[MiR-101-3p alleviates IL-1β-induced chondrocyte injury by targeting stanniocalcin 1]

Abstract

To explore the effects of miR-101-3p on IL-1β-induced chondrocyte injury and its underlying mechanisms. Methods: Chondrocytes were divided into 4 groups: a control group (NC group), a IL-1β group, a negative control group (IL-1β+miR-NC group), and a miR-101-3p group (IL-1β+miR-101-3p group), which were treated with IL-1β after transfecting with miR-101-3p mimic or negative mimic. The expressions of miR-101-3p-5p and stanniocalcin 1 (STC1) at different concentrations of IL-1β (1, 5, 10 ng/mL)-induced chondrocytes were detected by Western blotting and real-time PCR. MTT assay was used to detect cell proliferation rate, while caspases assay kits and flow cytometry were used to measure the cell caspase and apoptosis level. Western blotting assay was used to detect the expression levels of pro-inflammatory and ECM-related protein, such as matrix metalloproteinase 9 (MMP9) and collagen Type II. In addition, 3'-untranslated regions (UTR) of wild-type STC1 (STC1-3'-UTR-WT) or 3'-UTR of mutant STC1 (STC1-3'-UTR-MUT) were co-transfected with miR-101-3p mimic or miR-NC, respectively, while luciferase reporter assay was used to examine the regulative role of miR-101-3p in STC1. In order to detect whether STC1 was involved in the effect of miR-101-3p on chondrocytes, miR-NC (miR-NC group), miR-101-3p (miR-101-3p group), anti-NC (anti-NC group) and anti-miR-101-3p (anti-miR-101-3p group) were respectively transfected into the cells, and the expression of STC1 protein was detected by Western blotting. Subsequently, the cells were randomly divided into a miR-101-3P group (IL-1β+miR-101-3p group), an over-expression control group (IL-1β+miR-101-3p+ad-GFP group), and an over-expression STC1 group (IL-1β+miR-101-3p+ad-STC1 group) to investigate whether STC1 was involved in the role of miR-101-3p in chondrocyte. Similarly, MTT assay was used to detect cell proliferation rate, caspases assay kits and flow cytometry were used to measure the cell caspase and apoptosis level. Western blotting assay was used to detect the expression levels of pro-inflammatory and ECM-related protein MMP9 and collagen Type II. Results: Compared with the 0 ng/mL IL-1β, the expression of miR-101-3p was decreased in chondrocyte at different concentration of IL-1β (1, 5, 10 ng/mL) (all P<0.05), while the level of STC1 was increased (P<0.05). Compared with the NC group, the chondrocyte proliferation rate was down-regulated (P<0.05), while the apoptosis rate, the levels of caspases, IL-6 and TNF-α were increased in the IL-1β group (P<0.05). Moreover, the MMP9 levels were increased obviously, and the protein levels of collagen Type II were decreased in the IL-1β group compared with the NC group (both P<0.05). Compared with the IL-1β+miR-NC group, the proliferation rate was increased (P<0.05), whereas the apoptosis rates, the caspase-3/9 levels, the IL-6 and TNF-α levels were increased in the IL-1β+miR-101-3p group (all P<0.05). Then MMP9 levels were decreased obviously (P<0.05), and the protein levels of collagen Type II were increased in IL-1β+miR-101-3p group compared with the IL-1β+miR-NC group (both P<0.05). In addition, the double luciferase assay showed that the STC1 levels could be inhibited in the miR-101-3p group compared with the miR-NC group (P<0.05). STC1 levels were decreased in the miR-101-3p group compared with the miR-NC group (P<0.05), and the STC1 levels were increased in the anti-miR-101-3p group compared with those in the anti-NC group (P<0.05). The results of miR-101-3p+ad-STC1 group showed that compared with the miR-101-3p+ad-GFP group, the STC1 could reverse the effects of miR-101-3p on IL-1β-induced proliferation, apoptosis, inflammatory responses and ECM protein of chondrocytes. Conclusion: The regulation of miR-101-3p/STC1 signal pathway may have a role in reducing the IL-1β-induced chondrocyte injury.