Study On The Relationship Between BMPs/Smads Signaling Pathway And Hoxc8 Gene In The Process Of Osteogenic Differentiation

The multi-vertebral trait of sheep can improve performance of meat production,meanwhile the formation of multi-vertebral trait is closely related to the bone development process.Bone is one of the most important tissues in the composition of vertebrates,it plays a role in supporting,moving and protecting of the body.There are two ways of bone development:intramembranous ossification and endochondral ossification.Intramembranous ossification is the direct differentiation of mesenchymal cells into osteocytes;endochondral ossification is the differentiation of mesenchymal cells to form cartilage first,and then the cartilage is mineralized to form bone.The process of bone growth and development are regulated by many growth factors,cytokines and other factors,and at the same time,regulatory factors participate in the process of osteogenesis through related signal pathways.In the process of bone development,studies have found that Hoxc8 is not only an important developmental regulatory gene,but also the strict Hoxc8 expression pattern controls the positioning of the front and rear axis of the body and the formation of rib contours.Although it is known that Hoxc8 is related to osteogenesis,the signaling pathways that interact with it are still unclear.The BMPs/Smads signaling pathway,a signaling pathway that plays a key role in osteoblast differentiation and bone formation during bone development,may be related to Hoxc8.In this study whether the role of Hoxc8 is related to Smad1 in the Smads protein family,and then to the BMPs/Smads signaling pathway was observes,so that to understand the relationship between Hoxc8 and BMPs/Smads signaling pathways in the development of sheep bones,which will be further to lay the foundation for studying the causes mechanism of sheep polyvertebral traits and relative signaling pathways.1.The expression of related genes during the osteogenesis of adult stem cells from two different sources.Adipose-derived stem cells(ADSCs)and muscle-derived stem cells(MDSCs)were used to induce differentiation of osteoblasts in vitro,and they were all positive by staining examination,and the expression of osteogenic markers genes of OCN,Ⅰ collagen,Runx2 were detected in transcription level and protein level.At the transcription level,OCN and type Ⅰ collagen in ADSCs increased first and then stabilized,and the highest expression levels were up to 9.6 and 12.5 times respectively on the 9th day,while Runx2 first decreased and then increased;OCN and type Ⅰ in MDSCs Collagen also increased first and then stabilized,,the highest expression levels were up to 7 times and 6 times,respectively on the 9th day,while Runx2 first decreased and then increased.The expression at the protein level was consistent with that at the transcription level.2.Key factors of BMPs/Smads signaling pathway and Hoxc8 gene expression in the process of osteogenic differentiation induction.In the process of osteogenic differentiation,real-time quantitative PCR was used to detect the expression of important signal factors BMP2,BMP7,and Smad1 in the BMPs/Smads signaling pathway.It was observed that there was no singnificant change in the expression of BMP2 and BMP7,and the expression level of Smad1 and Hoxc8 was increased first and then decreased at the transcription level,and the expression level was the highest on the 9th day,and the protein expression was also consistent.3.The interaction between Smad1 and Hoxc8 gene.Smad1 overexpression and interference vectors were constructed,and transfected into ADSCs by liposome transfection method.Through transcription level detection,it was found that the expression of Smad1 in Smad1 overexpressing cells were 7 times than that of normal cells.The expression of Smad1 in Smad1 interference cells were 0.48 times than that of control cells.The protein level detection was as the same as the transcription level.The transcription level and protein level of Hoxc8 gene in Smad1 overexpression and interference cells were detected,and it was found that the expression level of Hoxc8 transcription level in cells overexpressing Smad1 was 0.85 times than that of control cells,while in Smad1 interference cells it was 2.42 times than that of control cells.At the same time,the detection at the protein level also showed the same trend,indicating that the expression of Smad1 was negatively correlated with the expression of Hoxc8.Through the detection of Smad1 expression in Hoxc8 gene overexpression and knockout of Smad1 cells,it was found that the expression of Smad1 did not change at the transcription level and proteome level,indicating that there was no effect of Hoxc8 gene on Smad1.The above studies indicated that expression change of Smad1 and Hoxc8 in the BMPs/Smads signaling pathway was in the same trend during the process of osteogenic differentiation.At the same time,Smad1 had a negative correlation with Hoxc8,while Hoxc8 had no effect on Smad1.It is speculated that the BMPs/Smads signaling pathway might be upstream of Hoxc8.This study preliminarily verified the mechanism of action between Hoxc8 gene and BMPs/Smads signaling pathway,and provided an experimental basis for future research on the mechanism of signaling pathways during osteogenesis.