MiR-101-3p通过靶向抑制斯钙素1减轻IL-1β诱导的软骨细胞损伤

目的：探究微小RNA-101-3p (microRNA-101-3p，miR-101-3p)对IL-1β诱导的骨关节软骨细胞损伤的影响及其 潜在的机制。方法：将骨关节软骨细胞随机分为对照组(NC组)、IL-1β组、阴性对照组(IL-1β+miR-NC组)，miR-101- 3p组(IL-1β+miR-101-3p组)，细胞转染后再用IL-1β(10 ng/mL)诱导软骨细胞。Real-time PCR和蛋白质印迹法检测不同 浓度的IL-1β(0，1，5，10 ng/mL)诱导的细胞中miR-101-3p和斯钙素1(stanniocalcin 1，STC1)的表达；采用MTT检测细 胞增殖；流式细胞仪检测细胞凋亡；caspases检测试剂盒检测含半胱氨酸的天冬氨酸蛋白水解酶3/9(caspase-3/9)的表 达；蛋白质印迹法检测炎性和细胞外基质(extracellular matrix，ECM)、金属蛋白酶9(matrix metalloproteinase 9，MMP9) 和II型胶原的表达。另外，将野生型STC1 的3'-非编码区(untranslated regions，UTR)(STC1-3'-UTR-WT)或者突变型STC1 的3'-UTR(STC1-3'-UTR-MUT)分别与miR-101-3p模拟物(miR-101-3p mimic)和对照物(miR-NC)共转染细胞，采用荧光素 酶报告实验检测miR-101-3p对STC1的调控作用。在miR-NC(miR-NC组)，miR-101-3p(miR-101-3p组)，anti-NC(anti-NC 组)和anti-miR-101-3p(anti-miR-101-3p组)分别转染细胞后，采用蛋白质印迹法检测细胞中STC1蛋白的表达。为检测 STC1是否参与miR-101-3p对软骨细胞的作用，将细胞随机分为miR-101-3p组(IL-1β+miR-101-3p组)、过表达对照组(IL- 1β+miR-101-3p+ad-GFP组)、过表达STC1组(IL-1β+miR-101-3p+ad-STC1组)。同样分别用MTT 检测细胞增殖；流式细胞 仪检测细胞凋亡；caspases检测试剂盒检测caspase-3/9的表达；蛋白印迹法检测炎性和ECM相关蛋白的表达。结果： 与0 ng/mL IL-1β相比，不同浓度IL-1β诱导的软骨细胞中miR-101-3p水平显著下降(均P<0.05)，STC1的水平则显著上 升(均P<0.05)。与NC组相比，IL-1β组软骨细胞增殖率下降(P<0.05)，凋亡率增加(P<0.05)，caspase-3/9，IL-6和TNF-α 的水平均显著上升(均P<0.05)，MMP9的表达显著上调(P<0.05)，II型胶原表达显著下调(P<0.05)。与IL-1β+miR-NC组 相比，IL-1β+miR-101-3p组软骨细胞增殖率上升(P<0.05)，凋亡率降低(P<0.05)；caspases，IL-6和TNF-α的水平均显著 下降(均P<0.05)；MMP9的表达下降(P<0.05)，II型胶原表达上调(P<0.05)。荧光素酶报告检测结果表明：与miR-NC 组相比，miR-101-3p组可靶向抑制STC1的水平(P<0.05)。蛋白质印迹法结果表明：与miR-NC组相比，miR-101-3p组 STC1水平下降(P<0.05)；与anti-NC组相比，anti-miR-101-3p组STC1水平上升(P<0.05)。与miR-101-3p+ad-GFP组相比， miR-101-3p+ad-STC1组STC1反转了miR-101-3p对IL-1β诱导的软骨细胞增殖、凋亡、炎性反应及ECM相关蛋白的影响。 结论：调节miR-101-3p/STC1通路能够保护IL-1β诱导的骨关节软骨细胞损伤。

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

Objective: 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 realtime 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 proinflammatory 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.