Identification of differentially expressed proteins of palate tissues by iTRAQ in fetal mice induced by TCDD or retinoic acid

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Objective To explore the common differentially expressed proteins in 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin(TCDD) and retinoic acid-induced cleft palate of fetal mice by isobaric tags for relative and absolute quantitation(iTRAQ) combined with mass spectrometry. Methods Thirty-six pregnant C57BL/6J mice were randomly divided into 3 groups, 12 cases in each group. C57BL/6J pregnant mice were given a gavage of TCDD 28 μg/kg or retinoic acid 80 mg/kg on gestational day 10.5(GD10.5) as experimental groups, while the control group received equivalent corn oil. Anatomical and histological changes of palates in fetal mice were observed on GD17.5. Total proteins were extracted from palates of fetal mice in each group on GD17.5. Differentially expressed proteins were identified in experimental groups as well as in control group by iTRAQ combined with two-dimensional liquid chromatography/tandem mass spectrometry. Western Blot was used for validation of the differentially expressed proteins of Annexin A1 and 14-3-3 sigma. All statistical analyses were measured with SPSS software(version 17.0). Chi-square test was used to compare the incidence of cleft palate. One-way ANOVA was carried out for comparison of the relative expression levels of three groups, homogeneity of variance was analyzed by Levene test, and Turkey HSD test was used for comparison between two groups. P values were judged as significant difference if they were less than 0.05. Results ①Model of cleft palate in fetal mice were successfully established with incidence of cleft palate of 97.1%(68/70)in TCDD group and 98.6%(70/71) in retinoic acid group, respectively(χ2=0.00, P>0.05), without significant difference between two groups. However, they were similar on the phenotype. ② A total of 2 996 proteins were identified. Compared with control group, 75 and 90 differentially expressed proteins were screened out from TCDD group and retinoic acid group respectively. There were 18 differentially expressed proteins in common both in two experimental groups. ③Western Blot assay indicated that the expression of Annexin A1 protein was 0.52±0.11 in control group, while in TCDD group was 0.99±0.34 and in retinoic acid group was 0.98±0.31, with significant difference between any of two experimental groups and control group(P<0.05). The expression of 14-3-3 sigma protein in control group was 0.55±0.15, while in TCDD group was 0.86±0.17 and in retinoic acid group was 0.93±0.13, with significant difference between any of two experimental groups and control group(P<0.05). These results were consistent with the results of iTRAQ experiment. Conclusions Using iTRAQ technology can quickly and effectively filtrate the common differentially expressed proteins in fetal mice with cleft palate induced by TCDD and retinoic acid. These proteins may have closely related relationship with the occurrence of cleft palate induced by TCDD or retinoic acid. Key words: Proteomics; Cleft palate; Disease models, animal; 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin; Retinoic acid