Transcriptome heterogeneity of congenital cleft palate model in congener New Zealand rabbits induced by dexamethasone

doi:10.7518/hxkq.2023.01.005

Abstract

Abstract:

Objective This work aimed to investigate the transcriptome heterogeneity of dexamethasone-induced congenital cleft palate in homozygous New Zealand rabbits and determine the molecular mechanism underlying the occurrence of congenital cleft palate. Methods Dexamethasone (1.0 mg per day) was administered intramuscularly to 20 New Zealand pregnant rabbits from day 14 to day 17 of gestation, and the palatal phenotype of all offspring of each pregnant rabbit was observed. Eight embryos with a 4∶4 ratio of cleft palate to non-cleft palate were selected and divided into the cleft palate group (CP) and non-cleft palate group (NCP). Their palatal tissues were collected for RNA sequencing. Results A total of 225 differentially expressed genes (Q<0.05) were found in the CP group compared with the NCP group, of which 120 genes were upregulated and 105 genes were downregulated. The GO and KEGG enrichment analyses of these differentially expressed genes were carried out. The results showed significant enrichment in GO classification, which included heterotrimeric G protein complex, extracellular matrix, transcription factor complex, and basement membrane. Meanwhile, GABA ergic synapse, morphine addiction, retrograde endocannabinoid signaling, glutamate synapse, serotonergic synapse, regulation of actin cytoskeleton, and the Apelin signaling pathway were significantly enriched in the KEGG pathway. Compared with the NCP group, the gene expression levels of ARHGEF6 (P<0.05) and ABI2 (P<0.001) decreased in the CP group, and APC increased (P<0.001); these results were confirmed by real-time polymerase chain reaction. Conclusion Abnormal expression levels of the ARHGEF6, APC, and ABI2 genes involved in the regulation of the actin cytoskeleton in the palatal synapse may be associated with the dexamethasone-induced congenital cleft palate in New Zealand rabbits.

Key words: dexamethasone, congenital cleft palate, transcriptomic analysis, cytoskeleton regulation

CLC Number:

R 782.2⁺1

Lin Lanling, Liu Haoyue, Luo Xiao, Zhang Chong, Jing Bingshuai, Shi Bing, Li Chenghao.. Transcriptome heterogeneity of congenital cleft palate model in congener New Zealand rabbits induced by dexamethasone[J]. West China Journal of Stomatology, 2023, 41(1): 37-42.

