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中华肺部疾病杂志(电子版)

中华肺部疾病杂志(电子版) ›› 2020, Vol. 13 ›› Issue (06) : 724 -730. doi: 10.3877/cma.j.issn.1674-6902.2020.06.002

论著

过表达LNCRNA 00593调控p53通路在非小细胞肺癌顺铂耐药中的作用机制
李峥1, 赵猛1, 葛鹏1, 张爱敏1, 任丽1,†()   
  1. 1. 300060　天津，天津医科大学肿瘤医院检验科
  • 收稿日期:2020-05-16 出版日期:2020-12-25
  • 通信作者: 任丽
  • 基金资助:
    国家自然科学基金青年科学基金项目(81602026); 天津医科大学肿瘤医院科研项目(Y1806)

Overexpression of LNCRNA 00593 regulates cisplatin resistance in non-small cell lung cancer through the p53 pathway

Zheng Li1, Meng Zhao1, Peng Ge1, Aimin Zhang1, Li Ren1,†()   

  1. 1. Department of Clinical Laboratory, Tumor Hospital, Tianjin Medical University, Tianjin 300060, China
  • Received:2020-05-16 Published:2020-12-25
  • Corresponding author: Li Ren
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李峥, 赵猛, 葛鹏, 张爱敏, 任丽. 过表达LNCRNA 00593调控p53通路在非小细胞肺癌顺铂耐药中的作用机制[J]. 中华肺部疾病杂志(电子版), 2020, 13(06): 724-730.

Zheng Li, Meng Zhao, Peng Ge, Aimin Zhang, Li Ren. Overexpression of LNCRNA 00593 regulates cisplatin resistance in non-small cell lung cancer through the p53 pathway[J]. Chinese Journal of Lung Diseases(Electronic Edition), 2020, 13(06): 724-730.

目的

探讨LNCRNA 00593调控非小细胞肺癌(NSCLC)顺铂耐药的作用机制。

方法

实时荧光定量PCR(quantitative real-time PCR, RT-qPCR)检测LNCRNA 00593在癌旁正常组织(normal)、肺腺癌(LUAD)和肺鳞癌(LUSC)、HEB细胞、A549细胞、H1299细胞、PC9细胞、H460细胞和calu3细胞中的表达量；CDPP(0 μM、5 μM、10 μM)作用H1299细胞，RT-qPCR检测LNCRNA 00593在顺铂治疗细胞中的表达量；10 μM CDPP处理H1299细胞0、12、24、36、48 h后，RT-qPCR检测细胞中LNCRNA 00593的表达量。CCK-8及流式细胞仪分析过表达及下调LNCRNA 00593对H1299细胞顺铂耐药的影响。RT-qPCR及Western blot检测过表达及下调LNCRNA 00593后，H1299细胞内p53的表达量，RNA-pull down验证LNCRNA 00593和p53的结合。p53通路抑制剂PFT-α作用细胞后，检测LNCRNA 00593对顺铂诱导的H1299细胞活力和凋亡的影响。

结果

LNCRNA 00593在NSCLC组织的表达水平明显低于癌旁正常组织(P<0.05)。LNCRNA 00593在NSCLCA549、H1299、PC9、H460、calu3细胞中的表达水平明显低于HEB细胞(P<0.01)。分别用0 μM、5 μM、10 μM CDPP处理H1299细胞后，LNCRNA 00593表达量随CDPP作用浓度依赖性升高(P<0.01)。用10 μM CDPP处理H1299细胞12、24、36、48 h后，LNCRNA 00593表达量随CDPP作用时间不断增加(P<0.01)。CDPP作用细胞后，下调LNCRNA 00593使H1299细胞活力明显上升，凋亡率明显下降，过表达LNCRNA 00593使H1299细胞活力下降，凋亡率明显上升。过表达LNCRNA 00593激活了p53通路，LNCRNA 00593能够与p53结合。PFT-α作用细胞后，下调了LNCRNA 00593对H1299细胞顺铂耐药的调控作用。

结论

过表达LNCRNA 00593通过p53通路调控NSCLC细胞的顺铂耐药。

关键词: LNCRNA 00593, p53, 非小细胞肺癌, 顺铂耐药
Objective

To investigate the mechanism of LNCRNA 00593 regulating cisplatin resistance in non-small cell lung cancer.

