Shen W-S, Xu X-Q, Zhai N-N, Zhou Z-S, Shao J, Yu Y-H
Department of Pain Clinic, Shaoxing Traditional Chinese Medical Hospital, Shaoxing, China.
Department of Anesthesiology, Shaoxing Traditional Chinese Medical Hospital, Shaoxing, China.
Gene Ther. 2017 Jun;24(6):353-360. doi: 10.1038/gt.2017.28. Epub 2017 Apr 25.
The present study aimed to investigate the potential role of microRNA-141-3p (miR-141-3p) in chronic inflammatory pain (CIP) by targeting the high-mobility group box1 (HMGB1) gene. In the in vitro study, BV2 microglial cells were selected and assigned into blank, lipopolysaccharide (LPS), miR-141-3p mimics, mimics control, miR-141-3p inhibitor, inhibitor control, miR-141-3p mimics+LPS, mimics control+ LPS, miR-141-3p inhibitor+LPS and inhibitor control+LPS groups. Ninety-six rats were randomly divided into 8 groups (12 rats in each group): blank control, model control, negative control (NC), miR-141-3p mimics+ complete Freund's adjuvant (CFA), mimics control+CFA, HMGB1 short hairpin RNA (shRNA)+CFA, HMGB1 NC+CFA and miR-141-3p mimics+HMGB1 shRNA+CFA groups. The quantitative real-time PCR, western blotting, enzyme-linked immunosorbent assay and pain behavioral test were used to measure the miR-141-3p and HMGB1 mRNA expressions, HMGB1 protein expression, inflammatory cytokines levels, and thermal and mechanical pain thresholds, respectively. Compared with the blank, mimics control, inhibitor control and miR-141-3p mimics+LPS groups, the miR-141-3p mimics group had increased miR-141-3p expression and interleukin (IL)-10 levels, and had decreased mRNA and protein expressions of HMGB1 and the levels of IL-1β, tumor necrosis factor-α (TNF-α) and IL-6, whereas the opposite trend were found in the LPS, miR-141-3p inhibitor, mimics control+LPS and inhibitor control+LPS groups. Compared with the LPS, miR-141-3p inhibitor, mimics control+LPS and inhibitor control+LPS groups, the miR141-3p+LPS group had an obviously decreased expression of miR-141-3p and IL-10, increased mRNA and protein expressions of HMGB1 and the levels of IL-1β, TNF-α and IL-6. Compared with the rats in the blank control group, the miR-141-3p expression, IL-10 level, and thermal and mechanical pain thresholds decreased significantly, whereas the mRNA and protein expressions of HMGB1, IL-1β, TNF-α and IL-6 increased significantly in rats in the NC, mimics control+CFA and HMGB1 NC+ CFA groups. The miR-141-3p expression was increased in rats in the miR-141-3p mimics+HMGB1 shRNA+CFA group. Our study demonstrated that miR-141-3p can alleviate the CIP by downregulating the downstream target gene HMGB1.
本研究旨在通过靶向高迁移率族蛋白B1（HMGB1）基因来探究微小RNA-141-3p（miR-141-3p）在慢性炎性疼痛（CIP）中的潜在作用。在体外研究中，选用BV2小胶质细胞并将其分为空白组、脂多糖（LPS）组、miR-141-3p模拟物组、模拟物对照组、miR-141-3p抑制剂组、抑制剂对照组、miR-141-3p模拟物+LPS组、模拟物对照组+LPS组、miR-141-3p抑制剂+LPS组和抑制剂对照组+LPS组。96只大鼠被随机分为8组（每组12只）：空白对照组、模型对照组、阴性对照组（NC）、miR-141-3p模拟物+完全弗氏佐剂（CFA）组、模拟物对照组+CFA组、HMGB1短发夹RNA（shRNA）+CFA组、HMGB1 NC+CFA组和miR-141-3p模拟物+HMGB1 shRNA+CFA组。分别采用定量实时聚合酶链反应、蛋白质免疫印迹法、酶联免疫吸附测定法和疼痛行为测试来检测miR-141-3p和HMGB1 mRNA表达、HMGB1蛋白表达、炎性细胞因子水平以及热痛和机械痛阈值。与空白组、模拟物对照组、抑制剂对照组和miR-141-3p模拟物+LPS组相比，miR-141-3p模拟物组的miR-141-3p表达和白细胞介素（IL）-10水平升高，HMGB1的mRNA和蛋白表达以及IL-1β、肿瘤坏死因子-α（TNF-α）和IL-6水平降低，而在LPS组、miR-141-3p抑制剂组、模拟物对照组+LPS组和抑制剂对照组+LPS组中则发现相反的趋势。与LPS组、miR-141-3p抑制剂组、模拟物对照组+LPS组和抑制剂对照组+LPS组相比，miR141-3p+LPS组的miR-141-3p和IL-10表达明显降低，HMGB1的mRNA和蛋白表达以及IL-1β、TNF-α和IL-6水平升高。与空白对照组的大鼠相比，NC组、模拟物对照组+CFA组和HMGB1 NC+CFA组大鼠的miR-141-3p表达、IL-10水平以及热痛和机械痛阈值显著降低，而HMGB1、IL-1β、TNF-α和IL-6的mRNA和蛋白表达显著升高。miR-141-3p模拟物+HMGB1 shRNA+CFA组大鼠的miR-141-3p表达升高。我们的研究表明，miR-141-3p可通过下调下游靶基因HMGB1来减轻慢性炎性疼痛。
