microRNA-4701-5p protects against interleukin-1β induced human chondrocyte CHON-001 cells injury via modulating HMGA1

Abstract

Background: miRNA-4701-5p has been reported to be a vital regulator in many diseases, including rheumatoid arthritis, and miRNA-4701-5p is evidenced to be participated in synovial invasion and joint destruction. In our report, we investigated the roles of miRNA-4701-5p in osteoarthritis (OA) and analyzed the molecular mechanism.

Methods: Interleukin-1β (IL-1β) was applied for stimulating human chondrocyte CHON-001 cells to establish an OA injury model. mRNA levels and protein expression were measured using qRT-PCR and western blot assay, respectively. The proliferation ability and cytotoxicity of CHON-001 cells were checked using MTT assay and lactate dehydrogenase activity. The inflammation of chondrocytes was accessed by the secretion levels of interleukin-6 (IL-6), interleukin-8 (IL-8) and tumor necrosis factor (TNF)-α. The apoptosis of chondrocytes was determined by flow cytometry assay. Bioinformatics software Starbase v2.0 analyzed the functional binding sites between miRNA-4701-5p and HMGA1 and the interaction was further confirmed using dual luciferase reporter analysis.

Results: miRNA-4701-5p was down-regulated in the IL-1β-stimulated chondrocytes and HMGA1 directly targeted miRNA-4701-5p. Up-regulation of miRNA-4701-5p could alleviate IL-1β-treated CHON-001 cells inflammation and apoptosis, and reversed the cell proliferation decrease and cytotoxicity increase after IL-1β treatment. Nevertheless, all the roles of miRNA-4701-5p overexpression in CHON-001 cells could be reversed by HMGA1 up-regulation.

Conclusions: miRNA-4701-5p could alleviate the inflammatory injury of IL-1β-treated CHON-001 cells via down-regulating HMGA1, indicating that miRNA-4701-5p/HMGA1 is a promising therapeutic target for OA.

Keywords: Chondrocytes; HMGA1; Osteoarthritis; miRNA-4701-5p.

Fig. 1

Expression of miRNA-4701-5p and HMGA1 in IL-1β induced CHON-001 cells. CHON-001 cells were exposed to 10 ng/ml IL-1β for 12 h. A CHON-001 cells proliferation was assessed by MTT assay. B The cytotoxicity of CHON-001 cells was accessed by LDH quantification. C, D Flow cytometry analysis of apoptotic cell. E, F Detection of cleaved-caspase3 expressions. G–I The secretion of IL-6, IL-8 and TNF-α in the supernatant of CHON-001 cells was determined by ELISA. J qRT-PCR analysis of miRNA-4701-5p level. K, L Western blot and qRT-PCR analysis of HMGA1 expression. **p < 0.01 versus control

Fig. 2

miRNA-4701-5p binds directly with HMGA1. A Bioinformatics software Starbase v2.0 analysis predicted the miRNA-4701-5p on HMGA1 3'UTR. B Dual luciferase reporter assay verified the relationship between miRNA-4701-5p and HMGA1. C The expression of miRNA-4701-5p in HEK293T cells was measured using qRT-PCR. **p < 0.01 versus mimic control

Fig. 3

miRNA-4701-5p negatively regulates HMGA1 expression in CHON-001 cells. CHON-001 cells were transfected with mimic control, miRNA-4701-5p mimic, control-plasmid, HMGA1-plasmid for 24 h, respectively. The expression and transcription levels of HMGA1 were determined using western blot assay and qRT-PCR, respectively. A The expression of miRNA-4701-5p was determined by qRT-PCR. B The transcription levels of HMGA1 were determined by qRT-PCR, respectively. C, D The protein expression and transcription levels of HMGA1 were determined using western blot assay and qRT-PCR, respectively. **p < 0.01 versus mimic control; ^##p < 0.01 versus control-plasmid; ^&&p < 0.01 versus miRNA-4701-5p mimic + control-plasmid

Fig. 4

miRNA-4701-5p enhanced IL-1β-stimulated CHON-001 cells viability by down-regulating HMGA1. CHON-001 cells were transfected with mimic control, miRNA-4701-5p mimic, control-plasmid, HMGA1-plasmid for 24 h, respectively. Then the cells were induced by 10 ng/ml IL-1β for 12 h. A qRT-PCR analysis of miRNA-4701-5p expression. B, C Western blot assay and qRT-PCR analysis of HMGA1 expressions in different groups. D CHON-001 cells proliferation ability was detected by MTT assay. E The cytotoxicity of CHON-001 cells was accessed by LDH quantification. **p < 0.01 versus control; ^##p < 0.01 versus IL-1β + mimic control; ^&&p < 0.01 versus IL-1β + miRNA-4701-5p mimic + control-plasmid

Fig. 5

miRNA-4701-5p reduced the apoptosis of IL-1β induced CHON-001 cells by downregulating HMGA1. A, B Flow cytometry analysis of apoptotic cell. C, D Detection of cleaved-caspase3 expression and cleaved-caspase3/GAPDH ratio using western blot assay. **p < 0.01 versus control; ^##p < 0.01 versus IL-1β + mimic control; ^&&p < 0.01 versus IL-1β + miRNA-4701-5p mimic + control-plasmid

Fig. 6

miRNA-4701-5p inhibited the inflammation of IL-1β- induced CHON-001 cells by downregulating HMGA1. The inflammation of CHON-001 cells was assessed using determining the secretion of IL-6 (A), IL-8 (B) and TNF-α (C) via ELISA assay. **p < 0.01 versus control; ^##p < 0.01 versus IL-1β + mimic control; ^&&p < 0.01 versus IL-1β + miRNA-4701-5p mimic + control-plasmid