| 胡珍,吴庭,孙弦,等.长链非编码 RNA变异性浆细胞瘤异位 1通过调控微小 RNA-214-3p/人凋亡抑制蛋白 X轴影响皮肤黑色素瘤细胞的顺铂耐药性[J].安徽医药,2022,26(6):1226-1231. |
| 长链非编码 RNA变异性浆细胞瘤异位 1通过调控微小 RNA-214-3p/人凋亡抑制蛋白 X轴影响皮肤黑色素瘤细胞的顺铂耐药性 |
| LncRNA PVT1 affects cisplatin resistance of cutaneous malignant melanoma cells by regulating miR-214-3p/XIAP axis |
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| DOI：10.3969/j.issn.1009-6469.2022.06.040 |
| 中文关键词: 黑色素瘤 抗药性，肿瘤 长链非编码 RNA变异性浆细胞瘤异位 1 微小 RNA-214-3p 人凋亡抑制蛋白 X 顺铂耐药性 |
| 英文关键词: Melanoma Drug resistance, neoplasm Long non-coding RNA plasmacytoma variant translocation 1 MiR-2143p Human X inhibitor of apoptosis protein Cisplatin resistance |
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| 中文摘要: |
| 目的探讨长链非编码 RNA(lncRNA)变异性浆细胞瘤异位 1(PVT1)调控微小 RNA(miR)-214-3p/人凋亡抑制蛋白 X(XIAP)对皮肤黑色素瘤( CMM)细胞的顺铂( DDP)耐药性的影响。方法该研究起止时间为 2020年 3—9月。体外培养人皮肤黑色素瘤 SK-MEL-1/DDP细胞，将细胞分为空白对照组( NG组，不做处理)、阴性转染组( NC组，转染 PVT1-inhibitor阴性对照)、抑制 PVT1表达组( PVT1-inhibitor组，转染 PVT1-inhibitor)、共转染组( PVT1-inhibitor-miR-214-3p-inhibitor组，转染 PVT1inhibitor同时转染 miR-214-3p-inhibitor)。采用实时荧光定量逆转录聚合酶链反应( qRT-PCR)检测 SK-MEL-1/DDP细胞 PVT1、 miR-214-3p水平； MTT法检测四组 SK-MEL-1/DDP细胞增殖能力及对 DDP的耐药性；流式细胞仪检测四组 SK-MEL-1/DDP细胞凋亡率；蛋白质印迹法( Western blotting)检测四组 SK-MEL-1/DDP细胞 XIAP、细胞增殖相关核抗原 Ki-67、凋亡相关蛋白 B细胞淋巴瘤 -2(Bcl-2)、 Bcl-2相关 X蛋白( Bax)表达水平。应用 TargetScan数据库预测 PVT1与 miR-214-3p的靶向关系，双荧光素酶报告基因实验验证。结果与 NG组、 NC组比较， PVT1-inhibitor组 SK-MEL-1/DDP细胞增殖抑制率［( 22.87±3.43)%比( 0.00± 0.00)%、(0.08±0.01)%］、凋亡率［( 40.05±6.06)%比( 15.69±2.35)%、(17.01±2.55)%］、 miR-214-3p及 Bax表达均显著升高( P< 0.05)PVT1、药物半数抑制浓度( IC50)值［( 15.13±0.45)μg/L比( 45.03±1.35)μg/L、(47.81±1.33)μg/L］、 Ki-67、Bcl-2蛋白、 XIAP mRNA及，其蛋白表达水平均显著降低( P<0.05)；与 PVT1-inhibitor组比较， PVT1-inhibitor+miR-214-3p-inhibitor组 SK-MEL-1/ DDP细胞增殖抑制率［(15.12±2.27)%比( 22.87±3.43)%］、凋亡率［(27.88±4.19)%比( 40.05±6.06)%］、 miR-214-3p及 Bax表达均显著降低( P<0.05)IC50［(23.98±0.72)μg/L比(15.13±0.45)μg/L］Ki-67、Bcl-2蛋白、 XIAP mRNA及蛋白表达水平均显著升高(P<0.05)。TargetScan，数据值库预测显示 miR-214-3p是 PVT1的潜在靶、基因，双荧光素酶报告基因实验证实二者存在靶向关系。 |
| 英文摘要: |
| Objective To investigate the effect of long non-coding RNA plasmacytoma variant translocation 1 (lncRNA PVT1) regulating microRNA-214-3p (miR-214-3p)/human X inhibitor of apoptosis protein (XIAP) on cisplatin (DDP) resistance of cutaneous malignant melanoma (CMM) cells.Methods The study period was from March 2020 to September 2020. SK-MEL-1/DDP cells were cultured in vitro and divided into blank control group (NG group, no treatment), negative transfection group (NC group, transfection withPVT1 inhibitor negative control), PVT1-inhibitor group (PVT1 inhibitor group, transfection with PVT1 inhibitor) and co-transfection group (PVT1-inhibitor-miR-214-3p-inhibitor group, transfection with PVT1 inhibitor and mir-214-3p-inhibitor). Real-time quantitative polymerase chain reaction (qRT-PCR) was used to detect the levels of PVT1 and miR-214-3p in SK-MEL-1/DDP cells; MTT assay was used to detect the proliferation and drug resistance of SK-MEL-1/DDP cells; flow cytometry was used to detect the apoptosis rate of SKMEL-1/DDP cells; Western blot was used to detect the expression levels of XIAP, cell proliferation associated nuclear antigen (Ki-67), B-cell lymphoma-2 (Bcl-2) and Bcl-2 associated X protein (Bax) in SK-MEL-1/DDP cells. The TargetScan database and double luciferase reporter gene experiment were used to predict and verify the targeting relationship between PVT1 and miR-214-3p.Results Com pared with those in the NG group and NC group, the proliferation inhibition rate [(22.87±3.43)% vs. (0.00±0.00)%, (0.08±0.01)%], apoptosis rate [(40.05±6.06)% vs. (15.69±2.35)%, (17.01±2.55)%], miR-214-3p and Bax protein expression of SK-MEL-1/DDP cells in PVT1 inhibitor group were significantly increased (P < 0.05), while PVT1, half inhibitory concentration (IC50) [(15.13±0.45) μg/L vs. (45.03±1.35) μg/L, (47.81±1.33) μg/L], Ki-67, Bcl-2 protein, XIAP mRNA and protein expression were significantly decreased (P < 0.05); compared with the PVT1 inhibitor group, the proliferation inhibition rate [(15.12±2.27) % vs. (22.87±3.43)%], apoptosis rate [(27.88±4.19)% vs. (40.05±6.06)%], miR-214-3p and Bax protein expression of SK-MEL-1/DDP cells in the PVT1-inhibitor+miR-2143p-inhibitor group were significantly decreased (P < 0.05), IC50 [(23.98±0.72) μg/L vs. (15.13±0.45) μg/L], while Ki-67, Bcl-2 protein, XIAP mRNA and protein expression were significantly increased (P < 0.05). The prediction of TargetScan database showed that miR214-3p was a potential target gene of PVT1, and the dual luciferase reporter gene experiment confirmed that there was a targeting relationship between miR-214-3p and PVT1.Conclusion Down-regulation of lncRNA PVT1 can up-regulate the expression of miR-2143p, inhibit XIAP expression, and reduce the resistance of CMM SK-MEL-1/DDP cells to DPP. |
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