Proteomic Analysis Of The Rat Model And Study On Some Genes Related To Talipes Equinovarus

ObjectiveCongenital talipes equinovarus (CTEV, also called clubfoot, CCF) is one of the most common musculoskeletal anomalies impacting the functions of the foot, with an incidence of between 0.64 and 8 per 1,000 births, and with a prevalence of 1 per 1,000 live births. Boys are affected twice as often as girls. Bilateral deformity is seen in more than half of the cases, and unilateral defects occur more often on the right side. The malformation typically manifest fixed in for-leg adduction, ankle suspiration, and calcaneal bone varus and with the secondly tibia distal end adtorsion. Although it may not influence the survival and reproducibility of the patients, the CTEV deformity can severely affect their quality of life.There are controversies on the etiology of the CTEV. Previous studies on the etiology, pathology and pathogenetics show that it is related to the bone development anomaly, neuromuscular pathological changes, soft tissue contraction, blood vessel abnormalities, heredity and intra-uterus development arrest. Many hypotheses about its etiology such as intra-uterus pressure increasing, bone tissue, vessel, connection tissue, muscle and nerve tissue development abnormality have been raised. Genetic epidemiology studies have strongly suggested genetic factors play key role in the development of CTEV.Proteins participate in functions and modulations of the cell, and protein decides the phenotype of the cells, tissues and organ. "Proteome" is the set of expressed protein complement of a genome. Proteomic analysis has been widely utilized in various heart fields of life science, which makes people comprehensively understand the mechanism of life from the indirect gene to the functional protein. "Expression proteomics" has attracted much attention in recent years, and the global alterations in protein expression are characterized by 2-DE and mass spectrometry, in combination with bioinformatics.Since it is difficult to perform biopsy for talipes equinovarus in human subjects, it is necessary to study the pathogenesy of the CTEV with the rat model with CTEV deformity. Here we analyze the differential expressions of clubfoot-related proteins of the spinal cord, ankle joint tissue and ankle joint bone in normal and model rat with CTEV deformities induced by ATRA (all-trans retinoic acid, ATRA) by 2-DE and mass spectrometry, in combination with bioinformatics, which may help to delineate the roles of particular proteins in the pathogenesis of CTEV.Apoptosis plays a key role in the fetal organ morphogenesis. During the embryonic development, coordination of the apoptosis and the mitotic maintain the constancy of the cells, and there derangement may induce congenital abnormality, which indicated that apoptosis is an important format of the teratosis factors inducing the abnormality. In this study, the function of the apoptosis in development of the spinal, bone and muscle tissues of the rat model with CTEV deformity was also discussed.Our previous results have found Xiap (X-Linked inhibitor of apoptosis protein) down-regulated in the fetus with CTEV deformities induced by ATRA. In this study, the association between X-Linked inhibitor of apoptosis protein and CTEV were further analyze. Further more, three SNPs (single nucleotide polymorphism, SNP) within XIAP were selected to assess whether it was associated with CTEV.MethodsPregnant Wister rats were randomly assigned to control and experimental groups. On day 10 of pregnancy, 135 mg/kg of ATRA in mineral oil suspension was given intragestically to the experimental groups; corresponding volume of mineral oil was given to the controls. On day 21 of gestation, the fetuses were killed, and the spinal cords, ankle joint tissues and the ankle joint bone were dissected.Total protein of spinal cord, ankle joint tissues and the ankle joint bone were prepared. Protein concentration of 2-DE samples was estimated according to a commercial Bradford reagent. For isoelectric focusing, PROTEAN IEF cell was used. The IPG strip was bathed in balance solution I and II. Then transferred onto 12% SDS polyacrylamide gel and electrophoresed. Following electrophoresis, the gels were stained, scanned and analyzed with the PDQuest 7.1.0 software package. Significant protein spots were selected for mass spectrometry and bioinformatics analysis.The