MicroRNA-9 regulates the development of knee osteoarthritis through the NF-kappaB1 pathway in chondrocytes
Authors: Ronghe Gu
Jie Yang
Simin Luo
Weiguo Huang
Ning Liu
Zhengang Zha
Journal: Medicine (2016)
Discipline: CLINICAL MEDICINE
Keywords: microRNA-9; knee osteoarthritis; NF-κB1; chondrocytes; proliferation
Affiliations: aDepartment of Orthopedics, The First Affiliated Hospital of Jinan University, Guangzhou bDepartment of Orthopedics, The First People's Hospital of Nanning, Nanning, China.

Abstract:  It has been suggested that microRNA-9 (miR-9) is associated with the development of knee osteoarthritis (OA). This study was aimed to investigate the association between the mechanism of miR-9 targeting nuclear factor kappa-B1 (NF-κB1) and the proliferation and apoptosis of knee OA chondrocytes.Cartilage samples were collected from 25 patients with knee OA and 10 traumatic amputees, and another 15 OA rat models, together with 15 rats without knee OA lesions were also established. MiR-9 expressions in both knee OA cartilage and normal cartilage samples were detected using quantitative real-time PCR. The expressions of related genes (NF-κB1, IL-6, and MMP-13) in the two groups were also detected. Dual luciferase reporter gene assay was employed to examine the effect of miR-9 on the luciferase activity of NF-κB1 3'UTR. Knee OA chondrocytes were transfected with miR-9 mimics, miR-9 inhibitor, and NF-κB1 siRNA, respectively, and changes in cellular proliferation and apoptosis were detected via MTT assay and flow cytometric analysis, respectively. Western blotting assay was used to detect the expressions of NF-κB1, interleukin-6 (IL-6), and matrix metalloproteinase-13 (MMP-13).According to results from human OA samples and rat OA models, miR-9 was significantly downregulated in knee OA cartilage tissues compared with normal cartilage tissues (P &lt; 0.01). The expressions of NF-κB1, IL-6, and MMP-13 in knee OA cartilage tissues were significantly higher than those in normal cartilage tissues (P &lt; 0.01). Dual luciferase reporter gene assay showed that miR-9 could bind to the 3'UTR of NF-κB1 and significantly inhibit the luciferase activity by 37% (P &lt; 0.01). Upregulation of miR-9 or downregulation of NF-κB1 could promote cell proliferation and suppress cell apoptosis.Conclusively, downregulated miR-9 can facilitate proliferation and antiapoptosis of knee OA chondrocytes by directly binding to NF-kB1, implying that stimulating miR-9 expressions might assist in treatment of knee OA. 

Retracted: No

PubPeer comments:
 **Wolters Kluwer Will Conduct An Investigation (Email From Product Manager)** **From: Victoria Ruhle (Victoria.Ruhle@wolterskluwer.com)** **Data: 2020-04-28 01:39** Dear Dr. ***, I hope this email finds you in good health. By way of introduction, my name is Victoria Ruhle and I am the Publisher of the journal Medicineu00ae. My colleagues forwarded me your concerns about the publisher article u201cMicroRNA-9 regulates the development of knee osteoarthritis through the NF-kappaB1 pathway in chondrocytesu201d. Thank you for raising your concerns. We will be conducting an investigation based on your comments and have contacted the authors for the original data for review by an editor. Best Regards, Victoria Ruhle Product Manager Health Learning, Research Practice Wolters Kluwer 2001 Market Street, Philadelphia, PA 19103 Telephone: (215) 521-8317 Email: Victoria.Ruhle@wolterskluwer.com , 
 1. In Figure 1, it s impossible to incubate three different antibodies in the same membrane by western blot. ![file] 2. The problem for WB also presented in OA tissues of SD rats in Figure 2. In addition, the lane width seems to be different for different groups. Only qRT-PCR and WB were conducted after ACLT-induced OA rat model being established. ![file] 3. In a previous study about miR-9, Makki et al. found overexpression of miR-9 in OA tissues and IL-1beta induced chondrocytes, which was opposite with the conclusion of this study. Reference: Makki MS, Haseeb A, Haqqi TM. MicroRNA-9 promotion of interleukin-6 expression by inhibiting monocyte chemoattractant protein-induced protein 1 expression in interleukin-1u03b2-stimulated human chondrocytes. Arthritis Rheumatol. 2015;67(8):2117-28. 4. Contrary to the results of this study, Du2018Adamo et al. demonstrated that overexpression of miR-9 in OA tissues induced a higher apoptosis rate and Caspase activity of chondrocytes compared with NC group. Reference: D Adamo S, Cetrullo S, Guidotti S, Borzu00ec RM, Flamigni F. Hydroxytyrosol modulates the levels of microRNA-9 and its target sirtuin-1 thereby counteracting oxidative stress-induced chondrocyte death. Osteoarthritis Cartilage. 2017;25(4):600-610. .3C? , 
 Fig 5A.  The apoptosis of chondrocytes was inhibited by miR-9.The apoptosis ability of chondrocytes at 48 hours after transfection was detected by the flow cytometric analysis. Flow cytometry. What could go wrong? I would expect to see **points** rather than hazy nebulae, but perhaps that s just me. ![file] ,