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Sci Rep
Sci Rep
Scientific Reports
2045-2322
Nature Publishing Group UK London

39300178
73036
10.1038/s41598-024-73036-0
Article
Proteomic analysis illustrates the potential involvement of motor proteins in cleft palate development
Huang Zijian 123
Zhang Chuzhao 123
Sun Meng 123
Ma Aiwei 123
Chen Liyun 123
Jiang Wenshi 123
Xu Mengjing 123
Bai Xujue 123
Zhou Jianda 4
Zhang Wancong martine2007@sina.com

123
Tang Shijie sjtang3@stu.edu.cn

123
1 https://ror.org/02gxych78 grid.411679.c 0000 0004 0605 3373 Department of Plastic Surgery and Burn Center, Second Affiliated Hospital, Shantou University Medical College, DongXiaBei Road, Shantou, 515000 Guangdong China
2 https://ror.org/02gxych78 grid.411679.c 0000 0004 0605 3373 Plastic Surgery Institute of Shantou University Medical College, DongXiaBei Road, Shantou, 515000 Guangdong China
3 Shantou Plastic Surgery Clinical Research Center, DongXiaBei Road, Shantou, 515000 Guangdong China
4 https://ror.org/05akvb491 grid.431010.7 Department of Plastic and Reconstructive Surgery, Central South University Third Xiangya Hospital, Changsha, 410013 Hunan China
19 9 2024
19 9 2024
2024
14 2186827 3 2024
12 9 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
Cleft palate (CP) is a congenital condition characterized by a complex etiology and limited diagnostic and therapeutic options. In this study, we delved into the molecular mechanisms associated with retinoic acid (RA)-induced CP in Kun Ming mice. Proteomic analysis of control and RA-induced CP samples at embryonic day 15.5 revealed 25 upregulated and 19 downregulated proteins. Further analysis identified these differentially expressed proteins (DEPs) as being involved in extracellular matrix organization, actin cytoskeleton, and myosin complex. Moreover, these DEPs were found to be enriched in pathways related to motor protein activity and extracellular matrix-receptor interaction. Protein-protein interaction network analysis identified 10 hub proteins, including motor proteins and ECM-related proteins, which exhibited higher expression levels in CP compared to control tissues. These findings provide insights into the molecular mechanisms underlying CP and highlight potential targets for diagnostic and therapeutic purposes.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-024-73036-0.

Keywords

Retinoic acid
Cleft palate
Proteomics
Differentially expressed proteins
Motor proteins
Subject terms

Biochemistry
Molecular biology
issue-copyright-statement© Springer Nature Limited 2024
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pmcIntroduction

Cleft palate (CP) is a prevalent congenital craniofacial birth defect, occurring at an average rate of one in every thousand newborns globally1–3. Despite being recognized to involve a combination of genetic factors and environmental influences4,5, the complex underlying causes and pathogenesis of CP remain poorly understood. To advance our understanding of CP, researchers have successfully established animal models using retinoic acid (RA)6. RA, a key metabolite of vitamin A, plays a significant role in organ development, cell proliferation, and differentiation7.

Studies have demonstrated that elevated levels of RA can induce a high rate of CP in mice8. Utilizing this CP model, integrated omics analyses have been conducted to investigate differentially expressed genes, proteins, and metabolites associated with CP. For instance, NSOFC, APOA, HPT, and CRP proteins were identified as potential serum biomarkers for prenatal diagnosis of nonsyndromic orofacial clefts9. Defective variants in MTHFR and RBP4 were found to contribute significantly to the incidence of non-syndromic cleft lip with or without CP10. In addition, deletion of Rbfox2 in neural crest cells leads to CP and defects in craniofacial bone development11.

Proteomic analysis is considered a powerful method for identifying molecular genetics characteristics12,13. In CP-related research, proteomic studies have shed light on the tight association between reduced levels of RBP4 and vitamin A and the occurrence of CP14. Additionally, results from proteomic analysis reveal differential expression of fibronectin, laminin, and collagen in CP tissue15.

