miR-30a inhibits the osteogenic differentiation of the tibia-derived MSCs in congenital pseudarthrosis via targeting HOXD8

Weihua Ye; Yiyong Huang; Guanghui Zhu; An Yan; Yaoxi Liu; Han Xiao; Haibo Mei

doi:10.1016/j.reth.2022.09.005

miR-30a inhibits the osteogenic differentiation of the tibia-derived MSCs in congenital pseudarthrosis via targeting HOXD8

Regen Ther. 2022 Oct 20:21:477-485. doi: 10.1016/j.reth.2022.09.005. eCollection 2022 Dec.

Authors

Weihua Ye¹, Yiyong Huang¹, Guanghui Zhu¹, An Yan¹, Yaoxi Liu¹, Han Xiao¹, Haibo Mei¹

Affiliation

¹ Department of Orthopaedics, Hunan Children's Hospital, Changsha 410007, Hunan Province, PR China.

Abstract

Background: Congenital pseudarthrosis of the tibia (CPT) is an uncommon congenital deformity and a special subtype of bone nonunion. The lower ability of osteogenic differentiation in CPT-derived mesenchymal stem cells (MSCs) could result in progression of CPT, and miR-30a could inhibit osteogenic differentiation. However, the role of miR-30a in CPT-derived MSCs remains unclear.

Methods: The osteogenic differentiation of CPT-derived MSCs treated with the miR-30a inhibitor was tested by Alizarin Red S staining and alkaline phosphatase (ALP) activity. The expression levels of protein and mRNA were assessed by Western blot or quantitative reverse transcription-polymerase chain reaction (RT-qPCR), respectively. The interplay between miR-30a and HOXD8 was investigated by a dual-luciferase reporter assay. Chromatin immunoprecipitation (ChIP) was conducted to assess the binding relationship between HOXD8 and RUNX2 promoter.

Results: CPT-derived MSCs showed a lower ability of osteogenic differentiation than normal MSCs. miR-30a increased in CPT-derived MSCs, and miR-30a downregulation promoted the osteogenic differentiation of CPT-derived MSCs. Meanwhile, HOXD8 is a direct target for miR-30a, and HOXD8 could transcriptionally activate RUNX2. In addition, miR-30a could inhibit the osteogenic differentiation of CPT-derived MSCs by negatively regulating HOXD8.

Conclusion: miR-30a inhibits the osteogenic differentiation of CPT-derived MSCs by targeting HOXD8. Thus, this study might supply a novel strategy against CPT.

Keywords: 3′-UTR, 3′-untranslated region; ADSCs, adipose-derived mesenchymal stem cells; ALP, alkaline phosphatase; ARS, Alizarin Red S; CPT, congenital pseudarthrosis of the tibia; ChIP, chromatin immunoprecipitation; Congenital pseudarthrosis of the tibia; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; HOXD8; HOXD8, Homeobox D8; MSCs, mesenchymal stem cells; OCN, osteocalcin; OPN, osteopontin; RT-qPCR, Quantitative reverse transcription PCR; RUNX2; RUNX2, runt-related transcription factor 2; SD, standard deviation; miR-30a; miRNAs, MicroRNAs; mut, mutant; wt, wild-type; α-MEM, α-minimum essential medium.