LncRNA-MEG3及Rac1/Limk1/Cofilin信号通路在先天性巨结肠中的作用机制

Mechanism of LncRNA-MEG3 and Rac1/Limk1/Cofilin signaling pathway in Hirschsprung disease

目的探讨母系表达基因3(maternally expressed gene 3,MEG3)及Rac1/Limk1/Cofilin信号通路在先天性巨结肠(Hirschsprung disease,HD)中的作用机制。方法应用qRT-PCR、Westernblot方法检测2018年1月至2021年1月在遵义医科大学附属医院治疗并确诊为HD的30例患儿HD狭窄段、30例移行段组织和14例正常肠管组织中MEG3和Rac1/Limk1/Cofilin表达情况,并通过建立MEG3过表达组、溶剂组、对照组及Rac1、Limk1、Cofilin抑制组细胞模型,观察Rac1、Limk1及Cofilin蛋白表达情况及细胞迁移和增殖能力。结果狭窄段、移行段MEG3、Limk1和Cofilin的RNA相对表达量较正常肠管组织低(P<0.05),而移行段与狭窄段比较,差异无统计学意义(P>0.05);Rac1的相对表达量比较:正常肠管>移行段>狭窄段,差异有统计学意义(P<0.05)。狭窄段、移行段Rac1、Limk1和Cofilin蛋白表达水平与正常肠管组织比较均明显降低(P<0.05),而移行段与狭窄段比较,差异无统计学意义(P>0.05)。MEG3过表达组MEG3的表达量明显高于对照组及溶剂组(P<0.05);MEG3过表达组Rac1、Limk1的相对表达量均明显高于对照组及溶剂组(P<0.05);MEG3过表达组Cofilin相对表达量与对照组、溶剂组比较无明显差异(P>0.05)。MEG3过表达组Rac1和Cofilin蛋白的相对表达量明显高于溶剂组和对照组(P<0.05),MEG3过表达组Limk1的表达量与对照组比较无明显差异(P>0.025);MEG3过表达组细胞的迁移数量明显高于对照组和溶剂组(P<0.05)。对照组Rac1、Limk1、Cofilin的蛋白表达量明显高于Rac1、Limk1和Cofilin抑制组(P<0.05),对照组细胞的迁移数量明显高于Rac1、Limk1、Cofilin抑制组(P<0.05)。结论MEG3可通过促进Rac1、Limk1和Cofilin基因表达影响神经嵴细胞的迁移,进而与先天性巨结肠发病有关。

Objective To explore the mechanism of maternally expressed gene 3(MEG3)and Rac1/Limk1/Cofilin signaling pathway in the pathogenesis of Hirschsprung disease(HD).Methods Between January 2018 and January 2021,the expressions of MEG33 and Rac1/Limk1/Cofilin were detected by quantitative reverse transcription-polymerase chain reaction(qRT-PCR)and Western blot in HD stenosis(n=30),transitional segment(n=30)and normal intestinal tissues(n=14).The cell models of MEG3 overexpression group,solvent group,control group and Rac1/Limk1/Cofilin inhibition groups were established for observing the expressions of Rac1/Limk1/Cofilin proteins and assessing the capabilities of cellular migration and proliferation.Results The expression levels of Rac1/Limk1/Cofilin were lower in stenotic and transitional segments than those in normal intestinal tissues(P<0.05).However,no significant differences existed between transitional and stenotic segments(P>0.05).The relative expression of Rac1 was higher in normal bowels than that in transitional and stenosis segments and the difference was statistically significant(P<0.05).The protein expression levels of Rac1 Rac1/Limk1/Cofilin in HD stenotic and transitional segments were significantly lower than those in normal intestinal tissues(P<0.05).However,no significant differences existed between transitional and stenotic segments(P>0.05).The expression of MEG3 was significantly higher in MEG3 overexpression group than that in control/solvent group(P<0.05).The relative expression levels of Rac1 and Limk1 were significantly higher in MEG3 overexpression group than those in control/solvent group(P<0.05).The relative expression of Cofilin in MEG3 overexpression group was not significantly different from that in control/solvent group(P>0.05).The expression levels of Rac1 and Cofilin were significantly higher in MEG3 overexpression group than those in solvent/control group(P<0.05).The expression levels of Limk1 in MEG3 overexpression group was not significantly different from that in control group(P>0.025).The number of cellular migration in MEG3 overexpression group was significantly higher than that in control/solvent group(P<0.05).Protein expression levels of Rac1/Limk1/Cofilin were significantly higher in control group than those in Rac1/Limk1/Cofilin inhibition group(P<0.05).The number of cellular migration was significantly higher in control group than that in Rac1/Limk1/Cofilin inhibition group(P<0.05).Conclusion MEG3 affects the migration of neural crest cells through promoting the expressions of Rac1/Limk1/Cofilin genes.It may be correlated with the pathogenesis of HD.