Kan Xuan, Sun Yanan, Lu Jianguang, Li Minghua, Wang Yu, Li Qiuying, Liu Ying, Liu Ming, Tian Linli
Department of Otolaryngology, Head and Neck Surgery, The Second Affiliated Hospital, Harbin Medical University, Harbin, Heilongjiang 150081, P.R. China.
Mol Med Rep. 2016 May;13(5):4315-20. doi: 10.3892/mmr.2016.5048. Epub 2016 Mar 28.
Dysregulation of a numerous microRNAs (miRNAs) has been implicated in laryngeal squamous cell carcinoma (LSCC). Among those miRNAs, miR‑21 and miR‑221 are co‑overexpressed and commonly target the phosphatase and tensin homolog protein (PTEN) that is located in the PTEN‑Akt signaling pathway. The present study investigated whether co‑inhibition of miR‑21 and miR‑221 induced synergistic apoptosis of human LSCC cells. Methyl thiazolyl tetrazolium (MTT) and terminal deoxynucleotidyl‑transferase‑mediated deoxynucleotide triphosphate nick end labeling (TUNEL) assays were used to observe the potential effect of miR‑21 and miR‑221 on cell viability and apoptosis in cells co‑transfected with anti‑miRNA oligonucleotide (AMO)‑21 and AMO‑221. The protein expression levels of PTEN, Akt and p53 were determined by western blotting. The cellular abundance of 6 pro‑apoptotic miRNAs transcribed by p53 mediation, consisting of miR‑15a, miR‑16‑1, miR‑26a, miR‑34a, miR‑143 and miR‑203, was measured with using reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR). MTT results indicate that in vitro co‑transfection of AMO‑21 and AMO‑221 leads to a decline in cell viability, compared with the transfection of AMO alone. This result was verified by the detection of apoptosis using TUNEL assays. Co‑transfection of AMO‑21 and AMO‑221 resulted in a marked reduction in Akt phosphorylation and enhanced expression of PTEN and p53 were observed; consequently, leading to an amplification of the transcription of 6 pro‑apoptotic miRNAs. The present findings confirmed that co‑inhibition of miR‑21 and miR‑221 synergistically triggers cell apoptosis in vitro. The altered PTEN‑Akt signaling and p53‑mediated amplification of the transcription of pro‑apoptotic miRNAs may be involved in the observed synergistic effect. The present study provides novel insights into the mechanism underlying apoptosis‑associated miRNA‑miRNA mutual regulation in LSCC.
众多微小RNA（miRNA）的失调与喉鳞状细胞癌（LSCC）有关。在这些miRNA中，miR-21和miR-221共同过度表达，且通常靶向位于PTEN-Akt信号通路中的磷酸酶和张力蛋白同源蛋白（PTEN）。本研究调查了miR-21和miR-221的共同抑制是否会诱导人LSCC细胞的协同凋亡。采用甲基噻唑基四氮唑（MTT）法和末端脱氧核苷酸转移酶介导的脱氧三磷酸核苷酸缺口末端标记（TUNEL）法，观察miR-21和miR-221对用抗miRNA寡核苷酸（AMO）-21和AMO-221共转染的细胞的细胞活力和凋亡的潜在影响。通过蛋白质印迹法测定PTEN、Akt和p53的蛋白表达水平。使用逆转录-定量聚合酶链反应（RT-qPCR）检测由p53介导转录的6种促凋亡miRNA的细胞丰度，这些miRNA包括miR-15a、miR-16-1、miR-26a、miR-34a、miR-143和miR-203。MTT结果表明，与单独转染AMO相比，体外共转染AMO-21和AMO-221会导致细胞活力下降。通过TUNEL检测凋亡验证了这一结果。AMO-21和AMO-221的共转染导致Akt磷酸化显著降低，并观察到PTEN和p53的表达增强；因此，导致6种促凋亡miRNA的转录增加。本研究结果证实，miR-21和miR-221的共同抑制在体外协同触发细胞凋亡。PTEN-Akt信号通路的改变以及p53介导的促凋亡miRNA转录的增加可能参与了观察到的协同效应。本研究为LSCC中与凋亡相关的miRNA-miRNA相互调控的机制提供了新的见解。
