LncRNA MALAT1对骨肉瘤细胞阿霉素耐药性的影响机制研究  

Effect mechanism of LncRNA MALAT1 on doxorubicin resistance in osteosarcoma cells

作　　者：梁福东 狄淑芳[1] 罗伟 齐江华[1] 刘利兵 LIANG Fudong;DI Shufang;LUO Wei;QI Jianghua;LIU Libing(First Department of Orthopaedics,Gansu Cancer Hospital,Lanzhou 730050,China)

机构地区：[1]甘肃省肿瘤医院骨软一科,兰州730050

出　　处：《中国药房》2025年第6期698-703,共6页China Pharmacy

基　　金：甘肃省自然科学基金资助项目(No.24JRRA1149)。

摘　　要：目的探讨长链非编码RNA(LncRNA)肺腺癌转移相关转录本1(MALAT1)与骨肉瘤(OS)细胞阿霉素(DOX)耐药性的关系及作用机制。方法将MG-63和MG-63/DOX细胞用不同浓度DOX(0、0.01、0.05、0.1、1μmol/L)处理后,以CCK-8法检测其存活率和半数抑制浓度(IC50);采用实时荧光定量聚合酶链反应法检测MG-63和MG-63/DOX细胞中LncRNA MALAT1表达。将MG-63/DOX细胞分为Control(对照)组、敲低LncRNA MALAT1的阴性对照(sh-NC)组、sh-MALAT1组、sh-MALAT1+抑制(anti)-NC组、sh-MALAT1+anti-miR-154-5p组。检测各组MG-63/DOX细胞中LncRNA MALAT1、miR-154-5p、细胞周期素D1(CCND1)mRNA相对表达量,采用CCK-8法、划痕实验、Transwell实验、流式细胞仪分别检测敲低LncRNA MALAT1对MG-63/DOX细胞增殖、迁移、侵袭、凋亡的影响;采用Western blot法检测MG-63/DOX细胞中增殖细胞核抗原(PCNA)、CCND1蛋白的表达;采用双荧光素酶报告基因实验检测LncRNA MALAT1与miR-154-5p、miR-154-5p与CCND1之间的相互作用。结果与0μmol/L DOX比较,0.01、0.05、0.1、1μmol/L DOX均能降低MG-63和MG-63/DOX细胞(0.01μmol/L DOX除外)的存活率(P<0.05),IC50分别为0.07、0.13μmol/L。sh-MALAT1组MG-63/DOX细胞存活率、迁移数、侵袭数、划痕愈合率、LncRNA MALAT1 mRNA表达、CCND 1 mRNA和蛋白表达、PCNA蛋白表达均显著低于sh-NC组、Control组,细胞凋亡率和miR-154-5p表达显著高于sh-NC组、Control组(P<0.05);相较于sh-MALAT1组,sh-MALAT1+anti-miR-154-5p组的上述生物学作用均被逆转(P<0.05)。在转染MALAT1-野生型(WT)和CCND1-WT的MG-63/DOX细胞中,miR-154-5p模拟物(mimic)组的荧光素酶活性均显著低于其阴性对照组(P<0.05)。结论敲低LncRNA MALAT1可以抑制OS细胞的DOX耐药性,其机制可能是通过靶向miR-154-5p/CCND1轴实现的。OBJECTIVE To investigate the relationship of long non-coding RNA(LncRNA)metastasis-associated lung adenocarcinoma transcript 1(MALAT1)and doxorubicin(DOX)resistance in osteosarcoma(OS)cells.METHODS MG-63 and MG-63/DOX cells were treated with different concentrations of DOX(0,0.01,0.05,0.1,1μmol/L),and survival rates and half maximal inhibitory concentration were determined using CCK-8 assay.The expressions of LncRNA MALAT1 in MG-63 and MG-63/DOX cells were detected by real-time quantitative fluorescence PCR.MG-63/DOX cells were divided into Control group,knocking down LncRNA MALAT1 negative control(sh-NC)group,sh-MALAT1 group,sh-MALAT1+anti-NC group,and sh-MALAT1+anti-miR-154-5p group.The expressions of LncRNA MALAT1,miR-154-5p and cyclin D1(CCND1)mRNA in MG-63/DOX cells of each group were detected.The effects of knocking down LncRNA MALAT1 on the proliferation,migration,invasion,and apoptosis of MG-63/DOX cells were detected by CCK-8 assay,scratch test,Transwell experiment and flow cytometry,respectively.The expression of proliferating cell nuclear protein(PCNA)and CCND1 protein in MG-63/DOX cells was detected by Western blot assay.Interactions between LncRNA MALAT1 and miR-154-5p,miR-154-5p and CCND1 were detected by dual luciferase reporter gene experiment.RESULTS Compared with 0μmol/L DOX,0.01,0.05,0.1 and 1μmol/L DOX could reduce the survival rates of MG-63 and MG-63/DOX cells(except for 0.01μmol/L DOX)(P<0.05),IC50 were 0.07 and 0.13μmol/L,respectively.The survival rate,cell migration number and invasion number of MG-63/DOX cells,scratch closure rate,mRNA expressions of LncRNA MALAT1,mRNA and protein expressions of CCND1,and PCNA protein expression in sh-MALAT1 group were significantly lower than sh-NC group and Control group;the apoptosis rate and miR-154-5p expression were significantly higher than sh-NC group and Control group(P<0.05).sh-MALAT1+anti-miR-154-5p group was able to reverse the aforementioned biological effects in sh-MALAT1 group(P<0.05).In MG-63/DOX cells transfected with both MALAT1-wi

关 键 词：长链非编码RNA 肺腺癌转移相关转录本1 微小RNA-154-5p 细胞周期蛋白D1 骨肉瘤 阿霉素 耐药性 

分 类 号：R979.14[医药卫生—药品] R738.1[医药卫生—药学]