Research On The Role And Mechanism Of TP53INP2 In The Osteogenic Differentiation Of Human Adipose Derived Stem/Stromal Cells

PURPOSE: Human adipose derived stem/stromal cells(hASCs)are commonly used as seed cells in bone tissue engineering,which have good osteogenic properties in vivo and in vitro.Tumor protein p53-induced nuclear protein 2(TP53INP2)regulates autophagy,diabetes,the development of tumour,regulation of obesity and adipogenic differentiation.However,whether TP53INP2 regulates osteogenic differentiation of hASCs has not been sufficiently studied.Therefore,the purpose of this research is to explore the role of TP53INP2 in the osteogenic differentiation of hASCs and its possible mechanism.METHODS: hASCs were isolated,cultured and identified in vitro,and the ability of multilineage differentiation was detected.The siRNA sequence with high efficiency of silencing TP53INP2 was screened by quantitative real-time PCR(qRT-PCR)and Western blot,and the model of cells with TP53INP2 silencing was constructed.After siRNA transfection,cells were cultured with osteogenic induction medium for 3 and 7 days,and the mRNA levels of RUNX2,ALP,OCN and COL1A1 were detected byqRT-PCR,and the expression of RUNX2 protein was detected by western blot.Appropriate concentration of lithium chloride(LiCl)was screened by Western blot,and was used to treat cells for 7 days.Then western blot was used to detect the β-catenin,GSK-3β,p-GSK-3β,which were related to Wnt/β-catenin signaling pathway,and the expression of RUNX2,TP53INP2.RESULTS: The hASCs were successfully isolated and cultured,and showed good ability of proliferation and multidifferentiation in vitro.The mRNA level of RUNX2 in siRNA-TP53INP2 cells decreased significantly after 3 days of osteogenic induction,and the mRNA levels of RUNX2,ALP,OCN and COL1A1 decreased significantly after 7 days of osteogenic induction.Western blots showed that RUNX2 protein expression decreased in siRNA-TP53INP2 cells at day 3,and 7 after osteogenic induction.The level of β-catenin,in siRNA-TP53INP2 cells was decreased at day 3 and 7after osteogenic induction.The results of LiCl concentration screening showed that 5mM concentration of LiCl could activate Wnt/β-catenin signaling pathway,and promote the osteogenic differentiation of hASCs,which showed the increase of RUNX2 expression,while high concentration of LiCl(≥15mm)inhibited the osteogenic differentiation of hASCs.In addition,the expression of β-catenin and RUNX2 protein in TP53INP2 silencing cells was partially restored after culturing with osteogenic induction medium containing 5mM LiCl.At last,the totalprotein level of LC3 and the LC3-II/LC3-I ratio in TP53INP2 silencing cells decreased after 3 or 7 days of osteogenic induction.CONCLUSIONS: Silencing TP53INP2 inhibited the osteogenic differentiation of hASCs.5mM concentration of LiCl promoted the osteogenic differentiation of hASCs,while high concentration of LiCl(≥15mM)inhibited the osteogenic differentiation of hASCs.The effect of TP53INP2 on the osteogenic differentiation of hASCs may be related to the activation of β-catenin signaling pathway and the regulation of autophagy.This laid a foundation for further study of the mechanism of TP53INP2 in the osteogenic differentiation of hASCs,and provided a possibility for targeted repair of bone defects by TP53INP2.