Studies have suggested a potential link between CP and motor proteins. For example, it has been reported that MEIS2 is associated with CP and cardiac septal defects, along with varying degrees of intellectual disability16. MEIS2 defects can result in a more severe phenotype, characterized by multiple congenital malformations, CP, congenital heart defects, and delayed gross motor and verbal development16. Mutations in the MYH3 gene have been identified as a cause of recessive CP in Limousine cattle17, and they have been associated with a spectrum of phenotypic manifestations in CP18. These associations highlight the involvement of MEIS2 and MYH3 in the development of CP and their potential impact on motor development. However, further research is needed to gain a deeper understanding of the specific mechanisms and pathways through which motor proteins contribute to CP development.

To investigate the role of proteins in the mechanisms of CP, we employed proteomics technology, specifically isobaric tags for relative and absolute quantitation (iTRAQ), to identify common differentially expressed proteins (DEPs) in RA-induced CP compared to normal control samples19. The objective of this study was to provide valuable references and insights for future investigations into the molecular mechanisms underlying CP development.

Materials and methods

Animal

Female Kun Ming mice, aged eight weeks, were housed in a temperature-controlled environment (22–25 °C, 45% humidity) at the Center Laboratory Animal Sciences, Shantou University Medical College. Mice were paired overnight with fertile male counterparts at a ratio of 2 females to 1 male. The day on which a vaginal plug was observed was designated as embryonic gestation day 0.5 (E0.5). On E10.5, the treatment group received oral administration of RA dissolved in sesame oil at a dosage of 70 mg/kg via gavage. On E15.5, the pregnant mice were euthanized humanely. The embryos were collected and their palatal tissues were dissected. Tissues from littermates from one female mouse were pooled together as one replicate for subsequent analysis. Three pairs of control and RA-treated pregnant females were utilized. For animal euthanasia, in brief, all pregnant mice were placed in the euthanasia chamber, and 100% CO2 gas was then introduced at a flow rate of 30–70% of the chamber volume per minute. When there was no corneal reflex, no detectable breathing, and no heartbeat for more than 5 min, the mice were confirmed dead. Euthanasia methods followed the requirements of the American Veterinary Medical Association’s Euthanasia Guidelines 2020 Edition. All efforts were made to minimize animal suffering. No anesthetic agents were used in this study. This study strictly adhered to ethical guidelines and received approval from the Animal Ethics Committee of Shantou University Medical College.

Sample preparation

Protein extraction was conducted by following a previous publication20. Briefly, tissues were gradually thawed at 4 °C and homogenized in 200 µL of water. Subsequently, 240 µL of pre-chilled methanol was added to the samples and mixed, followed by the addition of 800 µL of methyl tert-butyl ether (MTBE). The samples were then centrifuged at 14,000 ×g 10 °C for 15 min. The upper organic phase was carefully collected, and the solvent was evaporated using nitrogen gas.

To achieve mass-spectrometry (MS) resolution, 200 µL of a 90% isopropanol/acetonitrile solution was added to the dried samples. For the preparation of quality control (QC) samples, 10 µL from each sample was extracted and combined. The supernatant was subsequently sampled and analyzed through centrifugation at 14,000 × g and 10 °C for 15 min.

For further protein sample preparation, 100 µg of proteins from each sample solution were mixed with TEAB solution containing 0.1% SDS. The resulting mixture was incubated with 1 µL of trypsin at 37 °C for 4 h. Each sample was treated with 50 µg trypsin. Subsequently, a second trypsin addition at the same ratio extended the digestion process for an additional 8 h continuously. The resulting digested solution was frozen, and later re-dissolved using TEAB with a water-to-TEAB ratio of 1:1. The re-dissolved digested samples were then subjected to incubation with the iTRAQ® Reagent-8Plex Multiplex Kit (manufactured by AB Sciex Inc., USA) at a temperature of 25 °C. Each labeled reagent tube received 70 µL of isopropanol, which was thoroughly mixed for 1 min and then centrifuged to collect the contents at the bottom of the tube. The labeled samples were mixed and then subjected to centrifugation at 25 °C for 2 h, followed by lyophilization.

