Method: Plasmids for LSD1 RNAi and overexpression were constructed and packaged into lentiviruses. Then the lentiviruses were used to infect hASCs for LSD1 knockdown or overexpression. The difference of osteogenic differentiation between the knockdown group or overexpression group and control group was compared. Osteogenic differentiation was assessed by alizarin red staining and quantitative test of alkaline phosphatase activity. The expression of osteogenesis-associated genes such as alkaline phosphatase (ALP), osteocalcin (OC), Osterix (Osx) detected by Real-time PCR was also used to evaluate the cells’ osteogenic differentiation. hASCs treated with control or LSD1 siRNA were cultured in osteogenic differentiation medium or proliferation medium for 1 week before implantation into nude mice. Specimens of each group were harvested at six weeks after implantation. Then we used X-ray radiography and H&E staining to compare the difference of osteogenic differentiation between the knockdown group and control group. To explore the mechanism that LSD1 influences the osteogenic differentiation of hASCs, Co-immunoprecipitation (Co-IP) was used to explore the relationship between LSD1 and runt-related transcription factor 2 (Runx2), an essential transcription factor governs osteoblastic differentiation.
Result: LSD1 loss of function promotes osteogenic differentiation of hASCs in vitro and in vivo, while LSD1 gain of function inhibits hASCs diffrenentiation into osteoblast in vitro. LSD1 knockdown could significantly increase the expression of osteogenic genes such as ALP, OC and Osterix. LSD1 was associated with Runx2.
Conclusion: LSD1, a histone H3K4 demethylase, could repress the osteogenic differentiation of hASCs in vitro and in vivo. LSD1 regulates the expressions of osteogenesis-associated genes and interacts with Runx2.