LncRNA056298通过影响生长相关蛋白43的表达介导射频消融犬的神经重构

doi:10.6040/j.issn.1671-7554.0.2020.192

摘要/Abstract

摘要： 目的探讨射频消融术后内在自主神经重构的发生机制,为房颤复发机制提供理论依据。方法选取12只健康成年杂种犬,随机分为1月射频消融组、1月阴性对照组、6月射频消融组和6月阴性对照组,每组3只。1、6月对照组仅行开胸处理;1、6月射频消融组消融右上及右下神经节,分别饲养1、6个月后取消融周围1 cm的心房组织行高通量二代测序,寻找差异表达的长链非编码RNAs(lncRNAs),并预测其靶基因。选取健康成年杂种犬12只,对目的lncRNA进行过表达慢病毒构建,随机分为阴性对照组(右房肌内注射 LncRNA056298阴性对照慢病毒)及过表达慢病毒组(右房肌内注射LncRNA056298过表达慢病毒),每组6只。于注射前及注射慢病毒饲养10 d后检测心房有效不应期(AERP)、房颤诱发率,饲养10 d后将实验犬处死取右心房,采用qRT-PCR法和Western blotting法检测LncRNA056298和GAP43基因及蛋白水平的变化,采用免疫组织化学法检测乙酰胆碱转移酶(CHAT)、酪氨酸羟化酶(TH)、生长相关蛋白43(GAP43)及蛋白基因产物9.5(PGP9.5)等与内在自主神经重构相关的指标变化。结果通过生信分析,筛选出差异表达的LncRNA056298及其靶基因GAP43,过表达慢病毒组注射病毒10 d后,发现AERP较注射前明显缩短(95.000 vs 85.000, Z=-3.012,P=0.008),而阴性对照组注射病毒10 d后,AERP较注射前差异无统计学意义(92.500 vs 95.000,Z=0.842,P=0.600)。房颤诱发结果显示,过表达慢病毒组有3只犬诱发房颤,阴性对照组未诱发出房颤。免疫组化结果显示,过表达慢病毒组与阴性对照组相比,内在自主神经重构相关指标GAP43、TH、CHAT及PGP9.5表达均增加。结论 LncRNA056298可通过影响GAP43的表达影响内在自主神经重构的发生,内在自主神经重构的发生可能是影响射频消融后房颤复发的一个重要因素。

关键词: 心房颤动, 神经重构, 射频消融术, 长链非编码RNA, 长链非编码RNA-056298, 生长相关蛋白43

Abstract: Objective To investigate the mechanism of intrinsic autonomic remodeling after radiofrequency ablation in order to provide theoretical basis for mechanism of atrial fibrillation recurrence. Methods A total of 12 healthy adult mongrel dogs were randomly divided into 1-month radiofrequency ablation group, 1-month negative control group, 6-month radiofrequency ablation group and 6-month negative control group, with 3 dogs in each group. In the control groups, only thoracotomy was performed; in the ablation groups, the right upper and right lower ganglia were ablated, and atrial tissues 1 cm around the ablation were harvested after 1 month and 6 months of feeding for high-throughput second-generation sequencing to identify the differentially expressed long non-coding RNAs(lncRNAs)and predict their target genes. Another 12 healthy adult mongrel dogs were selected and randomly divided into the negative control group(intramuscular injection of LncRNA056298 negative control lentivirus into the right atrium)and overexpressing group(intramuscular injection of LncRNA056298 overexpressing lentivirus into the right atrium), with 6 dogs in either group. The atrial effective refractory period(AERP)and induction rate of atrial fibrillation were detected before lentivirus injection and 10 days after the injection. The right atria of the experimental dogs were collected to detect the mRNA and protein expressions of LncRNA056298 and GAP43 with qRT-PCR and Western blotting. The changes of metrics related to intrinsic autonomic remodeling were detected with immunohistochemistry, such as choline acetyltransferase(CHAT), tyrosine hydroxylase(TH), growth associated protein 43(GAP43)and protein gene product 9.5(PGP9.5). Results Differentially expressed lncRNA056298 and target gene GAP43 were screened out via bioinformatics analysis. Ten days after injection of lentivirus overexpressing lncRNA056298 into experimental animals, AERP was significantly shorter than before(95.000 vs 85.000, Z=-3.012, P=0.008). However, there was no significant difference in AERP before and after injection with negative control lentivirus(92.500 vs 95.000, Z=0.842, P=0.600). The results of atrial fibrillation induction exhibited that atrial fibrillation was induced in 3 dogs in the overexpressing group, whereas there was no atrial fibrillation induced in the negative control group. Immunohistochemistry indicated that compared with negative control group, the overexpressing group had significantly increased expressions of GAP43, TH, CHAT and PGP9.5. Conclusion LncRNA056298 can affect the occurrence of intrinsic autonomic remodeling by affecting the expression of GAP43. Intrinsic autonomic remodeling may be an important factor affecting the recurrence of atrial fibrillation after radiofrequency ablation.

Key words: Atrial fibrillation, Neural remodeling, Radiofrequency ablation, Long non-coding RNA, LncRNA056298, Growth associated protein 43

中图分类号:

R541.7

刘东路,王曦敏,李展,杜娟娟,李建华,马神洲,侯应龙. LncRNA056298通过影响生长相关蛋白43的表达介导射频消融犬的神经重构[J]. 山东大学学报 (医学版), 2020, 58(5): 27-37.

LIU Donglu, WANG Ximin, LI Zhan, DU Juanjuan, LI Jianhua, MA Shenzhou, HOU Yinglong. LncRNA056298 mediates the occurrence of neural remodeling recurrence in radiofrequency ablation dogs by affecting the expression of growth associated protein 43[J]. Journal of Shandong University (Health Sciences), 2020, 58(5): 27-37.

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