Sample separation was carried out using an AKTA Purifier 100 (GE, USA) with mobile phases A (10 mM KH2PO4, 25% ACN, pH 3.0) and B (10 mM KH2PO4, 500 mM KCl, 25% ACN, pH 3.0). The tryptic peptides were separated at a fluent flow rate of 1 mL/min and monitored at 214 nm. The elution buffer was collected every minute and the separated peptides were lyophilized before MS detection and desalted using the HyperSep C18 Cartridges (Thermo Fisher Scientific).

For MS analysis, an Easy-nLC machine (Thermo Fisher Scientific) was employed. Samples were loaded onto a capillary column (Acclaim PepMap100, 100 μm*2 cm, nanoViper C18, Thermo Fisher Scientific) and then separated by a capillary analytical column (EASY column, 10 cm, 18-A2) with a flow rate of 300 nL/min. Full MS scans were obtained using a Q Exactive platform (Thermo Fisher Scientific) in the mass range of 300–1800 m/z with a mass resolution of 70,000 (at 200 m/z ), and the AGC target value was set at 1e6, with a maximum injection time of 50 ms. The dynamic exclusion was set to 60 s. For the MS2 scan, the following parameters were used: activation type was higher-energy collisional dissociation (HCD), normalized collision energy was 30 eV, isolation window was 0.7 m/z, resolution was 17,500 (at 200 m/z), and underfill was 0.1%.

Mascot2.2 and ProteomeDiscoverer (v.1.4) were employed to map the raw data against the Swiss-Prot database (mouse) with Trypsin digestion specificity. The false discovery rate (FDR) was set to 0.01, and proteins with at least 2 peptides were subjected to quantification.

Identification of DEPs

To identify the DEPs between the control and RA-induced CP groups, we applied a rigorous criterion of fold change (FC, RA-treated/control) greater than 1.2 or lower than 0.8. Unpaired two-tailed Stduent’s t-test was performed and a P-value less than 0.05 was considered statistically significant. The volcano plot was generated by the R package “ggplot2”. The most significant 20 DEPs were plotted by heatmap, achieved by the R package “Pheatmap”.

Functional enrichment analysis

Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis21–23 were employed to annotate the DEPs24. This analysis was performed using the R packages “clusterProfiler”, “org.Mm.eg.db”, and “enrichplot”25.

Protein-protein Interaction (PPI) analysis

To investigate PPI among the DEPs, we utilized the STRING database (https://cn.string-db.org/). This database provided valuable insights into potential functional associations among the DEPs. To identify the top 10 hub proteins within the network, we employed the cytoHubba plugins integrated within the Cytoscape software (version 3.9.1)26.

Statistical analysis

The statistical analyses were conducted using R software (version 4.2.2). Unpaired two-tailed Student’s t-test was employed. P value less than 0.05 was considered statistically significant. The significance levels were denoted as follows: * for P < 0.05, ** for P < 0.01, and *** for P < 0.001.

Results

Induction of CP by RA in Kun Ming Mice

Among embryos exposed to RA, 22 out of 26 exhibited CP, while 4 embryos died before analysis, making it impossible to determine the occurrence of CP in the dead embryos. By comparison, none of the 27 embryos in the control group exhibited CP. Hematoxylin-Eosin (HE) staining confirmed that control animals exhibited normal palatal development, while RA-induced embryos displayed CP phenotype (Fig. 1A and B), suggesting the successful establishment of the CP model.

Fig. 1 RA induces CP formation at E15.5 in Kun Ming mice.A: Control embryos at E15.5. B: RA-induced embryos at E15.5. PS represents palatal shelves and T represents togue. Scale bar: 50 μm.

Identification of DEPs in CP

To gain a comprehensive insight into the proteins exhibiting significant expression differences in RA-induced CP, we conducted an analysis targeting proteins that met specific criteria. This analysis unveiled 25 proteins displaying significant upregulation and 19 proteins exhibiting significant downregulation (Fig. 2A and Table S1). The heatmap represents the expression patterns of these 40 DEPs (Fig. 2B).

