Fig. 5

Circ-ITCH promotes the ubiquitination degradation of HOXC10 through enhancing BRCA1 mRNA stability. (A) The interaction between BRCA1 protein and circ-ITCH was detected by RIP assay. Normal IgG served as a negative control. (B) The mRNA stability of BRCA1 was monitored after overexpressing or silencing circ-ITCH in the presence of transcription inhibitor actinomycin D. Human BM-MSCs were overexpressed circ-ITCH and silenced BRCA1, and then cultured in osteogenic differentiation medium under microgravity simulation for 14 days. (C) The levels of circ-ITCH and BRCA1 mRNA were detected by qRT-PCR. (D) The protein level of BRCA1 was detected by western blot. (E) The ubiquitination of HOXC10 in BM-MSCs were detected by Co-IP assay. (F) The interaction between BRCA1 and HOXC10 in BM-MSCs were assessed by Co-IP assay. Whole lysates or normal IgG served as an input or negative control, respectively. (G) The mineralization process in BM-MSCs was monitored by ARS staining. (H) The protein levels of osteogenic differentiation markers ALP, OPN and OCN in BM-MSCs were detected by western blot. *P < 0.05, **P < 0.01, and ***P < 0.001