The METTL14/YTHDF3 axis regulates EPSTI1-mediated osteogenic differentiation of human bone marrow mesenchymal stem cells.

BACKGROUND

Osteoporosis is a disease characterized by impaired bone microarchitecture and reduced bone mass. However, its molecular mechanisms are not fully understood.

METHODS

Reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blot (WB) were used to detect the expression of messenger RNA (mRNA) and proteins. Flow cytometry was used to quantify cell surface markers. Cell transfection was used for gene knockdown and overexpression. Cell counting kit-8 (CCK-8) and 5-ethynyl-2'-deoxyuridine (EdU) were used to assess cell viability and proliferation. Alkaline phosphatase (ALP) staining and alizarin red S (ARS) staining were used to evaluate ALP activity and osteogenic differentiation. RNA modification variant database 2.0 (RMVar 2.0), RNA modification base database 3.0 (RMbase V3.0), and sequence-based RNA adenosine methylation site predictor (SRAMP) were used to predict RNA modifications. RNA-protein interaction (RPI) database was used to predict RNA-protein interactions. RNA immunoprecipitation (RIP) and methylated RNA immunoprecipitation (MeRIP) were used to detect RNA-protein binding and RNA chemical modifications. Actinomycin D (Act D) was used to study mRNA stability. The encyclopedia of RNA interactomes (ENCORI) was used to predict interactions among RNA molecules.

RESULTS

Epithelial-stromal interaction 1 (EPSTI1) was highly expressed in osteoporosis. Knockdown of EPSTI1 promoted hBMSC viability, proliferation, and osteogenic differentiation, whereas its overexpression produced the opposite effects. Methyltransferase-like 14 (METTL14) and YTH N6-methyladenosine RNA binding protein 3 (YTHDF3) co-regulated the N6-methyladenosine (m6A) modification of EPSTI1. METTL14 overexpression enhanced hBMSC viability, proliferation, and osteogenic differentiation, but simultaneous overexpression of EPSTI1 partially offset these promoting effects.

CONCLUSION

METTL14 and YTHDF3 regulate EPSTI1 expression through its m6A modification, thereby modulating proliferation and osteogenic differentiation of hBMSCs.