基于LncRAN-HOTAIR探究益肺解毒方调控A549/DDP细胞耐药的机制

The mechanism of regulation of A549/DDP resistance by Yifei Jiedu Decoction was investigated based on LncRAN-HOTAIR

目的探讨益肺解毒方(YFJDF)作用于LncRAN-HOTAIR逆转人肺腺癌A549/DDP细胞耐药的机制。方法在研究过程中,采用通过血清药理学CCK8检测益肺解毒方对A549/DDP细胞的增殖抑制作用和IC_(50)效应;利用siRNA干扰技术和Lipofectamine 2000将HOTAIR-siRNA转染人肺腺癌A549/DDP细胞,并通过RT-PCR验证转染;随后借助流式细胞术检测确定细胞凋亡率;使用Western bloting法测量BCL-2、Bax的表达情况。结果在A549/DDP抑制率方面,益肺解毒方中剂量组(YFJDFM)为62%。与顺铂联合后,对A549/DDP的IC_(50)从45.091μg/ml降至4.89μg/ml。YFJDF组、DDP组、YFJDF+DDP组LncRNA-HOTAIR的相对表达量分别为(0.76±0.02)、(0.88±0.01)、(0.68±0.02),低于NC组(0.99±0.04)(P<0.01)。实验设定3个LncRNA-HOTAIR感染序列并与空白组(Control组)进行比较,转染后,予以PCR验证,验证结果显示siRNA2组LncRNA-HOTAIR的RNA相对表达量最低,并将siRNA2用于后续实验。YFJDF、siHOTAIR、YFJDF+siNC组中,各自对应的A549/DDP细胞凋亡率分别是24.08%、34.53%、46.72%,高于NC组(1.8±0.09),P<0.01;与siNC组(34.5±34.02)相比,YFJDF+siNC组的数值更高(P<0.01);YFJDF+siHOTAIR组A549/DDP的细胞凋亡率(29.89±3.92)与siHOTAIR组比较,P>0.05。YFJDF组、siHOTAIR组的BCL-2相对表达量(1.09±0.05)、(0.89±0.05),低于NC组(1.42±0.08),P<0.01;YFJDF+siNC组BCL-2的相对表达量(1.07±0.05),低于siNC组(1.38±0.05),P<0.01;YFJDF+siHOTAIR组BCL-2的相对表达量(0.85±0.21),与siHOTAIR组比较,P>0.05。YFJDF组和siHOTAIR组的Bax相对表达量分别为(0.57±0.05)、(0.81±0.06),高于NC组,P<0.01;与siNC组(0.37±0.05)相比,YFJDF+siNC组Bax(0.62±0.03)更高(P<0.05);YFJDF+siHOTAIR组Bax的相对表达量(0.78±0.21),与siHOTAIR组比较,差异无统计学意义(P>0.05)。结论益肺解毒方通过下调LncRNA-HOTAIR的表达,促进Bax表达,抑制BCL-2表达,进而诱导A549/DDP细胞凋亡,是逆转A549/DDP耐药的机制之一。

Objective To investigate whether Yifei Jiedu Decoction(YFJDF)can act on LncRAN-HOTAIR to reverse the drug resistance of human lung adenocarcinoma A549/DDP cell.Methods By serum pharmacology,CCK8 detected the proliferation inhibitory and IC_(50) effect of Yifei Jiedu Decoction on A549/DDP cell.HOTAIR-siRNA was transfected to human lung adenocarcinoma A549/DDP cell by Lipofectamine 2000 with siRNA interference technology,and it was verified by RT-PCR;Then the apoptosis rate was determined by flow cytometry;The expression of BCL-2 and Bax was measured by Western blotting.Results The proliferation inhibition rate of A549/DDP cells in Yifei Jiedu Decoction medium-dose group was 62%.After combined with cisplatin,the IC_(50) of cisplatin on A549/DDP cells decreased from 45.091 ug/ml to 4.89 ug/ml.The relative expression of LncRNA-HOTAIR in YFJDF group,DDP group and YFJDF+DDP group was(0.76±0.02)、(0.88±0.01)and(0.68±0.02),which were lower than that in NC group(0.99±0.04),P<0.01.Three LncRNA-HOTAIR infection sequences were set and compared with the Control group.PCR verification was conducted after transfection,and the verification results showed that siRNA2 group had the lowest RNA relative expression level,and siRNA2 was used in subsequent experiments.The apoptosis rates of A549/DDP cells in YFJDF group,siHOTAIR group,YFJDF+siNC group were 24.08%、34.53%、46.72%,which were higher than those in NC group(1.8±0.09),P<0.01.The apoptosis rate of A549/DDP cells in YFJDF+siNC group was higher than that in siNC group(34.5±34.02),P<0.01.The apoptosis rate of A549/DDP cells in YFJDF+siHOTAIR group was(29.89±3.92)compared with that in siHOTAIR group,P>0.05.The relative expression of BCL-2 in YFJDF+siHOTAIR group(1.09±0.05),(0.89±0.05)was lower than that in NC group(1.42±0.08),P<0.01;The relative expression of BCL-2 in YFJDF+siNC group(1.07±0.05)was lower than that in siNC group(1.38±0.05),P<0.01;The relative expression of BCL-2 in YFJDF+siHOTAIR group(0.85±0.21)was higher than that in siHOTAIR group(P>0.05).The relative expression of Bax in YFJDF group and siHOTAIR group were(0.57±0.05)and(0.81±0.06),higher than that in NC group(P<0.01);Compared with siNC group(0.37±0.05),Bax(0.62±0.03)was higher in YFJDF+siHOTAIR group(P<0.05);The relative expression of Bax in YFJDF+siHOTAIR group(0.78±0.21)was higher than that in siHOTAIR group(P>0.05).Conclusion Yifei Jiedu Decoction can increase the expression of Bax,decrease the expression of BCL-2 and induce the apoptosis of A549/DDP cells by decrease LncRNA-HOTAIR,which is one of the mechanisms of reversing the resistance of A549/DDP.