MicroRNA-495 downregulates AQP1 and facilitates proliferation and differentiation of osteoblasts in mice with tibial fracture through activation of p38 MAPK signaling pathway
Authors: Gang Wang
Hong Zhang
Zun-Wen Lin
Lei Zhu
Ben Liu
Qing-Jia Xu
Journal: Scientific Reports (2019)
Discipline: Multidisciplinary
Keywords: Osteoblasts; microRNA-495; aquaporin-1; p38 MAPK signaling pathway; tibial fractures
Affiliations: Department of Hand Surgery, Department of Foot and Ankle Surgery, Department of Orthopedics, Qilu Hospital of Shandong University, Jinan, 250012, P.R. China., Department of Orthopedics, The First Affiliated Hospital of Nanchang University, Nanchang, 330006, P.R. China., Department of Hand Surgery, Department of Foot and Ankle Surgery, Department of Orthopedics, Qilu Hospital of Shandong University, Jinan, 250012, P.R. China. .

Abstract:  Osteoblasts are implicated in the building of the vertebrate skeleton. The current study aimed to investigate the role of microRNA-495 (miR-495) in the osteoblasts of mice with tibial fractures and the underlying mechanism involving in aquaporin-1 (AQP1) and the p38 mitogen-activated protein kinase (p38 MAPK) signaling pathway. Initially, a microarray-based analysis was performed to screen the differentially expressed genes and miRNAs associated with tibial fracture. Following the establishment of a tibial fracture mouse model, the positive rate of the AQP1 protein in the fracture tissue was detected by immunohistochemistry (IHC). Next, to verify the binding site between miR-495 on AQP1, bioinformatics data were employed in addition to the application of a dual-luciferase reporter gene assay. The osteoblast cell line MC3T3-E1 was treated with miR-495 mimic, miR-495 inhibitor and Anisomycin to explore the potent effects of miR-495 on proliferation and differentiation of osteoblasts in mice with tibial fracture. The expression of miR-495, AQP1, p38 MAPK, PCNA, Cyclin D1, OCN, and OPN was subsequently evaluated by RT-qPCR and Western blot analysis. Cell viability, the number of calcium nodules and alkaline phosphatase (ALP) activity were detected by MTT assay, alizarin red staining, and ALP activity assay, respectively. Our results revealed that miR-495 was down-regulated while AQP1 was up-regulated in the mice with tibial fractures. AQP1 was verified as a target gene of miR-495. When the cells were treated with overexpressed miR-495 or activated p38 MAPK signaling pathway, elevated expression of PCNA, Cyclin, D1, OCN, and OPN along with an increased amount of calcium nodules, higher cell viability, and enhanced ALP activity was detected, while the expression of AQP1 was reduced. Collectively, the key findings of the present study support the notion that overexpressed miR-495 may activate the p38 MAPK signaling pathway to inhibit AQP1 and to promote the proliferation and differentiation of osteoblasts in mice with tibial fracture. 

