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Published on: February 28, 2017
LINC01089 governs the miR-1287-5p/HSPA4 axis to negatively regulate osteogenic differentiation of mesenchymal stem
1Department of Orthopedics, Xiangyang Hospital of Integrated Traditional Chinese and Western Medicine, Xiangyang, China.
Aims:
The involvement of long non-coding RNA (lncRNA) in bone marrow mesenchymal stem cell (MSC) osteogenic differentiation during osteoporosis (OP) development has attracted much attention. In this study, we aimed to disclose how LINC01089 functions in human mesenchymal stem cell (hMSC) osteogenic differentiation, and to study the mechanism by which LINC01089 regulates MSC osteogenesis.
Methods:
Quantitative reverse transcription polymerase chain reaction (RT-qPCR) and western blotting were performed to analyze LINC01089, miR-1287-5p, and heat shock protein family A (HSP70) member 4 (HSPA4) expression. The osteogenic differentiation of MSCs was assessed through alkaline phosphatase (ALP) activity, alizarin red S (ARS) staining, and by measuring the levels of osteogenic gene marker expressions using commercial kits and RT-qPCR analysis. Cell proliferative capacity was evaluated via the Cell Counting Kit-8 (CCK-8) assay. The binding of miR-1287-5p with LINC01089 and HSPA4 was verified by performing dual-luciferase reporter and RNA immunoprecipitation (RIP) experiments.
Results:
LINC01089 expression was reinforced in serum samples of OP patients, but it gradually diminished while hMSCs underwent osteogenic differentiation. LINC01089 knockdown facilitated hMSC osteogenic differentiation. This was substantiated by: the increase in ALP activity; ALP, runt-related transcription factor 2 (RUNX2), osteocalcin (OCN), and osteopontin (OPN) messenger RNA (mRNA) levels; and level of ARS staining. Meanwhile, LINC01089 upregulation resulted in the opposite effects. LINC01089 targeted miR-1287-5p, and the LINC01089 knockdown-induced hMSC osteogenic differentiation was repressed by miR-1287-5p depletion. HSPA4 is a downstream function molecule of the LINC01089/miR-1287-5p pathway; miR-1287-5p negatively modulated HSPA4 levels and attenuated its functional effects.
Conclusion:
LINC01089 negatively regulated hMSC osteogenic differentiation, at least in part, via governing miR-1287-5p/HSPA4 signalling. These findings provide new insights into hMSC osteogenesis and bone metabolism.
Insights
Long non-coding RNA LINC01089 inhibits human mesenchymal stem cell osteogenic differentiation by regulating the miR-1287-5p/HSPA4 pathway. This study reveals LINC01089
Area of Science:
- Stem cell biology
- Molecular biology
- Bone biology
Background:
- Long non-coding RNAs (lncRNAs) play critical roles in cellular differentiation.
- Osteoporosis (OP) is a bone disease characterized by impaired osteogenic differentiation of mesenchymal stem cells (MSCs).
- Understanding the regulatory mechanisms of MSC osteogenesis is crucial for developing OP treatments.
Purpose of the Study:
- To investigate the function of LINC01089 in human mesenchymal stem cell (hMSC) osteogenic differentiation.
- To elucidate the molecular mechanism by which LINC01089 regulates MSC osteogenesis.
Main Methods:
- Quantitative reverse transcription polymerase chain reaction (RT-qPCR) and western blotting to assess gene and protein expression.
- Assays for osteogenic differentiation: alkaline phosphatase (ALP) activity, alizarin red S (ARS) staining, and osteogenic gene marker analysis.
- Dual-luciferase reporter and RNA immunoprecipitation (RIP) assays to confirm molecular interactions.
Main Results:
- LINC01089 expression was elevated in OP patients and decreased during hMSC osteogenic differentiation.
- LINC01089 knockdown promoted hMSC osteogenic differentiation, evidenced by increased ALP activity, osteogenic gene expression, and ARS staining.
- LINC01089 directly targeted miR-1287-5p, and miR-1287-5p regulated the expression of its downstream target, HSPA4.
Conclusions:
- LINC01089 negatively regulates hMSC osteogenic differentiation through the miR-1287-5p/HSPA4 signaling pathway.
- These findings offer novel insights into the molecular mechanisms governing hMSC osteogenesis and bone metabolism.
- LINC01089 represents a potential therapeutic target for osteoporosis.
