LINC00462通过MYC/ABCC3轴影响肾透明细胞癌细胞糖酵解进而调控其对顺铂的敏感性  

LINC00462 regulates clear renal cell carcinoma cells sensitivity to cisplatin by affecting their glycolysis via the MYC/ABCC3 axis

作　　者：王晓玲 胡威威 孟莉丹 WANG Xiaoling;HU Weiwei;MENG Lidan(Department of Nephrology,Hengshui People's Hospital,Hengshui 053000,Hebei,China;Department of Geriatrics,Hengshui People's Hospital,Hengshui 053000,Hebei,China)

机构地区：[1]衡水市人民医院肾内科,河北衡水053000 [2]衡水市人民医院老年病一科,河北衡水053000

出　　处：《中国肿瘤生物治疗杂志》2023年第9期762-770,共9页Chinese Journal of Cancer Biotherapy

基　　金：河北省医学科学研究课题(No.20220452)。

摘　　要：目的:探讨LINC00462招募转录因子MYC激活ABCC3对肾透明细胞癌(ccRCC)顺铂敏感性的影响及其机制。方法:数据库分析ccRCC组织中ABCC3、MYC和LINC00462的表达及其相关性,并分析ABCC3基因的富集通路。常规培养人肾小管上皮细胞(HK-2)和ccRCC细胞(A-498、786-O和Caki-2),将si-LINC00462、oe-ABCC3、si-ABCC3、si-MYC、si-LINC00462-NC、oe-ABCC3-NC、si-ABCC3-NC和si-MYC-NC核酸序列分别转染A-498或786-O细胞,分为si-LINC00462组、si-LINC00462-NC组、oe-ABCC3组、oe-ABCC3-NC组、si-ABCC3组、si-ABCC3-NC组、si-MYC组、si-MYC-NC组;用2-脱氧-D-葡萄糖(2-DG)进行回复实验,构建oe-NC+PBS组、oe-ABCC3+PBS组、oe-ABCC3+2-DG组;为探究ccRCC细胞LINC00462/MYC/ABCC3轴对顺铂敏感性的影响,构建si-NC+oe-NC组、si-LINC00462+oe-NC组、si-LINC00462+oe-ABCC3组。qPCR法检测ABCC3、MYC和LINC00462在ccRCC细胞中的表达,CCK-8法检测细胞增殖活力,CCK-8法分析梯度浓度顺铂处理ccRCC细胞后IC50值,WB法检测糖酵解代谢途径相关蛋白的表达,Seahorse XP96法检测各处理组细胞的胞外酸化率(ECAR)和耗氧率(OCR),试剂盒检测细胞中丙酮酸、乳酸、ATP水平。双荧光素酶报告基因和染色质免疫共沉淀(ChIP)实验验证ABCC3与MYC间的结合关系,RNA结合蛋白免疫沉淀(RIP)实验验证LINC00462和MYC的结合关系。结果:数据库分析和qPCR实验结果显示,ABCC3在ccRCC组织和细胞中呈高表达,差异基因富集在糖酵解通路上。敲减或过表达ABCC3能够增加A-498细胞或降低786-O细胞对顺铂的敏感性,ABCC3可通过促进有氧糖酵解抑制A-498细胞对顺铂的敏感性,2-DG处理可以逆转过表达ABCC3对ccRCC细胞对顺铂敏感性的抑制作用。MYC可直接和ABCC3结合,LINC00462可招募转录因子MYC;敲低LINC00462可抑制ABCC3的表达,敲低LINC00462可抑制ccRCC细胞的有氧糖酵解,并提高其对顺铂敏感性;而进一步过表达ABCC3可逆转敲低LINC00462对ccRCC细胞有氧糖酵解的抑制作用和顺�Objective:To investigate the effect of LINC00462 binding to transcription factor MYC to activate ABCC3 on cisplatin sensitivity in clear cell renal cell carcinoma(ccRCC)and its potential mechanisms.Methods:The expression of ABCC3,MYC and LINC00462 and their correlation in ccRCC were analyzed,and the enrichment pathway of ABCC3 was also analyzed.Human renal tubular epithelial cells(HK-2)and ccRCC cells(A-498,786-O and Caki-2)were conventionally cultured.Nucleic acid sequences of si-LINC00462,si-LINC00462-NC,oe-ABCC3,oe-ABCC3-NC,si-ABCC3,si-ABCC3-NC,si-MYC and si-MYC-NC were transfected into A-498 or 786-O cells respectively,namely si-LINC00462 group,si-LINC00462-NC group,oe-ABCC3 group,oe-ABCC3-NC group,si-ABCC3 group,si-ABCC3-NC group,si-MYC group and si-MYC-NC group.2-deoxy-D-glucose(2-DG)was used in the rescue experiment,and oe-NC+PBS group,oe-ABCC3+PBS group and oe-ABCC3+2-DG group were established.To investigate the effects of LINC00462/MYC/ABCC3 axis on the cisplatin sensitivity of ccRCC cells,si-NC+oe-NC group,si-LINC00462+oe-NC gorup,and si-LINC00462+oe-ABCC3 group were constructed.The expression of ABCC3,MYC and LINC00462 in ccRCC cells was detected by qPCR.CCK-8 method was used to measure cell viability and the IC50 of cisplatin inhibiting the proliferation of ccRCC cells.WB was used to detect the expression of glycolytic metabolism-related proteins.Seahorse XP96 was used to analyze the extracellular acidification rate(ECAR)and oxygen consumption rate(OCR)in the cells of different treatment groups.The levels of pyruvate,lactic acid and ATP in the cells were detected by corresponding kits.The binding relationship between ABCC3 and MYC was verified by double luciferase reporter gene assay and chromatin immunoprecipitation(ChIP)assay,and the binding relationship between LINC00462 and MYC was verified using RNA-binding protein immunoprecipitation(RIP)experiment.Results:Database analysis and qPCR experiments showed that ABCC3 was highly expressed in ccRCC tissues and cells and was enriched in the glycolytic p

关 键 词：LINC00462 MYC ABCC3 肾透明细胞癌 A-498细胞 786-O细胞 糖酵解 顺铂敏感性 

分 类 号：R737.11[医药卫生—肿瘤] R730.53[医药卫生—临床医学]