微小RNA-495通过靶向信号转导子和转录激活子3（STAT3）来调节己糖胺酶刺激的大鼠脊髓神经元的细胞增殖和凋亡。

MiR-495 regulates cell proliferation and apoptosis in HO stimulated rat spinal cord neurons through targeting signal transducer and activator of transcription 3 (STAT3).

作者信息

Qiu Yunfeng, Zhao Ziru, Chen Qi, Zhang Bin, Yang Chuanjun

机构信息

Department of Orthopedic, Luhe Hospital Affiliated of Yangzhou University Medical College, Nanjing, China.

Department of Orthopedics, The Fourth Affiliated Hospital of Nanjing Medical University, Nanjing, China.

出版信息

Ann Transl Med. 2021 Mar;9(6):461. doi: 10.21037/atm-21-102.

DOI:10.21037/atm-21-102

PMID:33850858

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC8039649/

Abstract

BACKGROUND

MicroRNA-495 (miR-495) is a post-translational modulator that performs several functions, and it is involved in several disease states. On the other hand, the physiological functions of miR-495 in HO stimulated mouse spinal cord neuronal dysfunction have not yet been fully understood.

METHODS

In this study, we speculated that miR-495 may regulate the expression of STAT3 in the processes of neuronal proliferation and apoptosis following spinal cord injury (SCI). Cell viability was assessed with methyl thiazolyl tetrazolium (MTT) assay. Caspase-3 activity was assayed with ELISA. Cellular apoptotic changes were measured with TUNEL assay. Intracellular ROS production was determined by measuring uptake of dichlorodihydrofluorescein diacetate (DCFH-DA; PCR was used to assay the mRNA expression of STAT3 gene bearing predicted targeting positions for miR-495, while qRT-PCR was used to measure miR-495 mRNA.

RESULTS

The results demonstrated that treatment of SCNs with HO led to a significant decrease in cell survival, while it enhanced apoptosis. The HO treatment induced cell membrane dysfunction, and increased ROS levels and DNA damage. Interestingly, the expression of miR-495 was markedly suppressed when SCNs were exposed to HO. However, miR-495 overexpression reversed HO-induced cytotoxicity and apoptosis in SCNs. Moreover, HO exposure elevated protein and mRNA concentrations of STAT3 in SCNs. Bioinformatics analysis showed likely binding domains of miR-495 in the 3'-untranslated regions of STAT3 in SCNs. MiR-495 loss-of-function and gain-of-function significantly up-regulated and down-regulated both STAT3 mRNA and protein expressions, respectively, in SCNs.

CONCLUSIONS

miR-495 overexpression inhibited HO-induced SCN dysfunction. This mechanism was mediated through the down-regulation of STAT3 expression.

摘要

背景

微小RNA-495（miR-495）是一种具有多种功能的翻译后调节因子，参与多种疾病状态。另一方面，miR-495在过氧化氢刺激的小鼠脊髓神经元功能障碍中的生理功能尚未完全明确。

方法

在本研究中，我们推测miR-495可能在脊髓损伤（SCI）后的神经元增殖和凋亡过程中调节信号转导和转录激活因子3（STAT3）的表达。用噻唑蓝（MTT）法评估细胞活力。用酶联免疫吸附测定（ELISA）法检测半胱天冬酶-3活性。用末端脱氧核苷酸转移酶介导的缺口末端标记（TUNEL）法检测细胞凋亡变化。通过测量二氯二氢荧光素二乙酸酯（DCFH-DA）的摄取来测定细胞内活性氧（ROS）的产生；采用聚合酶链反应（PCR）检测含有miR-495预测靶向位点的STAT3基因的mRNA表达，而采用实时定量PCR（qRT-PCR）检测miR-495 mRNA。

结果

结果表明，用过氧化氢处理脊髓神经元（SCNs）导致细胞存活率显著降低，同时增强了细胞凋亡。过氧化氢处理诱导细胞膜功能障碍，增加ROS水平和DNA损伤。有趣的是，当SCNs暴露于过氧化氢时，miR-495的表达明显受到抑制。然而，miR-495过表达可逆转过氧化氢诱导的SCNs细胞毒性和凋亡。此外，过氧化氢暴露可提高SCNs中STAT3的蛋白质和mRNA浓度。生物信息学分析显示miR-495在SCNs中STAT3的3'非翻译区可能的结合域。miR-495功能丧失和功能获得分别显著上调和下调了SCNs中STAT3的mRNA和蛋白质表达。