目的 探讨microRNA-22对软骨细胞自噬的影响及其在骨关节炎(OA)发病中作用机制。方法 原代培养人健康、OA软骨细胞,荧光定量PCR检测microRNA-22在健康、OA软骨细胞中的表达水平;Western blot检测健康、OA软骨细胞中自噬相关蛋白LC3B-Ⅱ、Ⅰ的表达水平;软骨细胞转染microRNA-22模拟物、抑制物,平板克隆形成实验检测microRNA-22对软骨细胞活性的影响,Western blot检测软骨细胞自噬相关蛋白LC3B-Ⅰ、LC3B-Ⅱ及Ⅱ型胶原(Collagen Ⅱ)的表达水平。结果 荧光定量PCR检测结果显示,OA软骨细胞(14例)中microRNA-22表达含量(2.50 ± 0.39)显著增高(P＜0.01),约为健康软骨细胞(12例)(0.98 ± 0.25)的2.5倍。Western blot检测结果显示,与健康软骨细胞相比,OA软骨细胞自噬相关蛋白LC3B的Ⅱ类亚型含量降低;OA软骨细胞(4例)LC3B-Ⅱ/ LC3B-Ⅰ比值为1.25±0.13,健康软骨细胞(4例)LC3B-Ⅱ/ LC3B-Ⅰ比值为1.85 ± 0.21,差异具有统计学意义(P＜0.01)。平板克隆形成实验检测结果显示,与空白对照组(107.33±14.3)相比,软骨细胞转染miR-22模拟物组细胞克隆形成数量(75.67±11.36)降低,软骨细胞转染miR-22抑制物组(128.5±9.95)增高,差异有统计学意义(P＜0.05)。Western blot检测结果显示:与空白对照组细胞相比,miR-22模拟物组软骨细胞中LC3B-Ⅱ含量降低(1.50±0.20),LC3B-Ⅱ/ LC3B-Ⅰ比值降低(1.45±0.13),Ⅱ型胶原Collagen Ⅱ的表达水平降低(0.51±0.09);miR-22抑制物组软骨细胞中LC3B-Ⅱ含量增高(2.36±0.19),LC3B-Ⅱ/ LC3B-Ⅰ比值增高(2.30±0.08),同时Ⅱ型胶原Collagen Ⅱ的表达水平增高(1.31± 0.03),以上差异均有统计学意义(P＜0.05)。结论 microRNA-22抑制软骨细胞自噬,从而促进骨性关节炎的发生与进展。
Abstract
Objective To investigate the effects of microRNA-22 on autophagy in human articular chondrocytes and its role in OA pathogenesis. Methods Primary human articular chondrocytes were obtained from cartilage tissue donated by patients. Real time PCR was used to detect the expression level of microRNA -22 in healthy chondrocytes and osteoarthritis (OA) chondrocytes. Western blot was used to detect the expression of LC3B-Ⅱ、LC3B-Ⅰ in healthy chondrocytes and OA chondrocytes and the ratio of LC3B-Ⅱ/Ⅰ was calculated. After chondrocytes were transfected with miR-22 mimics and miR-22 inhibitor, colony formation assay was performed to evaluate cell viability and Western blot was used to detect the expression of LC3B-Ⅱ, LC3B-Ⅰ and Collagen Ⅱ. Results Real-time PCR suggested that the expression level of microRNA -22 in OA chondrocytes (2.50±0.39) was significantly higher compared with healthy chondrocytes (0.98±0.25). Western blot results showed that LC3B-Ⅱ was inhibited in OA chondrocytes. The ratio of LC3B-Ⅱ/Ⅰ in OA chondrocytes (1.25±0.13) was significantly lower compared with healthy chondrocytes (1.85±0.21). Results from colony formation assay demonstrated that chondrocytes transfected with miR-22 mimics formed less cell colonies (75.67±11.36) than NC group (107.33±14.3) while miR-22 inhibitor transfection promoted chondrocytes formed more cell colonies (128.5±9.95). Western blot results showed that miR-22 mimics transfection significantly reduced the expression level of LC3B-Ⅱ (1.50±0.20), the ratio of LC3B-Ⅱ/Ⅰ (1.45±0.13) and the expression level of Collagen Ⅱ (0.51±0.09) in chondrocyte while miR-22 inhibitor transfection significantly promoted the expression level of LC3B-Ⅱ (2.36±0.19), the ratio of LC3B-Ⅱ/Ⅰ (2.30±0.08) and the expression level of Collagen Ⅱ (1.31±0.03). Conclusions MicroRNA-22 plays a crucial role in OA pathogenesis through inhibiting chondrocytes autophagy and viability and could be a novel therapeutic target of OA.