Figures/Tables 7

Tab 1

Fig 1

Tab 2

Fig 2

Fig 3

Fig 4

Fig 5

References 24

1	Vandewalle J, Luypaert A, De Bosscher K, et al. Therapeutic mechanisms of glucocorticoids[J]. Trends Endocrinol Metab, 2018, 29(1): 42-54.
2	Shimba A, Ikuta K. Control of immunity by glucocorticoids in health and disease[J]. Semin Immunopathol, 2020, 42(6): 669-680.
3	Bonner JJ. The H-2 genetic complex, dexamethasone-induced cleft palate, and other craniofacial anomalies[J]. Curr Top Dev Biol, 1984, 19: 193-215.
4	Greene RM, Kochhar DM. Some aspects of corticosteroid-induced cleft palate: a review[J]. Teratology, 1975, 11(1): 47-55.
5	Evans RM. The steroid and thyroid hormone receptor superfamily[J]. Science, 1988, 240(4854): 889-895.
6	Hu X, Gao JH, Liao YJ, et al. Dexamethasone alters epithelium proliferation and survival and suppresses Wnt/β-catenin signaling in developing cleft palate[J]. Food Chem Toxicol, 2013, 56: 67-74.
7	Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2[J]. Genome Biol, 2014, 15(12): 550.
8	Li C, Lan Y, Jiang R. Molecular and cellular mechanisms of palate development[J]. J Dent Res, 2017, 96(11): 1184-1191.
9	Li R, Chen Z, Yu Q, et al. The function and regulatory network of Pax9 gene in palate development[J]. J Dent Res, 2019, 98(3): 277-287.
10	Yoshioka W, Tohyama C. Mechanisms of developmental toxicity of dioxins and related compounds[J]. Int J Mol Sci, 2019, 20(3): 617.
11	Baker NC, Sipes NS, Franzosa J, et al. Characterizing cleft palate toxicants using ToxCast data, chemical structure, and the biomedical literature[J]. Birth Defects Res, 2020, 112(1): 19-39.
12	Reynolds K, Kumari P, Sepulveda Rincon L, et al. Wnt signaling in orofacial clefts: crosstalk, pathogenesis and models[J]. Dis Model Mech, 2019, 12(2): dmm037051.
13	Weng M, Chen Z, Xiao Q, et al. A review of FGF signaling in palate development[J]. Biomed Pharmacother, 2018, 103: 240-247.
14	Sakuma C, Imura H, Yamada T, et al. Histological and immunohistochemical studies to determine the mechanism of cleft palate induction after palatal fusion in mice exposed to TCDD[J]. Int J Mol Sci, 2022, 23(4): 2069.
15	Lessard JL, Wee EL, Zimmerman EF. Presence of contractile proteins in mouse fetal palate prior to shelf elevation[J]. Teratology, 1974, 9(1): 113-125.
16	Chiquet M, Blumer S, Angelini M, et al. Mesenchymal remodeling during palatal shelf elevation revealed by extracellular matrix and F-actin expression patterns[J]. Front Physiol, 2016, 7: 392.
17	Chen J, Lan Y, Baek JA, et al. Wnt/beta-catenin signaling plays an essential role in activation of odontogenic mesenchyme during early tooth development[J]. Dev Biol, 2009, 334(1): 174-185.
18	Wang XM, Liu WL, Chen Y, et al. Lithium-induced overexpression of β-catenin delays murine palatal shelf elevation by Cdc-42 mediated F-actin remodeling in mesenchymal cells[J]. Birth Defects Res, 2021, 113(5): 427-438.
19	Oshima-Nakayama M, Yamada A, Kurosawa T, et al. Cdc42 is crucial for facial and palatal formation during craniofacial development[J]. Bone Rep, 2016, 5: 1-6.
20	Zhu C, Cheng C, Wang Y, et al. Loss of ARHGEF6 causes hair cell stereocilia deficits and hearing loss in mice[J]. Front Mol Neurosci, 2018, 11: 362.
21	Mamula D, Korthals M, Hradsky J, et al. Arhgef6 (alpha-PIX) cytoskeletal regulator signals to GTPases and Cofilin to couple T cell migration speed and persistence[J]. J Leukoc Biol, 2021, 110(5): 839-852.
22	Nusse R, Clevers H. Wnt/β-catenin signaling, disease, and emerging therapeutic modalities[J]. Cell, 2017, 169(6): 985-999.
23	Ranes M, Zaleska M, Sakalas S, et al. Reconstitution of the destruction complex defines roles of AXIN polymers and APC in β-catenin capture, phosphorylation, and ubi-quitylation[J]. Mol Cell, 2021, 81(16): 3246-3261.e11.
24	Chen J, Li H, Zhang B, et al. ABI2-mediated MEOX2/KLF4-NANOG axis promotes liver cancer stem cell and drives tumour recurrence[J]. Liver Int, 2022, 42(11): 2562-2576.

