| BackgroundRunt-associated transcription factor 2(Runx2)was a marker protein for osteogenic expression,and lysosomal associated transmembrane protein 5(LAPTM5)was closely related to lysosomal function,which was involved in the mineralization process of osteoblasts and plays a role in autophagy.Previous studies of our group have shown that Runx2 can transactivate the expression of LAPTM5,and a large number of literatures have confirmed that autophagy is correlated with the mineralization expression of osteoblasts.In this study,the expression ofRunx2/LAPTM5 during the induction of MC3T3-el mineralization was studied to explore the correlation between osteogenesis and autophagy.Method1.During the induction of MC3T3-el mineralization,alizarin red staining at d 0 to 21 and alkaline phosphatase staining at 0 to 14 d were detected,and osteogenic markers such as Runx2,ALP,OCN and expression of LAPTM5 were detected at d 0 to 14 by RT-qPCR and Western-Blot.2.Immunofluorescence expression of P62 and LC3 induced by MC3T3-el mineralization was detected on days 0-14.The changes of autophagosomes were observed under transmission electron microscopy,and the expressions of P62 and LC3 and other autophagy-related indexes were detected by RT-qPCR and WesternBlot.3.Overexpression and inhibition of Runx2/LAPTM5 were performed.The expressions of Runx2,ALP,LAPTM5 and P62 were detected by RT-qPCR and the expressions of Runx2,ALP,LAPTM5 and LC3 were detected by Western-Blot.Result1.During the induction of MC3T3-e1 mineralization,the number of alizarin red stained mineralized nodules increased with time from 0 to 21 days.The number of blue-purple granules also increased during alkaline phosphatase staining from 0 to 14 days.RT-qPCR and Western-Blot results of Runx2,ALP,OCN and LAPTM5 showed an upward trend from 0 to 14 days.2.The results of immunofluorescence and transmission electron microscopy from 0 to 14 d indicated that the expression of autophagy increased during 0 to 1 d and then decreased gradually.RT-qPCR of P62 and Western-Blot of P62,LC3 and other autophagy-related indexes during 0 to 14 d showed that the expression of autophagy increased during 1 to 2 d and then decreased gradually.3.After the overexpression of Runx2,the expression of osteogenic associated protein ALP was increased.The expression of autophagy-related protein LC3 also increased with the overexpression of Runx2,and P62 decreased accordingly.LAPTM5 increased accordingly.Interrupting Runx2 produced the opposite result.After LAPTM5 overexpression,the expression of osteogenic related protein ALP was increased.The expression of autophagy-related protein LC3 also increased with the overexpression of LAPTM5,while P62 decreased.The opposite results were found after interference with LAPTM5.The overexpression and interference of LAPTM5 had no significant effect on the expression of Runx2.Conclusion1.In this study,during the mineralization induction process of MC3T3-e1 cells,the expression of osteogenesis increased gradually with time from 0 to 14 days,and the expression of autophagy increased at the early stage and then decreased gradually.The expression of LAPTM5 increased during osteogenic mineralization.2.The expression of osteogenesis and autophagy increased after the overexpression of Runx2,while the opposite result appeared after the inhibition of Runx2.Overexpression of LAPTM5 also increased the expression of osteogenesis and autophagy,while inhibition of LAPTM5 showed the opposite result.In other words,RUNX2 and LAPTM5 may have a certain correlation with autophagy and osteogenesis,and RUNX2 can regulate the expression of LAPTM5.3.Autophagy at the early stage of osteoblast-induced mineralization is known to have a certain correlation with subsequent mineralization,and Runx2/LAPTM5 may play a bridging role in it. |
