Effect And Mechanism Of Sika Deer Prothymosin On Osteoblast Biological Activity In Mice

Objective: To investigate the effect of prothymosin α（PTMA）derived from sika deer on osteoblast growth,identify the mechanism of PTMA promoting osteoblast proliferation,and provide theoretical support for elucidating the biological activity and mechanism of PTMA.Methods: The total RNA and total protein of deer antler were extracted by modified Trizol method and lysis method respectively from the deer antler in primary stage（PS）,rapid growth stage（RG）and ossification stage（OS）.Transcriptome and proteome sequencing of Cornu Cervi Pantotrichum samples from the three periods were conducted using Illumina Hi Seq^TM 2000 data platform and i TRAQ technology.SPSS 22.0 software was used for joint analysis of the transcriptome data and proteome data.The structure of PTMA protein was predicted by bioinformatics analysis.Constructing a PTMA gene overexpression vector derived from sika deer by using a gene engineering method,and synthesizing an si RNA sequence targeting the PTMA gene in vitro by using an RNAi technology;The CCK8 method,flow cytometry,and mitochondrial membrane potential assay（JC-1）were used to determine the effect of overexpression and knockout of PTMA gene on the osteoblast activity in mice.Screening candidate genes and signaling pathways by a transcriptome sequencing method;The expression levels of candidate genes and proteins were detected by fluorescent quantitative PCR and western blot analysis,respectively.Results: 1.The transcriptome database and proteome database of primary,fast-growing and ossifying stages of deer antler were established,and 210,437 Unigene and2,261 protein were obtained.Compared with the primary stage,the differential genes and proteins were analyzed,and 11,294 and 4,924 significant differential genes and 236 and 211 differential proteins were obtained in PS vs RG group and PS vs OS group respectively.2.Joint analysis of transcriptome and proteome data showed that there were 54 and 51 DEGs encoding their corresponding DAPs in PS vs RG and PS vs OS groups,respectively.Pearson correlation analysis of these genes/proteins showed that there was a weak correlation between transcriptome and proteome data.After further screening of the obtained genes/proteins,eight candidate genes/proteins related to the growth and development of velvet antler were screened out.Among them,PTMA can be used as a candidate gene for further research.3.The structural analysis of sika deer PTMA protein showed that it encoded 110 amino acids,with a molecular weight of 12071.81 Da and a theoretical isoelectric point of 3.68.It is closely related to the PTMA of Sus scrofa,Lemur catta and Jaculus jaculus.4.According to the protein sequence of PTMA in the database,the eukaryotic overexpression vector PCDNA3.1（-）-PTMA was successfully constructed by changing its codon preference and optimizing its sequence without changing its sequence.The si RNA-PTMA fragment with the highest silencing efficiency was synthesized and screened for osteoblasts.The optimal conditions for overexpression and silencing PTMA gene were determined.5.When the PTMA gene is overexpressed in osteoblasts,the cell viability is significantly increased（P < 0.001）,and the cell viability is the highest when the transfection amount reaches 0.2 g.The results of flow cytometry and mitochondrial membrane potential detection showed that the apoptosis rate decreased with the increase of transfection amount,indicating that the overexpression of PTMA gene in osteoblasts inhibited cell apoptosis.6.After being transfected with si RNA-PTMA-2 into bone cells for 24 h,the cell viability is decreased,and the cell viability is the lowest when the transfection amount reaches 3 pmol.The results of flow cytometry and mitochondrial membrane potential detection showed that the apoptosis rate increased with the increase of transfection amount,and the silencing of PTMA gene in osteoblasts promoted apoptosis.7.Transcriptome sequencing was carried out on osteoblasts that overexpressed and knocked out PTMA gene and control group.The results of differential analysis showed that6522 and 722 differential genes were obtained in control group vs overexpression group and control group vs silence group respectively.The enrichment analysis of differential genes showed that the differential genes in the control group vs overexpression group mainly participated in the regulation of biological processes such as apoptosis;The differential genes screened by control group vs silent group are mainly involved in the regulation of apoptosis and other processes.Three candidate genes were further screened,including Bcl-2,Bax and Caspase-3.8.The expression of apoptosis-related genes at m RNA and protein level was detected by fluorescence quantitative PCR and western blot analysis.The results showed that the expression of apoptosis-promoting genes at m RNA and protein level decreased significantly with the increase of transfection amount,while the expression of Bcl-2 gene that inhibited apoptosis increased significantly.In osteoblasts with PTMA gene silencing,the expression of apoptosis-promoting gene at m RNA and protein level increased with the increase of transfection amount,while the expression of Bcl-2 gene inhibiting apoptosis decreased significantly.Conclusion: 1.It is a feasible method to establish the transcriptome database and proteome database of deer antler in different periods,and screen the candidate genes/proteins related to the growth and development of deer antler by multi-omics joint analysis.2.Overexpression of PTMA gene in mouse osteoblasts increased the cell survival rate,while knocking out PTMA gene decreased the cell survival rate.PTMA gene is an anti-apoptosis factor for osteoblasts.3.The overexpression of PTMA gene in osteoblasts may inhibit the apoptosis of osteoblasts by inhibiting the expression of Caspase-3 and Bax.4.PTMA plays an important role in regulating bone growth and development.