Endoscopy Center, Linyi Central Hospital, Linyi, China.
Eur Rev Med Pharmacol Sci. 2018 Sep;22(17):5546-5553. doi: 10.26355/eurrev_201809_15816.
The aim of this study was to investigate the expression of micro ribonucleic acid-411-5P (miR-411-5p) in non-small cell lung cancer (NSCLC), and to explore the effect of miR-411-5p on the biological behavior of NSCLC cells as well as the underlying molecular mechanism.
Quantitative Real Time- Polymerase Chain Reaction (qRT-PCR) was used to detect the expression level of miR-411-5p in NSCLC tissues and cells. MiR-411-5p mimics and relevant controls were transfected into NSCLC cells according to the instructions of Lipidosome 2000. Transfected cells were divided into the experimental group and the control group. The transfection efficiency of each group was detected by qRT-PCR. After miR-411-5p overexpression, methyl thiazolyl tetrazolium (MTT) assay, flow cytometry, and transwell assay were used to detect the biological changes of cells in each group. Bioinformatics predicted that pumilio homolog 1 (PUM1) was the target gene of miR-411-5p. Subsequently, the mRNA and protein expression level of PUM1 in each group was detected by qRT-PCR and Western blotting, respectively. Dual-luciferase reporter assay was used to validate the target regulatory relationship between miR-411-5p and PUM1.
The results of qRT-PCR showed that miR-411-5p was relatively lowly expressed in NSCLC tissues and cells. After miR-411-5p overexpression, MTT results revealed that the proliferation of NSCLC cells was decreased. Flow cytometry results indicated that the apoptosis rate of NSCLC cells was increased, and cell cycle was arrested in the G0-G1 phase. Meanwhile, the transwell assay demonstrated that the migration and invasion abilities of NSCLC cells were decreased. Bioinformatics predicted that PUM1 was the target gene of miR-411-5p. After miR-411-4p was overexpressed in NSCLC cells, qRT-PCR and Western blotting showed that both the mRNA and protein expression levels of PUM1 were up-regulated. Moreover, dual-luciferase reporter assay demonstrated that miR-411-5p could significantly inhibit the luciferase activity of wild-type PUM1-3'-untranslated region (3'-UTR). However, it exhibited no effect on the luciferase activity of cells transfected with mutant plasmids.
MiR-411-5p may be involved in regulating the biological function of NSCLC cells via targeting PUM1. In addition, miR-411-5p may serve as a potential target for the molecular therapy of NSCLC.
本研究旨在探讨微小 RNA-411-5P（miR-411-5p）在非小细胞肺癌（NSCLC）中的表达情况，并探讨 miR-411-5p 对 NSCLC 细胞生物学行为的影响及其潜在的分子机制。
采用实时荧光定量聚合酶链反应（qRT-PCR）检测 NSCLC 组织和细胞中 miR-411-5p 的表达水平。根据 Lipidosome 2000 的说明书，将 miR-411-5p 模拟物及其相应对照物转染至 NSCLC 细胞中。将转染后的细胞分为实验组和对照组。采用 qRT-PCR 检测各组的转染效率。miR-411-5p 过表达后，采用噻唑蓝（MTT）比色法、流式细胞术和 Transwell 实验检测各组细胞的生物学变化。生物信息学预测 pumilio 同源物 1（PUM1）是 miR-411-5p 的靶基因。随后，采用 qRT-PCR 和 Western blot 分别检测各组 PUM1 的 mRNA 和蛋白表达水平。双荧光素酶报告基因实验验证 miR-411-5p 与 PUM1 之间的靶基因调控关系。
qRT-PCR 结果显示，miR-411-5p 在 NSCLC 组织和细胞中表达水平较低。过表达 miR-411-5p 后，MTT 结果显示 NSCLC 细胞的增殖能力降低。流式细胞术结果表明，NSCLC 细胞的凋亡率增加，细胞周期被阻滞在 G0-G1 期。同时，Transwell 实验表明 NSCLC 细胞的迁移和侵袭能力降低。生物信息学预测 PUM1 是 miR-411-5p 的靶基因。过表达 miR-411-5p 后，qRT-PCR 和 Western blot 结果显示 PUM1 的 mRNA 和蛋白表达水平均上调。此外，双荧光素酶报告基因实验证实 miR-411-5p 可显著抑制野生型 PUM1-3'非翻译区（3'-UTR）的荧光素酶活性，但对转染突变质粒的细胞的荧光素酶活性无影响。
miR-411-5p 可能通过靶向 PUM1 参与调节 NSCLC 细胞的生物学功能。此外，miR-411-5p 可能成为 NSCLC 分子治疗的潜在靶点。
