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Long non-coding RNA MALAT1 enhances angiogenesis during bone regeneration by regulating the miR-494/SP1 axis

Abstract

Bone regeneration is a coordinated process involving connections between blood vessels and osteocytes. Angiogenesis and osteogenesis are tightly connected throughout the progression of bone regeneration. This study aimed to explore the underlying mechanism of metastasis-associated lung adenocarcinoma transcript 1 (MALAT1)-regulated angiogenesis during bone regeneration. Gene and protein expression was detected by quantitative real-time PCR and western blot assay. Vascular endothelial growth factor (VEGFA) secretion was assessed by enzyme-linked immunosorbent assay. To evaluate the effect of osteogenic differentiation, alkaline phosphatase (ALP) and alizarin red staining assays were performed. Proliferation was detected by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Migration and angiogenesis were measured using Transwell and tube formation assays. A dual luciferase reporter assay was performed to confirm the binding relationship among MALAT1, miR-494, and specificity protein 1 (SP1). Expression levels of MALAT1, SP1, and VEGFA were elevated and miR-494 was suppressed in MC3T3-E1 cells after culture in osteogenic medium. MALAT1 knockdown suppressed the osteogenic differentiation of MC3T3-E1, since ALP activity, mineralized nodules, and expression of the osteodifferentiated markers runt-related transcription factor 2 and osterix were restrained. In addition, MALAT1 silencing inhibited angiogenesis during bone regeneration, as the proliferation, migration, and capillary tube formation of human umbilical vein endothelial cells were blocked. Furthermore, miR-494 was directly targeted by MALAT1 and regulated the SP1/Toll-like receptor 2 (TLR2)/bone morphogenetic protein 2 (BMP2) axis by targeting SP1. Furthermore, miR-494 overexpression inhibited angiogenesis and osteogenic differentiation. Moreover, SP1 overexpression or miR-494 inhibition rescued the regulatory effect of sh-MALAT1 on angiogenesis and osteogenic differentiation. Taken together, these findings indicate that MALAT1 promotes angiogenesis and osteogenic differentiation by targeting miR-494 and activating the SP1/TLR2/BMP2 pathway, suggesting a novel target for bone regeneration therapy by promoting angiogenesis.

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Fig. 1: Osteogenic differentiation is suppressed in response to silencing MALAT1.
Fig. 2: Knockdown of MALAT1 represses angiogenesis in vitro.
Fig. 3: MALAT1 mediates the SP1/TLR2/BMP2 axis by targeting miR-494.
Fig. 4: MiR-494 overexpression inhibits angiogenesis and osteogenic differentiation.
Fig. 5: SP1 overexpression or miR-494 knockdown attenuates the inhibition of sh-MALAT1 on angiogenesis and osteogenic differentiation.

Data availability

All data generated or analyzed during this study are included in this published article.

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Funding

This work was supported by Demonstration of special local scientific and technological innovation projects guided by the central government of Anhui Province (YDZX20183400004841).

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A.D.: concepts, design, experimental studies, data analysis, statistical analysis, preparation, editing, review; C.-H.L.: concepts, experimental studies; C.-Y.Y.: data acquisition, data analysis; H.-T.Z.: data analysis; and Z.-H.Z.: supervision, design.

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Correspondence to Zhi-Hong Zhang.

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Ding, A., Li, CH., Yu, CY. et al. Long non-coding RNA MALAT1 enhances angiogenesis during bone regeneration by regulating the miR-494/SP1 axis. Lab Invest 101, 1458–1466 (2021). https://doi.org/10.1038/s41374-021-00649-8

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