颅神经损伤面颈部肌萎缩纤维化中环状RNA差异表达分析

Analysis of Differential Expression of Circular RNA in Muscular Atrophy and Fibrosis of Facial and Neck Cranial Nerve Injury

目的检测颅神经损伤后面颈部骨骼肌萎缩纤维化过程中环状RNA(circular RNA,circRNA)的差异表达,探讨circRNA在面颈部失神经骨骼肌萎缩纤维化中的潜在功能。方法使用12周龄C57BL/6雄性小鼠共50只,分为对照组(仅行右侧副神经探查术)、失神经支配1周组、失神经支配2周组、失神经支配3周组及失神经支配4周组,每组10只;失神经支配各组建立颅神经损伤失神经支配肌萎缩纤维化模型(均切除5 mm右侧副神经)。各组分别取术侧胸锁乳突肌进行Masson染色观察骨骼肌形态,高通量转录组测序技术(RNA-seq)检测各组小鼠失神经支配胸锁乳突肌的circRNA差异表达,并行生物信息学分析。结果Masson染色验证骨骼肌组织萎缩纤维化显著,造模成功;鉴定失神经骨骼肌具有差异性表达的circRNA共1386个,其中上调686个,下调700个,与对照组比较,失神经支配1、2、3、4周组分别上调83、138、314、151个,下调103、149、204、244个;GO功能分析显示,这些差异表达circRNA的亲本基因在失神经损伤早期与肌细胞分化和发育等相关,失神经损伤晚期则与肌细胞凋亡、结缔组织增生等相关。KEGG分析显示TGF-β、Apelin信号通路、FoxO信号通路等与纤维化调控密切相关的信号通路在各实验组均有不同程度的富集。结论面颈部颅神经损伤肌萎缩纤维化过程中circRNA具有差异性表达,可能参与喉返神经等颅神经的失神经肌萎缩纤维化进程。

Objective To detect the differential expression of circular RNA(circRNA)in the process of skeletal muscle atrophy and fibrosis after cranial nerve injury in the face and neck,and explore the potential function of circRNA in the denervated skeletal muscle atrophy and fibrosis of the face and neck.Methods A total of 5012-week-old C57BL/6 male mice were used and were divided into 5 groups,with 10 mice in each group control group,denervation for 1 week group,denervation for 2 weeks group,denervation for 3 weeks group,and denervation for 4 weeks group.A cranial nerve injury denervated muscle atrophy and fibrosis model were established taking the sternocleidomastoid muscle.Masson staining was used for morphological observation of skeletal muscle.The differentially expressed(DE)circRNAs were screened by high-throughput transcriptome sequencing technology(RNA-seq)in each group of denervated sternocleidomastoid muscle,bioinformatics analysis was performed.Results Masson staining verified the success of the modeling.In this study,1386 differentially expressed circRNAs in denervated skeletal muscle were identified,in which 686 were up-regulated and 700 were down-regulated.There were differential expressions at different times of denervation.Comparing to the control group,denervation for 1 week group,for 2 weeks group,for 3 weeks group and for 4 weeks group up-regulated 83,138,314 and 151 circRNAs,and down-regulated 103,149,204 and 244 circRNAs,respectively.GO functional analysis showed that these differentially expressed circRNA parent genes were related to the differentiation and development of muscle cells in the early stage of denervation injury,and were related to muscle cell apoptosis and connective tissue proliferation in the late stage of denervation injury.KEGG analysis showed that signal pathways closely related to fibrosis regulation,such as TGF-β,Apelin signaling pathway,and FoxO signaling pathway,were enriched in different degrees in each experimental group.Conclusion CircRNA is differentially expressed in muscular atrophy and fibrosis of facial and neck cranial nerve injury,which may be involved in the process of denervated muscle atrophy and fibrosis of cranial nerves such as recurrent laryngeal nerve.