Methods

Quantitative real-time PCR (RT-qPCR) was used to detect the expression of LNCRNA 00593 in normal paracancer tissues (Normal), lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC), HEB cell, A549 cell, H1299 cell, PC9 cell, H460 cell and calu3 cell. H1299 cells were treated with different concentrations of CDPP (0 μM, 5 μM, 10 μM), the expression of LNCRNA 00593 in cisplatin-treated cells was detected by RT-qPCR. After treating H1299 cells with CDPP for 0 h, 12 h, 24 h, 36 h, 48 h, the expression of LNCRNA 00593 in the H1299 cells was detected by RT-qPCR. The effects of overexpression and down-regulation of LNCRNA 00593 on cisplatin resistance of H1299 cells were analyzed by CCK-8 and flow cytometry. The expression of p53 in H1299 cells was detected by RT-qPCR and Western blot after overexpression and down-regulation of LNCRNA 00593, and the binding of LNCRNA 00593 and p53 was verified by RNA-pull down. The effects of LNCRNA 00593 on the viability and apoptosis of H1299 cells induced by cisplatin were detected after PFT-α treatment.

Results

The expression level of LNCRNA 00593 in non-small cell lung cancer tissues was significantly lower than that in adjacent normal tissues (P<0.05). The expression levels of LNCRNA 00593 in A549, H1299, PC9, H460 and calu3 cells were significantly lower than that of HEB cells (P<0.01). After treating H1299 cells with 0 μM, 5 μM, and 10 μM CDPP respectively, the expression of LNCRNA 00593 increased in a concentration-dependent manner (P<0.01). After treating H1299 cells with 10 μM CDPP for 12 h, 24 h, 36 h, 48 h, the expression of LNCRNA 00593 increased in a time-dependent manner (P<0.01). After CDPP treatment, down-regulation of LNCRNA 00593 significantly increased H1299 cell activity and decreased apoptosis rate, while overexpression of LNCRNA 00593 significantly decreased H1299 cell activity and increased apoptosis rate. Overexpression of LNCRNA 00593 activated the p53 pathway, and LNCRNA 00593 can bind to p53. The regulation of cisplatin resistance of H1299 cells by LNCRNA 00593 was down-regulated in PFT-α-treated cells.

Conclusion

Overexpression of LNCRNA 00593 regulates cisplatin resistance in non-small cell lung cancer cells through the p53 pathway.

Key words: LNCRNA 00593, p53 pathway, Non-small cell lung cancer, Cisplatin resistance
图1 LNCRNA 00593在NSCLC组织和细胞中的表达量；注：A：RT-qPCR检测LNCRNA 00593在NSCLC组织和癌旁正常组织中的表达量，*表示和Normal比较；B：RT-qPCR检测LNCRNA 00593在HEB、A549、H1299、PC9、H460和calu3细胞中的表达量，*和HEB细胞比较，*P<0.05；**P<0.01
图2 LNCRNA 00593在顺铂治疗的H1299细胞中的表达量；注：A：Western blot检测不同浓度CDPP处理H1299细胞Cleaved-PARP的表达量；*和0 μM比较，*P<0.05；**P<0.01；B：Western blot检测10 μM CDPP处理H1299细胞12 h、24 h、36 h、48 h后Cleaved-PARP的表达量*和0 h比较，*P<0.05；**P<0.01
图3 LNCRNA 00593调控H1299细胞的顺铂耐药；注：A：CCK-8检测CDPP作用细胞后下调或过表达LNCRNA 00593对H1299细胞活力的影响；B：流式细胞仪检测CDPP作用细胞后下调或过表达LNCRNA 00593对H1299细胞凋亡率的影响
图4 LNCRNA 00593对顺铂治疗的H1299细胞p53通路的影响；注：A：Western blot检测CDPP作用细胞后下调或过表达LNCRNA 00593对p53表达的影响；1：siRNA NC组；2：LNCRNA 00593 siRNA组；3：pcDNA-3.1(+)组；4：pcDNA-LNCRNA 00593组B：RT-qPCR检测CDPP作用细胞后下调或过表达LNCRNA 00593对p53表达的影响，RNA-pull down及Western blot验证LNCRNA 00593与p-53结合；1：Bio-LNCRNA 00593；2：LNCRNA 00593(No bio)；3：Input
图5 LNCRNA 00593通过p53通路调控H1299细胞的顺铂耐药；注：A：CCK-8检测CDPP作用细胞后LNCRNA 00593通过p53通路对H1299细胞活力的影响；B：流式细胞仪检测CDPP作用细胞后LNCRNA 00593通过p53通路对H1299细胞凋亡率的影响
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