Fig. 2 Identification of DEPs in CP. (A) Volcano plot illustrating the DEPs. Red dots represent significantly upregulated proteins, while blue dots represent significantly downregulated proteins. (B) Heatmap visualizing the expression of 40 DEPs in CP samples. NP refers to normal palatal samples, while RP refers to samples with RA-induced palatal samples.

GO and KEGG enrichment analysis of DEPs in CP

To gain a comprehensive understanding of the functions associated with these DEPs in CP formation, we conducted enrichment analyses utilizing the GO and KEGG databases. For these analyses, a significance threshold of P < 0.05 was applied. The GO Biological Processes (BP) analysis revealed significant enrichment in processes such as extracellular matrix organization, extracellular structure organization, and external encapsulating structure organization (Fig. 3A). These findings suggest pivotal roles of the DEPs in the formation and maintenance of these structures. Furthermore, the Cellular Component (CC) analysis demonstrated enrichment in collagen-containing extracellular matrix, actin cytoskeleton, and myosin complex (Fig. 3A), indicating potential involvement in the organization and functioning of these specific cellular components. Additionally, the Molecular Function (MF) analysis revealed significant enrichment in extracellular matrix structural constituents, actin filament binding, and actin binding (Fig. 3A), implying specific molecular functions related to extracellular matrix organization and actin dynamics. Moreover, the KEGG analysis highlighted pathways associated with motor protein and ECM-receptor interaction (Fig. 3B), suggesting these DEPs may participate in cellular processes associated with motor protein activity and interactions between the extracellular matrix and cell receptors.

Fig. 3 GO and KEGG analysis of DEPs in CP. (A) Bubble plots showing the GO BP, CC, and MF enrichment analysis data of the identified 40 DEPs. (B) Bubble plot indicating the KEGG pathway enrichment analysis result of the 40 DEPs in CP.

PPI analysis reveals key hub proteins in CP

To gain a thorough understanding of PPIs pertinent to CP, we conducted a network analysis utilizing the STRING database. The resulting network (Fig. 4A) revealed a densely interconnected set of proteins, suggesting potential functional associations among them. To identify the core proteins with pivotal roles in CP, we employed the CytoHubba plugin within the Cytoscape software. This analysis identified the top 10 hub proteins: Tnnc2, Myl1, Myh8, Ckm, Myh3, Myl4, Col2a1, Col9a1, Matn3, and Acan. Notably, these hub proteins included motor proteins such as Tnnc2, Myl1, Myh8, Ckm, Myl4, and Myh3. Additionally, we observed that Col2a1, Col9a1, Matn3, and Acan were enriched in the ECM-receptor interaction pathway (Fig. 4B), suggesting their potential involvement in cellular processes related to interactions between the extracellular matrix and cell receptors. Interestingly, these 10 hub proteins displayed elevated expression levels in CP (Fig. 4C), indicating their potential as informative biomarkers for this condition.

Fig. 4 Identification of hub proteins by PPI analysis. (A) PPI network of DEPs associated with CP. (B) The top 10 hub proteins using the cytoHubba plugin in Cytoscape software. (C) Heatmap visualization of the expression levels of the top 10 hub proteins.

In summary, our findings suggest that the presence of motor proteins and their distinctive expression patterns may hold significance in understanding the pathogenesis of CP.

Discussion

CP is a prevalent congenital birth defect known for its multifactorial etiology involving genetic and environmental factors27. To induce CP in Kun Ming mouse embryos, we utilized RA. Our findings revealed a significant CP induction rate among RA-exposed embryos, similar to the prior research on RA-induced myogenic tongue28. The confirmation of CP occurrence via histological examination using HE staining further validates the successful establishment of our experimental model, serving as an appropriate platform for investigating the intricate molecular mechanisms underlying CP formation.

To identify the DEPs associated with CP, we conducted proteomic analyses comparing control embryos and RA-induced CP embryos. This analysis unveiled a total of 40 DEPs, with 25 proteins showing significant upregulation and 19 proteins exhibiting significant downregulation in the RA-treated group compared to the control group. These findings underscore the potential critical roles of these DEPs in the development and progression of CP.

