Potential mechanisms of microRNA-141-3p to alleviate chronic inflammatory pain by downregulation of downstream target gene HMGB1: in vitro and in vivo studies.

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-1ß, 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-1ß, 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-1ß, 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.

Leucine-rich repeat-containing G-protein-coupled receptor 5 is associated with invasion, metastasis, and could be a potential therapeutic target in human gastric cancer.

BACKGROUND: Leucine-rich repeat-containing G-protein-coupled receptor 5 (Lgr5), which is identified as a novel intestinal stem cell marker, is overexpressed in various tumours. In this study, we explore Lgr5 expression in gastric carcinoma and analyse its role in invasion, metastasis, and prognosis in carcinoma. METHODS: A combination of immunohistochemistry, western blotting, and quantitative reverse transcription-polymerase chain reaction were used to detect mRNA and protein expression levels of Lgr5 and matrix metalloproteinase 2 (MMP2). Small interfering RNA against Lgr5 was designed, synthesised, and transfected into AGS cells. The effects of Lgr5 siRNA on cell invasion were detected by transwell invasion chamber assay and wound healing assay. RESULTS: Leucine-rich repeat-containing G-protein-coupled receptor 5 expression was significantly higher in gastric carcinomas than in normal mucosa. Leucine-rich repeat-containing G-protein-coupled receptor 5 expression positively correlated with the depth of invasion, lymph node metastasis, distance of metastasis, and MMP2 expression levels. Multivariate analysis showed that Lgr5 had an independent effect on survival, and that it positively correlated with MMP2. Leucine-rich repeat-containing G-protein-coupled receptor 5 siRNAs inhibited Lgr5 mRNA and protein expression. Transwell assays indicated that these siRNAs resulted in significantly fewer cells migrating through the polycarbonate membrane, and wound healing assay also indicated that siRNAs decreased the migration of cells. Inhibition of Lgr5 resulted in a significant decrease in MMP2 and ß-catenin levels compared with those in controls. CONCLUSIONS: Leucine-rich repeat-containing G-protein-coupled receptor 5 was correlated with invasion and metastasis. Leucine-rich repeat-containing G-protein-coupled receptor 5 inhibition could serve as a novel therapeutic approach.