MiRNA-133a-5p inhibits the expression of osteoblast differentiation-associated markers by targeting the 3′ UTR of RUNX2

Wei Zhang, Yonggang Wu, Yasuyuki Shiozaki, Yoshihisa Sugimoto, Tomoyuki Takigawa, Masato Tanaka, Akihiro Matsukawa, Toshihumi Ozaki

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

Recent studies have recognized the involvement of microRNAs (miRNAs) in the development of osteoporosis, which regulate the balance between osteogenesis and osteoclasis. In this study, we investigated the regulation by miRNA-133a-5p on the osteoblast differentiation-associated markers in the mouse osteoblast-like MC3T3-E1 cells by RUNX2. First, we manipulated the miRNA-133a level in the MC3T3-E1 cells with 20 or 40 nM miR-133a-5p mimics, miR-133a-5p inhibitor, or scramble miRNA. Then, we quantified with real-time polymerase chain reaction (qRT-PCR) the expression of Collagen I, osteocalcin (OCN), and osteopontin (OPN) in the miR-133a-5p-manipulated MC3T3-E1 cells. And the confocal microscopy was also utilized to confirm the regulation by miR-133a-5p on the expression of the three molecules. We also investigated the extracellular matrix (ECM) mineralization and the alkaline phosphatase (ALP) activity in the miR-133a-5p-manipulated MC3T3-E1 cells. In addition, we explored the possible targeting by miR-133a-5p on RUNX2, which was a well-recognized promoter to osteoblast differentiation, with luciferase reporter, qRT-PCR, and Western blotting assay. Results demonstrated that the miRNA-133a-5p mimics markedly reduced, whereas the miRNA-133a-5p inhibitor significantly promoted the expression of Collagen I, OCN, and OPN, the ECM mineralization, and the ALP activity in MC3T3-E1 cells. The alignment analysis demonstrated a high homology between miRNA-133a-5p and the 3′ UTR of RUNX2. Moreover, the luciferase reporter assay demonstrated that miRNA-133a-5p targeted the 3′ UTR of RUNX2, and inhibited the expression of RUNX2 in both mRNA and protein levels. In conclusion, we identified the inhibition by miRNA-133a-5p to the expression of osteoblast differentiation markers, to the ECM mineralization, and to the ALP activity in MC3T3-E1 cells, by targeting the 3′ UTR of RUNX2. Our study suggests that miRNA-133a-5p might be an important target to inhibit osteoblast differentiation in osteoporosis.

Original languageEnglish
Pages (from-to)199-209
Number of pages11
JournalDNA and Cell Biology
Volume37
Issue number3
DOIs
Publication statusPublished - Mar 1 2018

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Differentiation Antigens
3' Untranslated Regions
Osteoblasts
MicroRNAs
Extracellular Matrix
Alkaline Phosphatase
Osteopontin
Osteocalcin
Luciferases
Osteoporosis
Real-Time Polymerase Chain Reaction
Collagen
Osteogenesis
Confocal Microscopy
Western Blotting
Messenger RNA

Keywords

  • miRNA-133a-5p
  • osteoblast differentiation
  • osteoporosis
  • RUNX2

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics
  • Cell Biology

Cite this

MiRNA-133a-5p inhibits the expression of osteoblast differentiation-associated markers by targeting the 3′ UTR of RUNX2. / Zhang, Wei; Wu, Yonggang; Shiozaki, Yasuyuki; Sugimoto, Yoshihisa; Takigawa, Tomoyuki; Tanaka, Masato; Matsukawa, Akihiro; Ozaki, Toshihumi.

In: DNA and Cell Biology, Vol. 37, No. 3, 01.03.2018, p. 199-209.

Research output: Contribution to journalArticle

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AU - Takigawa, Tomoyuki

AU - Tanaka, Masato

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