探讨 miR-221 在糖尿病周围神经病变中的作用及其相关分子机制。

Investigation of the role of miR-221 in diabetic peripheral neuropathy and related molecular mechanisms.

机构信息

Department of Anesthesiology, The First Affiliated Hospital of Soochow University, Suzhou, China.

Department of Anesthesiology, Xuzhou Children's Hospital, Xuzhou Medical University, China.

出版信息

Adv Clin Exp Med. 2021 Jun;30(6):623-632. doi: 10.17219/acem/131217.

DOI:10.17219/acem/131217

PMID:34018345

Abstract

BACKGROUND

Diabetic peripheral neuropathy (DPN) is one of the most common complications of diabetes, but the molecular mechanisms of DPN are still unclear.

OBJECTIVES

To investigate the role of miR-221 in DPN and the related molecular mechanisms.

MATERIAL AND METHODS

Streptozotocin (STZ) was used to establish an in vivo DPN model. An in vitro DPN model was established using high glucose-induced SH-SY5Y cells. The pain condition of rats was measured by evaluating the 50% paw withdrawal threshold (PWT) and paw withdrawal latency (PWL). Serum exosomes were extracted and identified. Expression of miR-221 in serum exosomes and serum SOCS3 expression were determined using reverse-transcription quantitative polymerase chain reaction (RT-qPCR). Western blotting was used to measure the protein levels of SOCS3, bradykinin (BK) and prostaglandin E2 (PEG2). The dual luciferase reporter assay was performed to confirm SOCS3 3'-UTR as a target of miR-221. The serum or cell supernatant levels of PEG2, BK, interleukin (IL)-6, IL-1β, and tumor necrosis factor alpha (TNF-α) were measured using enzyme-linked immunosorbent assay (ELISA).

RESULTS

Induction of the lenti-miR-221 inhibitor significantly decreased the expression of miR-221 in DPN rats. Both 50% PWT and PWL values were markedly decreased in DPN rats. When miR-221 was inhibited, the 50% PWT and PWL values were both significantly increased. Knockdown of miR-221 significantly increased the expression of SOCS3 and decreased the expression of NF-κB. Furthermore, knockdown of miR-221 remarkably decreased the expression of PEG2, BK, IL-6, IL-1β, and TNF-α in both STZ-treated DPN rats and high glucose-induced SH-SY5Y cells, which was reversed by inhibition of SOCS3. The dual luciferase reporter assay showed that miR-221 directly targeted and negatively regulated SOCS3.

CONCLUSIONS

Inhibition of miR-221 can reduce pain and decrease expression of inflammatory factors through targeting SOCS3 in DPN.

摘要

背景

糖尿病周围神经病变（DPN）是糖尿病最常见的并发症之一，但 DPN 的分子机制仍不清楚。

目的

探讨 miR-221 在 DPN 中的作用及其相关分子机制。

材料与方法

采用链脲佐菌素（STZ）建立体内 DPN 模型，高糖诱导 SH-SY5Y 细胞建立体外 DPN 模型。通过评估 50%足底撤回阈值（PWT）和足底撤回潜伏期（PWL）来测量大鼠的疼痛状况。提取血清外泌体并进行鉴定。采用逆转录定量聚合酶链反应（RT-qPCR）测定血清外泌体中 miR-221 的表达和血清 SOCS3 的表达。采用 Western blot 测定 SOCS3、缓激肽（BK）和前列腺素 E2（PEG2）的蛋白水平。采用双荧光素酶报告基因实验证实 SOCS3 3'-UTR 是 miR-221 的靶标。采用酶联免疫吸附试验（ELISA）测定血清或细胞上清液中 PEG2、BK、白细胞介素（IL）-6、IL-1β和肿瘤坏死因子-α（TNF-α）的水平。

结果

诱导 lenti-miR-221 抑制剂可显著降低 DPN 大鼠 miR-221 的表达。DPN 大鼠的 50%PWT 和 PWL 值均明显降低。抑制 miR-221 后，50%PWT 和 PWL 值均明显升高。敲低 miR-221 可显著增加 SOCS3 的表达并降低 NF-κB 的表达。此外，在 STZ 处理的 DPN 大鼠和高糖诱导的 SH-SY5Y 细胞中，敲低 miR-221 可显著降低 PEG2、BK、IL-6、IL-1β和 TNF-α的表达，而 SOCS3 抑制可逆转这种作用。双荧光素酶报告基因实验表明，miR-221 可直接靶向并负调控 SOCS3。