Effect Of STAT3 On Osteogenic Differentiation Of MC3T3-E1 In Vitro

Objective: Signal transducer and activator of transcription 3(STAT3)plays a pivotal role in osteoblastic differentiation.However,the exact role of STAT3 in osteogenic differentiation of the pre-osteoblastic cell line MC3T3-E1 is still controversial.some researchers thought that STAT3 positively affects osteoblastic differentiation while some hold a contrary opinion.The study evaluated the effect of STAT3 inhibitor cryptotanshinone(CPT),STAT3 silencing and STAT3 overexpression on the osteoblasts proliferation,differentiation and mineralization.These findings may provide a basis for the development of more efficient and controllable protocols for osteoblastic differentiation and facilitate their use in regenerative medicine.In addition,our results provide novel insights i nto the effect of the STAT3 antagonist CPT on modulation of osteogenesis.Methods:(1)MC3T3-E1 cells treated with CPT were experimental groups while the cells without CPT were control groups.Quantitative real time polymerase chain reaction(RT-q PCR)was used to detect STAT3 m RNA expression at 3,6,9 d in the two groups;Cell proliferation assay detected by cell counting kit-8(CCK-8);The effect of STAT3 on early and late differentiation of osteoblasts in the two groups were quantitatively detected by alkaline phosphatase(ALP)activity test;Alizarin red staining was used to detect the formation of calcium nodules in each group after 14 d,21 d of culture.Western blot was used to detect expression of osteoblast-specific marker proteins ALP,collegan I(Col I)and osteocalcin(OCN)on early and late differentiation of osteoblasts in the two groups;(2)Transfected MC3T3-E1 cells with p EX-3-AMP-STAT3,p EX-3-AMP plasmids and STAT3 si RNA,NC si RNA.RT-q PCR was also used to detect the expression of STAT3 m RNA in the four transfection groups;Western blot was used to detect the protein expression of Col I,ALP and OCN in the four transfection groups at 6 d.Results:(1)At the time points 3 d,6 d,9 d the relative expression of STAT3 mRNA in CPT groups were significantly lower than the control groups(p<0.05);CCK-8 results revealed cell proliferation increased gradually in both groups,at every time point CPT groups did not show significant difference as compared to the negative controls(p>0.05); The eradication of STAT3 by CPT,exhibited a typical decrease in ALP activity at 3 d,6 d,9 d compared to untreated control cells(p<0.05);At 14 d,alizarin red staining showed no mineralized nodules in the CPT groups and control groups while extensive matrix mineralization could be observed in the two groups at 21 d,there was a significant difference between the two groups in the number of calcium nodules(p<0.05);After CPT intervention,the relative protein expression levels of ALP,Col I,OCN were significantly lower,and the difference was statistically significant(p<0.05);(2)Compared with negative control si RNA and p EX-3-AMP vector,the presence of STAT3 si RNA significantly reduced the expression of STAT3 while the presence of p EX-3-AMP-STAT3 significantly up-regulated STAT3 in MC3T3-E1(p<0.05);After the transfection at 6 d,MC3T3-E1 cells transfected with p EX-3-AMP-STAT3 showed higher expression of ALP and Col I(p<0.05),while the difference in OCN protein level among the four groups was not significant(p>0.05).Conclusion: STAT3 positively regulates osteogenic differentiation of MC3T3-E1.