To unravel the functional implications of these DEPs, we performed GO and KEGG enrichment analyses. The GO analysis highlighted the enrichment of BPs related to extracellular matrix organization, extracellular structure organization, and external encapsulating structure organization. This suggests the involvement of the DEPs in the formation and maintenance of these structural components, crucial for normal palate development. The CC analysis revealed enrichment in collagen-containing extracellular matrix, actin cytoskeleton, and myosin complex, indicating the role of DEPs in the organization and functioning of these cellular components. Additionally, the MF analysis showed enrichment in extracellular matrix structural constituent, actin filament binding, and actin binding, suggesting specific molecular functions related to extracellular matrix organization and actin dynamics. Moreover, KEGG analysis indicated the enrichment of pathways associated with motor protein and ECM-receptor interaction, suggesting the DEPs may be involved in cellular processes related to motor protein activity and interactions between the extracellular matrix and cell receptors. These findings provide valuable insights into the potential mechanisms and pathways underlying CP development.

Through PPI analysis, we identified the top 10 hub proteins, including Tnnc2, Myl1, Myh8, Ckm, Myh3, Rps16, Col2a1, Col9a1, Matn3, and Acan. Notably, these hub proteins included motor proteins such as Tnnc2, Myl1, Myh8, Ckm, Myl4, and Myh3. Additionally, Col2a1, Col9a1, Matn3, and Acan were found to be enriched in the EM-receptor interaction pathway. It has been reported that motor proteins, especially the heavy chain of non-muscle myosin IIA, play a key role in CP29. Myh9 was also thought to be a biomarker for no syndromic cleft lip30,31, and nonsyndromic orofacial clefts32,33. Among these hub proteins, COL2A1 variations were associated with a high risk of CP34. COL9A1 mutations were linked to Stickler syndrome that can display CP35. MATN3 is located in a locus regulating palatal fusion36. Although direct evidence from animal models showing that these genes are causative to CP formation is lacking, these genetic analyses support the notion that the abnormal expression of these 10 hub proteins may contribute to RA-induced CP development, underscoring their potential as informative biomarkers for this condition.

It is noteworthy that our conclusion is not verified by additional methods such as Western blot or immunohistochemistry. Additionally, the function of the identified DEPs in CP formation needs to be further investigated by knockout and/or transgenetic overexpression assays using animal models.

In conclusion, our study provides insights into the molecular mechanisms associated with CP development. The identified DEPs, enriched functional categories, and hub proteins shed light on the potential roles of motor proteins and extracellular matrix components in CP pathogenesis. These findings contribute to a better understanding of the underlying biology and may pave the way for the development of novel diagnostic and therapeutic approaches for CP.

Supplementary Information

Supplementary Information 1.

Supplementary Information 2.

Author contributions

Zijian Huang, Chuzhao Zhang, Meng Sun and Aiwei Ma collected the results and wrote the paper, Liyun Chen, Wenshi Jiang, Mengjing Xu, Xujue Bai and Jianda Zhou conducted statistical analysis, Wancong Zhang and Shijie Tang proposed the ideas and drafted the framework of the paper. All authors reviewed the manuscript.

Funding

This study was funded by the National Natural Science Foundation of China (82071101), Guangdong Basic and Applied Basic Research Foundation (2021A1515011142, 2023A1515012343, 2022A1515220099), Guangdong University Innovation Team Project (2021KCXTD047), Provincial science and technology innovation strategy special project funding program (200114165897946, 210714106901245, STKJ202209067, STKJ2023004), 2022 Shantou University Graduated Student Innovation Program (Shandafa[2022]207).

Data availability

The datasets used or analyzed during this study are available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

This study strictly adhered to ethical guidelines and received approval from the Animal Ethics Committee of Shantou University Medical College.

Competing interests

The authors declare no competing interests.

Conflict of interest

The authors have declared that no competing interests exist.

Declaration on animal experimentation

This study is reported in accordance with ARRIVE guidelines.

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Zijian Huang, Chuzhao Zhang, Meng Sun and Aiwei Ma contributed equally to this work.
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