胞核转位,增强C225对细胞的增殖抑制作用.
목적 구건인DNA의뢰단백격매최화아기(DNA-PKcs)기인RNA간우만병독재체,관찰재비소세포폐암세포주중침묵DNA-PKcs대C225유도적표피생장인자수체(EGFR)핵전위급C225억제세포증식적영향.방법 구건만병독간우재체(LV-si-DNA-PKcs)감염A549세포.형광정량취합매련반응(FQ-PCR)검측DNA-PKcs mRNA표체;Western blot검측DNA-PKcs화세포핵EGFR단백적표체.세포계수시제합(CCK-8)법검측C225대세포적증식억제작용.결과 LV-si-DNA-PKcs감염적A549세포견록색형광,감염솔체95％이상;LV-si-DNA-PKcs유효간우DNA-PKcs단백급mRNA표체수평(P＜0.05).가C225전미견세포핵EGFR표체.C225처리1h,LV-si-DNA-PKcs세포주견세포핵EGFR표체,지속용약4、24 h세포핵EGFR표체무명현증가;재LV-si-control세포주급공백조,C225처리1、4、24h후세포핵EGFR표체축점증다.C225처리48 h후,LV-si-DNA-PKcs세포주적세포존활솔개시저우기타2조세포,72 h세포존활솔명현강저[(40.04±2.89)％비(55.82±7.11)％、(52.67±1.43)％,F=9.392,P＜0.01].결론 억제A549세포중DNA-PKcs표체,재일정정도상억제료C225유도적EGFR세포핵전위,증강C225대세포적증식억제작용.
Objective Intrinsic and acquired resistances to cetuximab (C225),the anti-epidermal growth factor receptor (EGFR) monoclonal antibody,limit its clinical application.After nuclear translocation,EGFR combines with DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and then functions as a co-transcriptional and regulatory factor to enhance resistance to C225.This study was to discuss the effect of Lentivirus-mediated small interfering RNA (siRNA) interference targeting DNA-PKcs on EGFR translocation induced by C225 and cellular proliferation inhibition of C225 in lung cancer cell line.Methods The lentiviral vector expressing DNA-PKcs small interfering RNA (LV-si-DNA-PKcs) was constructed to suppress DNA-PKcs expression in A549 cell line.The targeted gene expression was detected by real-time fluorescent quantitative polymerase chain reaction (FQ-PCR).The contents of DNA-PKcs and EGFR were measured by Western blotting.Cell counting kit-8 (CCK-8) was used to determine the inhibitory effect of C225 on cell proliferation at different time points.Results A549 cells infected with LV-siDNA-PKcs were GFP-positive and the delivery efficiency was more than 95％.After lentivirus mediated siRNA transfection,the DNA-PKcs expressions on mRNA and protein levels in LV-si-DNA-PKcs infected cells were significantly reduced compared with control (LV-si-control) and blank groups (P ＜ 0.05).Before C225 treatment,there were no nuclear EGFR expressions in the 3 group cells.After 1 hour C225 treatment,nuclear EGFR expression increased in LV-si-DNA-PKcs infected cells,but it kept stable after 4 and 24 hours.In control and blank groups,nuclear EGFR expressions continued rising after C225 treatment.With C225 treatment for 48 hours,cell survival rate in LV-si-DNA-PKcs infected cells began falling and significantly decreased in 72 hours compared to other groups [(40.04 ± 2.89) ％ vs.(55.82 ± 7.11) ％ and (52.67 ±1.43)％,F=9.392,P＜0.01].Conclusion Our findings suggest that suppression of DNA-PKcs gene expression in A549 cell line may reduce nuclear EGFR expression induced by C225 and enhance the inhibitory effect of C225 on cell proliferation.