Cloning And The Preliminary Study On Functions Of S100A16 Gene CDNA From Sika Deer

The morbidity of malignancies are increasing year by year,which has become the leading cause of death for human beings,more and more attentions has been paid to the treatment of malignancies.S100A16 is a special member of S100 gene family,which is closely related to cancer.Early studies showed that S100A16 was highly expressed in malignant cells,but later studies found that it plays different roles in different malignant cells.It indicates that S100A16 has special expression mechanism and function in malignancies.Growing sika deer antler has become an important model for malignancies research due to its rapid growth rate.However,the study of S100A16 in deer is rarely reported,so the study on S100A16 gene of sika deer is of great significance.In this study,we designed primers based on the S100A16 gene sequences of cattle and sheep in GenBank,and successfully obtained the S100A16 gene by RT-PCR and molecular cloning technology using the cDNA of antler from sika deer.Differences of gene expression level of S100A16 within the mesenchymal tissue of sika deer antler between early,mid and late growth stage were analyzed using Q-PCR tech.In this study,pEGFP-C1-S100A16 was constructed and transfected into 293T cells.The effect of S100A16 on the expression of NOTCH1,ZEB1,ZEB2,NANOG,CDKN1A,PSMD9 and TP53 in 293T cells were analyzed by Q-PCR.The results show that:(1)The overall length of the CDS of S100A16 gene from sika deer is 312 bp and it encodes 103 amino acids.The protein translated by S100A16 gene of sika deer is a stable protein in the cell,it contains 11 phosphorylation sites,a transmembrane domain,and no signal peptide.The protein contains a classical EF-hand like the S100 protein family whose Ca2+binding loop consists of 12 amino acids at the C-terminal,and a EF-hand at the N-terminal whose Ca2+ binding loop consists of 15 amino acids.A binding site of FGF-1 protein is found at the C-terminal of the protein.The secondary structure of the protein is mainly composed of ?-helixes and random coils,and the tertiary structure shows that the protein has two binding sites of ions.The functional structure and cellular localization of S100A16 protein in sika deer are similar to those in human.(2)The amino acid sequence encoded by S100A16 gene of the sika deer has 100%homologies with the Eastern European red deer,and followed by sheep and cattle which both are 98%.The phylogenetic tree was constructed with the sequence of S100A16 protein from sika deer and other species.The result shows that the S100A16 protein is conservative in evolution and accords with the characteristics of functional proteins.(3)The expression level of S100A16 in the mesenchyme was the lowest during the mid-growth of pilose antler from sika deer.This gene plays an important role in the growth of pilose antler.(4)The pEGFP-C1-S100A16 of sika deer was constructed successfully.The efficiency of transfection in 293T cells reached 70%-80%,and the expression was successful.(5)Compared with 293T cells transfected with pEGFP-C1-S100A16 and pEGFP-C1,the expression level of ZEB1 and ZEB2 was significantly down regulated(P<0.01),the expression level of NOTCH1,PSMD9,NANOG and CDKN1A was significantly down regulated(P<0.05),while the expression level of TP53 was not changed(P>0.05).Overexpression of sika deer S100A16 gene in 293T cells can down-regulate the expression of some oncogenes.