J-L Wang1, X Wei, A-G Wang, Y Bai, X-J Wu. 1. Department of Orthopedics, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China. rende726274@126.com.
Abstract
OBJECTIVE: Osteogenic differentiation plays a crucial role in maintaining general bone homeostasis. Long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) act as important regulators during the osteogenesis process. This study aimed to elucidate the mechanism of Potassium voltage-gated channel subfamily Q member 1 overlapping transcript 1 (KCNQ1OT1) in osteogenic differentiation. MATERIALS AND METHODS: Quantitative Real-time polymerase chain reaction (qRT-PCR) was conducted to detect the expression of KCNQ1OT1, osteonectin (OCN), osteopontin (OPN), runt-related transcription factor 2 (RUNX2), miR-320a, mothers against DPP homolog 5 (Smad5). Protein levels of OCN, OPN, RUNX2 and Smad5 were measured by Western blot assay. Alkaline phosphatase (ALP) activity detection assay was performed to examine the ALP activity. The interactions among KCNQ1OT1, miR-320a and Smad5 were determined by dual-luciferase reporter assay. RESULTS: KCNQ1OT1, OCN, OPN and RUNX2 expression were enhanced in human bone marrow-derived mesenchymal stem cells (hBMSCs) treated with osteogenic medium (OM). KCNQ1OT1 positively regulated OCN, OPN and RUNX2 expression and ALP activity of hBMSCs. Furthermore, KCNQ1OT1 directly bound to miR-320a, and KCNQ1OT1 knockdown reduced OCN, OPN and RUNX2 expression and ALP activity by suppressing miR-320a expression. Moreover, Smad5 was a target of miR-320a, and miR-320a inhibition abated the effects of Smad5 silencing in OCN, OPN and RUNX2 expression and ALP activity of hBMSCs. Also, KCNQ1OT1 knockdown reduced OCN, OPN and RUNX2 expression by targeting miR-320a/Smad5 axis. CONCLUSIONS: KCNQ1OT1 promoted osteogenic differentiation of hBMSCs by regulating Smad5 expression via sponging miR-320a.
OBJECTIVE: Osteogenic differentiation plays a crucial role in maintaining general bone homeostasis. Long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) act as important regulators during the osteogenesis process. This study aimed to elucidate the mechanism of Potassium voltage-gated channel subfamily Q member 1 overlapping transcript 1 (KCNQ1OT1) in osteogenic differentiation. MATERIALS AND METHODS: Quantitative Real-time polymerase chain reaction (qRT-PCR) was conducted to detect the expression of KCNQ1OT1, osteonectin (OCN), osteopontin (OPN), runt-related transcription factor 2 (RUNX2), miR-320a, mothers against DPP homolog 5 (Smad5). Protein levels of OCN, OPN, RUNX2 and Smad5 were measured by Western blot assay. Alkaline phosphatase (ALP) activity detection assay was performed to examine the ALP activity. The interactions among KCNQ1OT1, miR-320a and Smad5 were determined by dual-luciferase reporter assay. RESULTS:KCNQ1OT1, OCN, OPN and RUNX2 expression were enhanced in human bone marrow-derived mesenchymal stem cells (hBMSCs) treated with osteogenic medium (OM). KCNQ1OT1 positively regulated OCN, OPN and RUNX2 expression and ALP activity of hBMSCs. Furthermore, KCNQ1OT1 directly bound to miR-320a, and KCNQ1OT1 knockdown reduced OCN, OPN and RUNX2 expression and ALP activity by suppressing miR-320a expression. Moreover, Smad5 was a target of miR-320a, and miR-320a inhibition abated the effects of Smad5 silencing in OCN, OPN and RUNX2 expression and ALP activity of hBMSCs. Also, KCNQ1OT1 knockdown reduced OCN, OPN and RUNX2 expression by targeting miR-320a/Smad5 axis. CONCLUSIONS:KCNQ1OT1 promoted osteogenic differentiation of hBMSCs by regulating Smad5 expression via sponging miR-320a.
Authors: Carmen Lanzillotti; Monica De Mattei; Chiara Mazziotta; Francesca Taraballi; John Charles Rotondo; Mauro Tognon; Fernanda Martini Journal: Front Cell Dev Biol Date: 2021-04-09