基因名称	引物类型	引物序列
ARHGEF6	Forward	5’-GCAGCGGAGATGACGGAGAATG-3’
	Reverse	5’-CTTCCCACCAGCCTCCCTCTTC-3’
ABI2	Forward	5’-GGGTGGCTGATTACTGCGAGAAC-3’
	Reverse	5’-TGCTAAGGACTGGGTGGTGTAGG-3’
APC	Forward	5’-GCAGCACTCCACAACATCATTCAC-3’
	Reverse	5’-ACTCCCAACAGGTTTCACAGTAAGC-3’
GAPDH	Forward	5’-TGGTGAAGGTCGGAGTGAAC-3’
	Reverse	5’-ATGTAGTGGAGGTCAATGAATGG-3’

样品名称	样品类型	浓度/（mg·L^-1）	体积/μL	总量/ng	RIN值	结论质检
NCP1	RNA	870	40	34.88	9.6	合格
NCP2	RNA	525	40	21	9.9	合格
NCP3	RNA	840	40	33.6	9.7	合格
NCP4	RNA	745	40	29.8	10.0	合格
CP1	RNA	415	40	16.6	10.0	合格
CP2	RNA	555	40	22.2	10.0	合格
CP3	RNA	870	40	34.8	10.0	合格
CP4	RNA	510	40	20.4	10.0	合格

[1]	Li Ma, Bing Shi, Qian Zheng. Influence of dexamethasone on the cell polarity and PAR complex of the embryonic epithelial cells in the palate [J]. West China Journal of Stomatology, 2018, 36(1): 9-16.
[2]	Pang Xiaoxiao, Li Li, Ma Li, Zheng Qian, Li Chenghao. Preliminary study on E-cadherin expression in dexamethasone-induced palatal cleft in mouse [J]. West China Journal of Stomatology, 2015, 33(6): 581-584.
[3]	Li Chunjie, Zhao Hongwei, Li Longjiang, Li Shuangjun, Wu Yuan, Liao Xuejuan, Pan Jian.. Systematic review on control of swelling and trismus after extraction of impacted mandibular third molar by Dexamethasone pericoronal injection [J]. West China Journal of Stomatology, 2013, 31(3): 267-271.
[4]	Pan Yan1, Qi Xiangmin2, Yin Meng2. The effect of interleukin-1β and Dexamethasone on keratinocyte growth factor expression level of cultured oral fibroblasts [J]. West China Journal of Stomatology, 2011, 29(06): 636-639.
[5]	LIAO Li -shu1，2, ZHENG Qian1，3, SHI Bing1，3, LU Sheng -jun2，4, ZHANG Rui1，2, MENG Tian1. Study on the in vitro effects of dexamethasone at different concentrations on the growth of murine embryonic palatal mesenchymal cell [J]. West China Journal of Stomatology, 2011, 29(02): 206-209.
[6]	HE Wei1，2, LU Sheng -jun1，3, LI Cheng -hao1，3, ZHOU Jing-lin1, MENG Tian1, ZHENG Qian1，3, SHI Bing1，3. Effects of dexamethasone and vitamin B12 on expression of fibroblast growth factor 10 and fibroblast growth factor receptor 2b during early palatogenesis [J]. West China Journal of Stomatology, 2010, 28(05): 551-555.
[7]	CHAI Mao-zhou1，2, LI Cheng -hao1，2, HE Yong -hong1, SHI Bing2. The preliminary study on transforming growth factor-beta 3, activin receptor-like kinase 5 expression in 2, 3, 7, 8-tetrachloro-p-dibenzodioxin and Dexamethasone induced palatal cleft in mice [J]. West China Journal of Stomatology, 2010, 28(04): 356-360.
[8]	HAN Shao- xing, YANG Fan, ZENG Hong. Determination of the Content of Dexamethasone Acetate in Stomatology Ulcer Pasta [J]. West China Journal of Stomatology, 2007, 25(03): 278-279.
[9]	HUANGLei1, SHI Bing2, SUNJin-hu3, WANG Yan2. Influence of Dexamethasone on Fusion of Embryonic Palatal Medial Edge Expithelium in Mouse Palatal Shelvesin vitro [J]. West China Journal of Stomatology, 2005, 23(02): 103-105.
[10]	. [J]. , 1998, 16(04): 0-.