Filamin B knockdown impairs differentiation and function in mouse pre-osteoblasts via aberrant transcription and alternative splicing

Abstract

Objective: Filamin B (FLNB) encodes an actin-binding protein that is known to function as a novel RNA-binding protein involved in cell movement and signal transduction and plays a pivotal role in bone growth. This study aimed to investigate possible FLNB function in the skeletal system by characterizing the effecs of FLNB knockdown in mouse preosteoblast cells. Methods: Stable FLNB MC3T3-E1 knockdown cells were constructed for RNA-seq and alternative splicing event (ASE) analysis of genes involved in osteoblast differentiation and function that may be regulated by FLNB. Standard transwell, MTT, ALP, qPCR, Western blot, and alizarin red staining assays were used to assess functional changes of FLNB-knockdown MC3T3-E1 cells. Results: Analysis of differentially expressed genes (DEGs) in FLNB knockdown cells revealed enrichment for genes related to osteoblast proliferation, differentiation and migration, such as ITGA10, Cebpβ, Grem1, etc. Alternative splicing (AS) analysis showed changes in the predominant mRNA isoforms of skeletal development-related genes, especially Tpx2 and Evc. Functional asslysis indicated that proliferation, migration, and differentiation were all inhibited upon FLNB knockdown in MC3T3-E1 cells compared to that in vector control cells. Conclusions: FLNB participates in regulating the transcription and AS of genes required for osteoblast development and function, consequently affecting growth and development in MC3T3-E1 cells.

Keywords

• Filamin B
• MC3T3-E1
• Alternative splicing
• RNA-Sequencing