spinal column and the foot of the rat model with talipes equinovarus and the muscle of the CTEV and normal children were routinely fixed in 4% neutral formalin and embedded in paraffin. Sections were cut from paraffin blocks. Apoptosis detection was performed according to the manual of the DeadEndTM Fluometric TUNEL System.The total RNA of spinal cord, ankle joint bone and the tibia-fibulae musculature of the rat model with talipes equinovarus and the muscle of the CTEV patients and corresponding controls was refined respectively. The expressions of xiap and coL2a1 in the three tissues were analyzed to explore the association between these genes and CTEV.Immunohistochemical staining was used to analyze the expression of Xiap in the spinal cords, bone and muscle of the rat and muscle of the CTEV children. Western Blot was used to analyze the expression of Xiap in the tibia-fibulae musculature.Peripheral leukocytes genomic DNA of the 84 core families was isolated. Three SNPs (rs894737, rs2071448, rs2071450, rs2277371) were chosen from the region of XIAP gene and amplified by PCR. The genotypes of the SNPs were identified by restrictive enzyme. The genotypic frequencies and allele frequencies were analyzed by statistical software.ResultsIncluding 112 for normal controls and 232 for disease fetus, totally 344 rat fetus were obtained. The incidence of deformity was 80.98% and the incidence for the isolated rat model with talipes equinovarus was 29.27%. The total proteome of rat fetus were separated through pH3-10 and pH5-8 2-DE with good reproducibility and resolution. Compared with those of the normal controls, image analysis with PDQuest7.1.0 showed the differentially expressed protein spots, among of which 22 distinct protein spots were selected for MALDI-TOF mass spectrometry analysis. The peptide mass fingerprints were searched by using protein databases including PeptIdent, MS-Fit, and Mascot. The results showed that the expressions of the 18 proteins such as collagen alpha-1 (II) (Col2a1) changed.In rat model, the rates of the apoptosis in the spinal, vertebra and muscle of the CTEV deformity fetuses were 5.4, 10 and 3.7 times of those in the normal fetuses. By analyzing the apoptosis of muscle, bone and spinal tissues, we found that the apoptotic cells in these tissues are significantly higher from the rat model than those from normal controls (P<0.01, x~2 test).Compared to those from the normal controls, xiap mRNA in almost all the rat model showed low levels of expression in muscle, bone and spinal tissues, especially in the muscle. Col2a1 mRNA in almost all the rat model showed low levels of expression in bone. Compared to those from the normal children, XIAP mRNA in almost all the CTEV children showed low levels of expression in muscle. Immunohistochemical analysis of Xiap showed it down-regulated in the model rat muscle, bone and spinal tissue, especially in the cartilage, and it also down-regulated in the CTEV children. Western Blot analysis showed it down-regulated in the tibia-fibulae musculature in rat model.The lengths of PCR products were consistent with prediction. Rs17334732 and rs17334725 polymorphisms were confirmed while rs17330630 polymorphism was not existed. The statistics result showed there was no significant differences in the genotype and allele frequency of SNP rs17334725 (X~2= 4.7872, P>0.05) and there was significant differences in the genotype and allele frequency of SNP rs17334732 (X~2= 11.7556, P<0.05) .Conclusions1 The proteome of the protein patterns of spinal cord, ankle joint tissue and ankle joint bone of the rat model with talipes equinovarus and normal rat fetuses is firstly obstained.2 Proteomic difference between the rat model with talipes equinovarus and normal rat fetuses was presented, and the alteration of the 18 differentially expressed proteins such as collagen alpha-1 (II) (Col2a1) are found. The down-regulation of the Col2a1 gene is related with CTEV.3 Apoptosis rates were significantly different in the spinal, bone and muscle tissues from the rat model compared to those from normal controls. Some genes related to the apoptosis might play key roles in the development of CTEV.4 Based on the proteomic analysis, the down-regulations of the X-Linked inhibitor of apoptosis protein in the rat model and the CTEV children are found. The PCR-RFLP results also proved that XIAP does correlate to the development of CTEV.