地塞米松诱导同系新西兰仔兔先天性腭裂的转录组异质性

doi:10.7518/hxkq.2023.01.005

华西口腔医学杂志 ›› 2023, Vol. 41 ›› Issue (1): 37-42.doi: 10.7518/hxkq.2023.01.005

地塞米松诱导同系新西兰仔兔先天性腭裂的转录组异质性

林兰玲(), 刘皓月, 罗枭, 张翀, 景兵帅, 石冰, 李承浩()

口腔疾病研究国家重点实验室国家口腔疾病临床医学研究中心四川大学华西口腔医院唇腭裂外科，成都 610041

收稿日期:2022-07-05 修回日期:2022-09-08 出版日期:2023-02-01 发布日期:2023-02-21
通讯作者: 李承浩 E-mail:2535806717@qq.com;leechenghao_cn@163.com
作者简介:林兰玲，硕士，E-mail：2535806717@qq.com
基金资助:
四川省科技厅重点研发计划(2022ZDYF2641)

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

Lin Lanling(), Liu Haoyue, Luo Xiao, Zhang Chong, Jing Bingshuai, Shi Bing, Li Chenghao.()

State Key Laboratory of Oral Diseases & National Clinical Research Center for Oral Diseases & Dept. of Cleft Lip and Palate Surgery, West China Hospital of Stomatology, Sichuan University, Chengdu 610041, China

Received:2022-07-05 Revised:2022-09-08 Online:2023-02-01 Published:2023-02-21
Contact: Li Chenghao. E-mail:2535806717@qq.com;leechenghao_cn@163.com
Supported by:
Key Research and Development Plan of Sichuan Science and Technology Department(2022ZDYF2641)

摘要/Abstract

摘要：

目的研究地塞米松诱导同系新西兰仔兔先天腭裂的转录组异质性，进一步探究先天性腭裂发生的分子机制。方法妊娠第14天到第17天给20只新西兰孕兔肌肉注射地塞米松（每天1.0 mg），观察每只孕兔所有子代腭部表型。选取生产了8只胚胎、腭裂胚胎与非腭裂胚胎为4∶4的孕兔，分为腭裂组（CP）与非腭裂组（NCP），收集其腭部组织进行转录组测序分析。结果 CP组与NCP组相比，共有225个差异基因（Q<0.05），其中120个基因上调，105个基因下调。差异性表达基因的GO和KEGG富集分析表明，异三聚体G蛋白复合物、细胞外基质、转录因子复合物、基底膜的GO分类呈显著富集；GABA能突触、吗啡成瘾、逆行内源性大麻素信号、谷氨酸突触、含血清素的神经突触、肌动蛋白细胞骨架的调节、Apelin信号通路等在KEGG通路中显著富集。实时荧光定量分析法验证表明，与NCP组相比，CP组ARHGEF6（P<0.05）、ABI2（P<0.001）基因表达水平下降，APC基因表达水平上升（P<0.001）。结论参与腭突中肌动蛋白细胞骨架调节的基因ARHGEF6、APC、ABI2的表达水平异常可能和地塞米松诱导新西兰兔先天性腭裂的形成相关。

关键词: 地塞米松, 先天性腭裂, 转录组学分析, 细胞骨架调节

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

中图分类号:

R 782.2⁺1

林兰玲, 刘皓月, 罗枭, 张翀, 景兵帅, 石冰, 李承浩. 地塞米松诱导同系新西兰仔兔先天性腭裂的转录组异质性[J]. 华西口腔医学杂志, 2023, 41(1): 37-42.

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.

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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

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地塞米松诱导同系新西兰仔兔先天性腭裂的转录组异质性

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

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