关键词
microRNA-22 /
自噬 /
骨关节炎 /
软骨细胞
Key words
MicroRNA-22 /
autophagy /
osteoarthritis /
chondrocyte
{{custom_sec.title}}
{{custom_sec.title}}
{{custom_sec.content}}
参考文献
[1] Abramoff B, Caldera F E. Osteoarthritis: pathology, diagnosis, and treatment options[J]. Med Clin North Am, 2020,104(2):293-311.
[2] Hunter D J, March L, Chew M. Osteoarthritis in 2020 and beyond: a Lancet commission[J]. Lancet, 2020,396(10264):1711-1712.
[3] Vina E R, Kwoh C K. Epidemiology of osteoarthritis: literature update[J]. Curr Opin Rheumatol, 2018,30(2):160-167.
[4] Levine B, Kroemer G. Biological functions of autophagy genes: a disease perspective[J]. Cell, 2019,176(1-2):11-42.
[5] Urbanska K, Orzechowski A. The secrets of alternative autophagy[J]. Cells, 2021,10(11): 3241.
[6] Luo P, Gao F, Niu D, et al. The role of autophagy in chondrocyte metabolism and osteoarthritis: a comprehensive research review[J]. Biomed Res Int, 2019,2019:5171602.
[7] Bartel D P. MicroRNAs: genomics, biogenesis, mechanism, and function[J]. Cell, 2004,116(2):281-297.
[8] Rani V, Sengar R S. Biogenesis and mechanisms of microRNA-mediated gene regulation[J]. Biotechnol Bioeng, 2022,119(3):685-692.
[9] Cao Y, Tang S, Nie X, et al. Decreased miR-214-3p activates NF-κB pathway and aggravates osteoarthritis progression[J]. EBioMedicine, 2021,65:103283.
[10] Yan S, Wang M, Zhao J, et al. MicroRNA-34a affects chondrocyte apoptosis and proliferation by targeting the SIRT1/p53 signaling pathway during the pathogenesis of osteoarthritis[J]. Int J Mol Med, 2016,38(1):201-209.
[11] Yang R, Zhang D, Yu K, et al. Detection of miR-22, miR-140 and bone morphogenetic proteins (BMP)-2 expression levels in synovial fluid of osteoarthritis patients before and after arthroscopic debridement[J]. Med Sci Monit, 2018,24:863-868.
[12] Iliopoulos D, Malizos K N, Oikonomou P, et al. Integrative microRNA and proteomic approaches identify novel osteoarthritis genes and their collaborative metabolic and inflammatory networks[J]. PLoS One, 2008,3(11):e3740.
[13] Favero M, Belluzzi E, Ortolan A, et al. Erosive hand osteoarthritis: latest findings and outlook[J]. Nat Rev Rheumatol, 2022,18(3):171-183.
[14] Abdel-Aziz M A, Ahmed H, El-Nekeety A A, et al. Osteoarthritis complications and the recent therapeutic approaches[J]. Inflammopharmacology, 2021,29(6):1653-1667.
[15] Mahmoudian A, Lohmander L S, Mobasheri A, et al. Early-stage symptomatic osteoarthritis of the knee - time for action[J]. Nat Rev Rheumatol, 2021,17(10):621-632.
[16] Sandell L J, Aigner T. Articular cartilage and changes in arthritis. An introduction: cell biology of osteoarthritis[J]. Arthritis Res, 2001,3(2):107-113.
[17] Jiang Y. Osteoarthritis year in review 2021: biology[J]. Osteoarthritis Cartilage, 2022,30(2):207-215.
[18] 刘旭剑, 王东来, 李增怀, 等. miR-29a对于膝关节骨性关节炎大鼠滑膜损伤中的保护作用研究[J]. 现代生物医学进展, 2021,21(19):3649-3653.
[19] Meng C Y, Zhao Z Q, Bai R, et al. MicroRNA22 regulates autophagy and apoptosis in cisplatin resistance of osteosarcoma[J]. Mol Med Rep, 2020,22(5):3911-3921.
[20] Li G, Wang G, Ma L, et al. miR-22 regulates starvation-induced autophagy and apoptosis in cardiomyocytes by targeting p38α[J]. Biochem Biophys Res Commun, 2016,478(3):1165-1172.
[21] Kaushik S, Tasset I, Arias E, et al. Autophagy and the hallmarks of aging[J]. Ageing Res Rev, 2021,72:101468.
[22] Wang S, Deng Z, Ma Y, et al. The role of autophagy and mitophagy in bone metabolic disorders[J]. Int J Biol Sci, 2020,16(14):2675-2691.
[23] Andersson J K, Hagert E, Brittberg M. Cartilage injuries and posttraumatic osteoarthritis in the wrist: a review[J]. Cartilage, 2021,13(1_suppl):156S-168S.
基金
海南省自然科学基金青年基金项目(819QN379);国家自然科学基金面上项目(82172407)
PDF(1279 KB)