Retracted: No

PubPeer comments:
 This paper is part of a set of at least 14 papers with shared authors (always including one with the last name Galdiero), spanning 1995-2008, that all appear to show the same gels. The porin gels appear to all represent the same sample (Salmonella typhimurium porins), but the LPS gels represent Salmonella, Pasteurella multocida, or Pasteurella haemolytica). Sometimes fewer or more lanes are visible, and in each paper the data are represented as novel. Here are the papers: * Mariateresa Vitiello et al., Immunology and Cell Biology (1995), DOI: 10.1038 icb.1995.70 - https: pubpeer.com publications 17EC2E40E190E2FCC6F181A2293361 * M. Galdiero et al., Immunology (1995), PMID: 8567029 - https: pubpeer.com publications DECE082D02578F84676293D3A3D307 * L. Sommese et al., J Med Microbiology (1996), DOI: 10.1099 00222615-45-1-40 - https: pubpeer.com publications 7DF0A919C0BB60C8DD14EA1AA8DF34 * M Galdiero et al., Immunology (1998), DOI: 10.1046 j.1365-2567.1998.00492.x - https: pubpeer.com publications BE8182500B61343C93C4D083288F5A * Giuseppe Iovane et al., Veterinary Immunology and Immunopathology (1998), DOI: 10.1016 S0165-2427(98)00183-4 - https: pubpeer.com publications 6B20332BB1333F6A9128F81ADE8047 * M. Galdiero et al., Clin Exp Immunol (1999), DOI: 10.1046 j.1365-2249.1999.00904.x - https: pubpeer.com publications 7489649618C468805E4E8A2DA683BB * Antonella Marcatili et al., Research in Microbiology (2000), DOI: 10.1016 S0923-2508(00)00142-X - https: pubpeer.com publications AE562BC01FEC2640B955C330A0B013 * M. Galdiero et al., Microbiology (2001), DOI: 10.1099 00221287-147-10-2697 - https: pubpeer.com publications 511A96912036B49FECC6CEB6F1CBF2 * Massimiliano Galdiero et al., Infection and Immunity (2002), DOI: 10.1128 iai.70.2.558-568.2002 - https: pubpeer.com publications A8765584CC85E943BCA5CB79901DAC * M. Galdiero et al., Journal of Infection (2003), DOI: 10.1053 jinf.2002.1068 - https: pubpeer.com publications FED37BABEA925C28B2CF7BF86CE5B8 * M. Galdiero et al., Clin Microbiol Infect (2003), DOI: 10.1046 j.1469-0691.2003.00728.x - https: pubpeer.com publications FC98EF7F5F87C7AE81F88F12D9C8F4 * M. Vitiello et al., Cytokine (2004), DOI: 10.1016 j.cyto.2004.03.010 - https: pubpeer.com publications 47A3D51AB3A1C226D75197C36D5753 * M. Galdiero et al., Med Microbiol Immunol (2005), DOI: 10.1007 s00430-003-0209-7 - https: pubpeer.com publications 850E4A39A93C3F04AC922264AFC923 * M. Vitiello et al., Cytokine (2008), DOI: 10.1016 j.cyto.2007.11.021 - https: pubpeer.com publications 1EB6BE6DC91E81C414E128642A2FA9 Here is a compilation of most of these gels, as made by Edentulina Martensi, for which I thank this PubPeer user. ![file] ![file] ![file] , 
 Adding some more papers to the ones found by Dr. Bik and me. Dr. Bik:  This paper is part of a set of at least 14 papers with shared authors (always including one with the last name Galdiero), spanning 1995-2008, that all appear to show the same gels. The porin gels appear to all represent the same sample (Salmonella typhimurium porins), but the LPS gels represent Salmonella, Pasteurella multocida, or Pasteurella haemolytica). Sometimes fewer or more lanes are visible, and in each paper the data are represented as novel. Here is a compilation of most of these gels, as made by Edentulina Martensi, for which I thank this PubPeer user. Here are the papers: - Mariateresa Vitiello et al., Immunology and Cell Biology (1995), DOI: 10.1038 icb.1995.70 - https: pubpeer.com publications 17EC2E40E190E2FCC6F181A2293361 - M. Galdiero et al., Immunology (1995), PMID: 8567029 - https: pubpeer.com publications DECE082D02578F84676293D3A3D307 - L. Sommese et al., J Med Microbiology (1996), DOI: 10.1099 00222615-45-1-40 - https: pubpeer.com publications 7DF0A919C0BB60C8DD14EA1AA8DF34 - M Galdiero et al., Immunology (1998), DOI: 10.1046 j.1365-2567.1998.00492.x - https: pubpeer.com publications BE8182500B61343C93C4D083288F5A - Giuseppe Iovane et al., Veterinary Immunology and Immunopathology (1998), DOI: 10.1016 S0165-2427(98)00183-4 https: pubpeer.com publications 6B20332BB1333F6A9128F81ADE8047 - M. Galdiero et al., Clin Exp Immunol (1999), DOI: 10.1046 j.1365-2249.1999.00904.x - https: pubpeer.com publications 7489649618C468805E4E8A2DA683BB - Antonella Marcatili et al., Research in Microbiology (2000), DOI: 10.1016 S0923-2508(00)00142-X - https: pubpeer.com publications AE562BC01FEC2640B955C330A0B013 - M. Galdiero et al., Microbiology (2001), DOI: 10.1099 00221287-147-10-2697 - https: pubpeer.com publications 511A96912036B49FECC6CEB6F1CBF2 - Massimiliano Galdiero et al., Infection and Immunity (2002), DOI: 10.1128 iai.70.2.558-568.2002 - https: pubpeer.com publications A8765584CC85E943BCA5CB79901DAC - M. Galdiero et al., Journal of Infection (2003), DOI: 10.1053 jinf.2002.1068 - https: pubpeer.com publications FED37BABEA925C28B2CF7BF86CE5B8 - M. Galdiero et al., Clin Microbiol Infect (2003), DOI: 10.1046 j.1469-0691.2003.00728.x - https: pubpeer.com publications FC98EF7F5F87C7AE81F88F12D9C8F4 - M. Vitiello et al., Cytokine (2004), DOI: 10.1016 j.cyto.2004.03.010 - https: pubpeer.com publications 47A3D51AB3A1C226D75197C36D5753 - M. Galdiero et al., Med Microbiol Immunol (2005), DOI: 10.1007 s00430-003-0209-7 - https: pubpeer.com publications 850E4A39A93C3F04AC922264AFC923 - M. Vitiello et al., Cytokine (2008), DOI: 10.1016 j.cyto.2007.11.021 - https: pubpeer.com publications 1EB6BE6DC91E81C414E128642A2FA9 ![file] ![file] ![file]  Here my edit: - M. Galdiero et al., Immunobiology (2000) DOI: 10.1016 S0171-2985(00)80030-3 https: pubpeer.com publications DBF917296422998A5EC5E6A50170A9 - G. Iovane et al., FEMS Immunology and Medical Microbiology (1998) DOI: 10.1111 j.1574-695x.1998.tb01108.x https: pubpeer.com publications 6A9702D24AB5F8283FCB210414880C - Marilena Galdiero et al., INFECTION AND IMMUNITY (2004), DOI: 10.1128 IAI.72.2.1204u20131209.2004 https: pubpeer.com publications 81F27E6CD12B2FCE200A53F1EDE8A9 In particular, in the lattest, of the rows in Figure 1 has a lot of similarities in common with another one in another paper , but there is one big difference (green arrow). Here compared: ![file] ,