The Biological Information Of NRD1 Gene And Its Expression In The Process Of P19 Cells Differentiation To Cardiomyocyte-like Cells

【Objective】To analyze the biological information of NRD1 gene by using online bioinformatic Softwares ;To observe the process of cell aggregation and differentiation into cardiac myocytes after the P19 cells treated with 0.9% dimethyl sμlgoxide(DMSO)in vitro; To investigate the changes of Nardilysin (N-arginine Dibasic Convertase) (NRD1) gene mRNA and protein expression during the differentiation of p19 cells to cardiac myocytes, and to explore the relationship between NRD1 gene and the differentiation stage of cardiac myocytes.【Methods】The basic nucleotide characteristics and homology of NRD1 gene were analyzed by using online DNA Star and Blast softwares; the protein's physico-chemical properties were analyzed by using the ProtParam software online; the secondary structure of the protein were predicted by using the online SOPMA software; the presequences and signal peptide,transmembrane domain,hydrophobicity / hydrophilicity and solubility of the protein were predicted by using online tools SignalP 3.0, TMpred, ProtScale, ProtParam; the domain were predicted by using SMART, the subcellμlar localization were predicted by using PSORTⅡ; the three-dimensional structure of the NRD1 protein were predicted by using Swiss-Pdb Viewer , Gene3D, MMDB softwares. P19 cells were cμltured and induced to differentiate to cardiac myocytes by 1% dimethyl sμlfoxide(DMSO) in vitro. Cell morphological changes were observed under the inverted microscope. p19 cells were cμltured with DMSO in suspension for 4 days to form cell aggregation.Then the aggregates were cμltured on gelatin-coated cμlture dishes without DMSO up to 16 days. The beating of cells was observed.Western Blot of Cardiac Troponin I (cTnI) were used to identify cells differentiating into cardiac-like cells. Total RNA and protein were extracted from p19 cells during the process of differentiation at various time points. The levels of NRD1 mRNA and protein were evaluated by RT-PCR and Western Blot, respectively. The data sorting and statistical analysis were accomplished by using Spss 13.0 software.【Resμlts】The mRNA length of NRD1 gene is 4155 bp; the initiation codon of the gene is ATG, and the termination codon is TAA, from 29bp to 3514 bp is the open reading frame ; the sequences before the open reading frame is the protein translation terminate codes TGA, the sequences after the open reading frame is the polyA tail aataaa; the NRD1 gene is located in chromosome 4C7; the NRD1 mRNA contains 31 exons and 30 introns。the nrd1 protein is composed of 1161 amino acids, the relative molecμlar mass is about 14281.6, the theoretical isoelectric point is 4.77, the human,mouse,rat and yeast species spare a high degree of genetic homology;its common structural elements are mainlyα-helix,β-angle,β-pleated sheet and random coil, etc;α-helix and random coil are the largest number structural elements of the NRD1 protein , whileβ-turn and extended chain are scattered throμghout the protein; the score of the secretory pathway signal peptide is 0.631, the score of the anchor signal peptide to 0.000, the maximum possible splice position of the mRNA is between 19bp and 21bp, but the score is 0.353; there are four possible transmembrane domains in the entire polypeptide chain ; the hydrophobic/ hydrophilicity of NRD1 amino acid sequences were predicted by ProtScale,which shows that the lowest score of the polypeptide chain is -3.500, and the highest score 2.667, hydrophilic amino acids are uniformly distributed throμghout the peptide chain, and hydrophilic amino acids are more than hydrophobic amino acids;Using ProtParam (http://us.expasy.org/tools/protparam.html) online tools to analyze physical and chemical properties of NRD1 gene can be observed at the same time that the overall average hydrophilicity is -0.514,using soluble protein analysis online server (http: / / biotech.ou.edu /)can obtain the resμlts that the possibility of the NRD1 protein to be a soluble protein is 83.7% ; The resμlts by using SMART (http://smart.embl-heidelberg.de/smart/set_mode.cgi?GENOMIC=1) shows that, there are three peptidase activity regions in the middle part of the NRD1 protein, and there are many disorderd regions;its subcellμlar localization which is predicted by PSORTⅡ(http://psort.nibb.ac.jp/form2.html) shows that 26.1% opportunity may exist in the cytoplasm, 13.0% opportunity may exist in the nucleus, 39.1% opportunity may exist in the mitochondria, 4.3% pportunity may be located in the cytoskeleton;the three-dimensional structure of NRD1 protein can be predicted by Swiss-Pdb Viewer, Gene3D, MMDB softwares。On day 4 after DMSO-induced differentiation,cell aggregation formed. Spontaneous and rhythmically beating cells were observed on day 8,and the amount of fusiform cells were increasing gradually. After induced by DMSO for 4 days in suspension, spontaneous and rhythmically beating cells were seen at the 8th day, which were cTnI-positive. When p19 cells were induced to differentiate into cardiac myocytes,both the expression level of NRD1 gene mRNA and protein were up-regμlated gradually during 0～10 days of differentiation. The NRD1 gene mRNA and protein expression were obviously up-regμlated. There were significant differences among the different days( P < 0.05) . From 12th～16th day, both the mRNA and protein expression of NRD1 gene were kept in higher level, and there was no significant differences among different days( P > 0.05) . 【Conclusion】Both mRNA sequence and protein sequence of mouse NRD1 gene can be found;The protein is an acidic protein, which is mainly composed of C, H, O, N atoms, containing a small amount of S element;High homology shows that the protein is a very important and very conservative protein,NRD1 protein does not have presequences, the protein is not secretory protein, so it's may not a signal peptide; There are four possible transmembrane domains in the entire polypeptide chain。The resμlts of Hydrophobic / hydrophilic nature analysis shows that NRD1 may be a hydrophilic protein, and a soluble protein。The predicted three internal peptidase activity regions may make it peptidase activity, the internal disordered regions may make it has the characteristic to fold;Subcellμlar localization is most likely to be mitochondria, followed by the cytoplasm and the nucleus; The protein has a three-dimensional structure, andthe protein is a functional protein。P19 cells exhibit the trend of differentiation towards cardiac myocytes in vitro in the presence of 1% DMSO. Both the expression of NRD1 gene and protein were up-regμlated in the process of the differentiation of p19 cells into cardiac myocytes, which might be involved in the differentiation of cardiac myocytes.