title: Role of miR-146a in human chondrocyte apoptosis in response to mechanical pressure injury in vitro.
abstract: MicroRNA (miR)-146a is known to be overexpressed in osteoarthritis (OA). However, the role of miR-146a in OA has not yet been fully elucidated. In the present study, we applied mechanical pressure of 10 MPa to human chondrocytes for 60 min in order to investigate the expression of miR-146a and apoptosis following the mechanical pressure injury. Normal human chondrocytes were transfected with an miR-146a mimic or an inhibitor to regulate miR-146a expression. Potential target genes of miR-146a were predicted using bioinformatics. Moreover, luciferase reporter assay confirmed that Smad4 was a direct target of miR-146a. The expression levels of miR-146a, Smad4 and vascular endothelial growth factor (VEGF) were quantified by quantitative reverse transcription PCR and/or western blot analysis. The effects of miR-146a on apoptosis were detected by Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) flow cytometry. The results indicated that mechanical pressure affected chondrocyte viability and induced the early apoptosis of chondrocytes. Mechanical pressure injury increased the expression levels of miR-146a and VEGF and decreased the levels of Smad4 in the chondrocytes. In the human chondrocytes, the upregulation of miR-146a induced apoptosis, upregulated VEGF expression and downregulated Smad4 expression. In addition, the knockdown of miR-146a reduced cell apoptosis, upregulated Smad4 expression and downregulated VEGF expression. Smad4 was identified as a direct target of miR-146a by harboring a miR‑146a binding sequence in the 3'-untranslated region (3'-UTR) of its mRNA. Furthermore, the upregulation of VEGF induced by miR‑146a was mediated by Smad4 in the chondrocytes subjected to mechanical pressure injury. These results demonstrated that miR-146a was overexpressed in our chondrocyte model of experimentally induced human mechanical injury, accompanied by the upregulation of VEGF and the downregulation of Smad4 in vitro. Moreover, our data suggest that miR-146a is involved in human chondrocyte apoptosis in response to mechanical injury, and may contribute to the mechanical injury of chondrocytes, as well as to the pathogenesis of OA by increasing the levels of VEGF and damaging the transforming growth factor (TGF)-β signaling pathway through the targeted inhibition of Smad4 in cartilage.
authors.affiliation: Department of Orthopedics, Tangdu Hospital, Fourth Military Medical University, Xi'an, Shaanxi 710038, P.R. China.
authors.forename: Lei
authors.initials: L
authors.lastname: Jin
authors.affiliation: Department of Orthopedics, Tangdu Hospital, Fourth Military Medical University, Xi'an, Shaanxi 710038, P.R. China.
authors.forename: Jian
authors.initials: J
authors.lastname: Zhao
authors.affiliation: Department of Orthopedics, Tangdu Hospital, Fourth Military Medical University, Xi'an, Shaanxi 710038, P.R. China.
authors.forename: Wensen
authors.initials: W
authors.lastname: Jing
authors.affiliation: Department of Orthopedics, Tangdu Hospital, Fourth Military Medical University, Xi'an, Shaanxi 710038, P.R. China.
authors.forename: Shiju
authors.initials: S
authors.lastname: Yan
authors.affiliation: Department of Orthopedics, Tangdu Hospital, Fourth Military Medical University, Xi'an, Shaanxi 710038, P.R. China.
authors.forename: Xin
authors.initials: X
authors.lastname: Wang
authors.affiliation: Department of Orthopedics, Tangdu Hospital, Fourth Military Medical University, Xi'an, Shaanxi 710038, P.R. China.
authors.forename: Chun
authors.initials: C
authors.lastname: Xiao
authors.affiliation: Department of Orthopedics, Tangdu Hospital, Fourth Military Medical University, Xi'an, Shaanxi 710038, P.R. China.
authors.forename: Baoan
authors.initials: B
authors.lastname: Ma
mesh_terms: D006801:Humans; D017209:Apoptosis / Q000235:genetics; D002358:Cartilage, Articular / Q000378:metabolism; D049109:Cell Proliferation / Q000235:genetics; D019902:Chondrocytes / Q000378:metabolism / Q000473:pathology; D005786:Gene Expression Regulation; D066298:In Vitro Techniques; D035683:MicroRNAs / Q000096:biosynthesis / Q000235:genetics; D010003:Osteoarthritis / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D012333:RNA, Messenger / Q000096:biosynthesis; D013314:Stress, Mechanical; D042461:Vascular Endothelial Growth Factor A / Q000096:biosynthesis; D014947:Wounds and Injuries / Q000378:metabolism / Q000473:pathology; D003668:Pressure Ulcer / Q000378:metabolism / Q000473:pathology
chemical_list: D035683:MicroRNAs; C542542:MIRN146 microRNA, human; D012333:RNA, Messenger; D042461:Vascular Endothelial Growth Factor A; C467484:VEGFA protein, human