miR-142-5p 通过靶向肺癌细胞中的 HOXD8 逆转对吉非替尼的耐药性。
MiR-142-5p reverses the resistance to gefitinib through targeting HOXD8 in lung cancer cells.
机构信息
Department of Internal Medicine, Linyi Cancer Hospital, Linyi, China.
出版信息
Eur Rev Med Pharmacol Sci. 2020 Apr;24(8):4306-4313. doi: 10.26355/eurrev_202004_21011.
OBJECTIVE
To investigate the role and potential mechanism of micro ribonucleic acid (miR)-142-5p in the acquired resistance to gefitinib in lung cancer cells.
MATERIALS AND METHODS
The drug resistance of PC9/G cells was detected via methyl thiazolyl tetrazolium (MTT) assay. Expression levels of miR-142-5p and HOXD8 in PC9 and PC9/G cells were detected via quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) and Western blotting. PC9/G cells were transfected with miR-142-5p mimic, while PC9 cells were transfected with miR-142-5p inhibitor. Subsequently, expression changes of HOXD8 were determined using qRT-PCR and Western blotting, cell sensitivity to gefitinib was detected through MTT assay, and the apoptosis was detected via flow cytometry. Moreover, Dual-Luciferase reporter assay was conducted to determine the relationship between HOXD8 and miR-142-5p. Finally, potential involvement of HOXD8 in miR-142-5p-regulated gefitinib sensitivity was confirmed via rescue tests.
RESULTS
PC9/G cells were more significantly resistant to gefitinib compared with its parental PC9 cells. MiR-142-5p was down-regulated in PC9/G cells, while that of HOXD8 was up-regulated in PC9/G cells. Transfection of miR-142-5p mimic could inhibit the expression level of HOXD8 in PC9/G cells and reverse its resistance to gefitinib. Conversely, transfection of miR-142-5p inhibitor could upregulate HOXD8 in PC9 cells and promote its resistance to gefitinib. According to the Dual-Luciferase reporter assay, miR-142-5p could suppress the expression of HOXD8 through the targeted binding to the HOXD8 3'UTR. Moreover, miR-142-5p could regulate mitochondrial apoptosis pathway by targeting HOXD8. Finally, rescue tests confirmed that miR-142-5p regulated the sensitivity of PC9 cells to gefitinib by acting on the target gene HOXD8.
CONCLUSIONS
Down-regulation of miR-142-5p induces the resistance of lung cancer PC9 cells to gefitinib by upregulating HOXD8.
目的
探讨微小 RNA（miR）-142-5p 在肺癌细胞获得性对吉非替尼耐药中的作用及潜在机制。
材料与方法
采用噻唑蓝（MTT）比色法检测 PC9/G 细胞的耐药性。采用实时定量逆转录聚合酶链反应（qRT-PCR）和 Western blot 检测 miR-142-5p 和 HOXD8 在 PC9 和 PC9/G 细胞中的表达水平。用 miR-142-5p 模拟物转染 PC9/G 细胞，用 miR-142-5p 抑制剂转染 PC9 细胞。然后，通过 qRT-PCR 和 Western blot 检测 HOXD8 的表达变化，通过 MTT 检测细胞对吉非替尼的敏感性，通过流式细胞术检测细胞凋亡。此外，还进行了双荧光素酶报告基因实验来确定 HOXD8 与 miR-142-5p 之间的关系。最后，通过挽救实验证实了 HOXD8 在 miR-142-5p 调节吉非替尼敏感性中的潜在作用。
结果
与亲本 PC9 细胞相比，PC9/G 细胞对吉非替尼的耐药性明显增强。miR-142-5p 在 PC9/G 细胞中下调，而 HOXD8 在 PC9/G 细胞中上调。转染 miR-142-5p 模拟物可抑制 PC9/G 细胞中 HOXD8 的表达水平并逆转其对吉非替尼的耐药性。相反，转染 miR-142-5p 抑制剂可上调 PC9 细胞中 HOXD8 的表达并促进其对吉非替尼的耐药性。根据双荧光素酶报告基因实验，miR-142-5p 可通过靶向结合 HOXD8 3'UTR 抑制 HOXD8 的表达。此外，miR-142-5p 可通过靶向 HOXD8 调节线粒体凋亡途径。最后，挽救实验证实，miR-142-5p 通过作用于靶基因 HOXD8 调节 PC9 细胞对吉非替尼的敏感性。
结论
miR-142-5p 的下调通过上调 HOXD8 诱导肺癌 PC9 细胞对吉非替尼的耐药性。
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