胞外囊泡中linc-VLDLR表达与食管癌发生及耐药的关系

Expression of long noncoding RNA(linc-VLDLR) in extracellular vesicles and its relation to the development and multidrug resistance of esophageal cancer

目的探讨胞外囊泡(EVs)中linc-VLDLR的表达与食管癌的发生及耐药的关系。方法以不同浓度阿霉素(ADM)作用于食管鳞癌Eca109细胞24h,采用MTT法检测ADM对Eca109细胞的半数抑制浓度(IC_(50))。以IC_(50)浓度为基准设置3个ADM浓度干预Eca109细胞24h,提取培养液中EVs。荧光定量RT-PCR检测EVs中linc-VLDLR mRNA表达。以EVs干预Eca109细胞48h,随后以不同浓度ADM再作用于细胞24h,MTT法检测IC_(50);EVs干预Eca109细胞48h,流式细胞术检测细胞周期;荧光定量RT-PCR检测Eca109细胞中linc-VLDLR及三磷酸腺苷结合转运蛋白G2(ABCG2)mRNA表达。结果 ADM作用Eca109细胞24h的IC_(50)为0.44±0.02μg/ml,选取0、0.2、0.4、0.8μg/ml ADM浓度作用Eca109细胞24h后提取上清液中的EVs(EVs1–4)。EVs4中linc-VLDLR表达水平明显高于EVs1–3(P<0.01)。EVs1–4干预Eca109细胞48h,EVs4组IC_(50)值明显高于EVs1–3及对照组(P<0.05)。流式细胞术检测显示EVs4组Eca109细胞增殖指数(PI)明显高于EVs1–3及对照组(P<0.01)。EVs4干预Eca109细胞48h后,Eca109细胞中linc-VLDLR及ABCG2基因表达水平明显高于EVs1–3及对照组(P<0.05)。结论 linc-VLDLR及ABCG2基因在食管癌细胞中高表达,参与了食管癌耐药形成。耐药细胞释放的EVs可以上调食管癌细胞中ABCG2表达,调控细胞耐药性,其作用与EVs上携带的linc-VLDLR基因有关。

Objective To investigate the relationship between the expression of linc-VLDLR in extracellular vesicles（EVs） and the development and drug resistance of esophageal carcinoma. Methods Fifty percent of inhibitory concentration（IC（50）） of adriamycin（ADM） for Eca109 cells was detected by MTT assay, after the treatment of esophageal squamous cell carcinoma Eca109 cell line with different concentrations of ADM for 24 h. The culture medium was treated with 3 concentrations of ADM based on the IC（50） for 24 h for extracting EVs in Eca109 cells. linc-VLDLR mRNA expression in EVs was detected by q RT-PCR assay. IC（50） of ADM for Eca109 cells intervened by EVs for 48 h was detected by MTT assay. Cell cycle was detected by FCM and linc-VLDLR and ABCG2 mRNA expressions in Eca109 cells were detected by q RT-PCR after the treatment of the EVs for 48 h. Results IC（50） of ADM acting on Eca109 cells for 24 h was 0.44±0.02μg/ml, so ADM concentrations of 0.2, 0.4, 0.8μg/ml were choosed in the following studies. EVs were extracted from the supernatant after the treatment of 0, 0.2, 0.4, 0.8μg/ml ADM for 24 h and were labeled as EVs1, 2, 3 and 4 respectively. LincVLDLR mRNA expression in EVs4 was significantly higher than that in EVs1-3（P〈0.01）. ADM IC（50） for Eca109 cells in EVs4 group was significantly higher than that in other groups after the treatment of EVs1-4 on Eca109 cells for 48 h（P〈0.05）. Flow cytometry results showed that the proliferation index of Eca109 cells in EVs4 group was significantly higher than that in EVs1-3 and control groups（P〈0.01）. Linc-VLDLR and ABCG2 mRNA expression levels in Eca109 cells of EVs4 group were significantly higher than these of EVs1-3 and control groups（P〈0.05）. Conclusions High expression of linc-VLDLR and ABCG2 gene in esophageal cancer cells is involved in the formation of esophageal cancer resistance. EVs released by drug-resistant cells can upregulate the expression of ABCG2 in esophageal cancer cells and regulate the drug resistance of esophageal cancer cells, which is related to the linc-VLDLR gene carried by EVs.