MyomiRs在维甲酸诱导舌发育异常中对舌肌分化调控的机制研究

The research on the mechanisms of the muscle-specific microRNAs' regulation in the tongue's differentiation of the retinoic acid induced tongue dysplasia

目的:检测维甲酸(Retinoic acid,RA)诱导小鼠胚胎腭裂模型中胎鼠舌体发育过程中肌相关microRNAs(MyomiRs)、成肌调节因子(Myogenic regulatory factors,MRFs)以及Pax基因的表达变化,探究MyomiRs在舌肌分化过程中的调控作用,推测RA致胎鼠腭裂伴发舌异常的可能机制。方法:建立RA诱导小鼠胚胎腭裂模型,分别在E13.5、E14.5、E15.5收集胎鼠舌体组织,用SYBR GreenⅠ实时定量PCR检测舌体中MRFs和Pax基因的表达;用Taq Man探针实时定量PCR检测舌体中MyomiRs的表达。结果:胎鼠舌体发育过程中,正常组miR-1和miR-206相对表达量均持续上升,RA诱导组二者变化趋势与正常组相似,但相对表达量均低于正常组,miR-1的结果在E14.5和E15.5具有统计学意义(P<0.01),miR-206的结果在E13.5具有统计学意义(P<0.05)。正常组和RA诱导组胎鼠舌体中Myo D和Myf5相对表达量都在E14.5达到峰值,随后下降。RA诱导组Myo D的表达在E14.5显著低于正常组(P<0.05),在E15.5显著高于正常组(P<0.01);RA诱导组Myf5的表达在E15.5显著低于正常组(P<0.05)。正常组和RA诱导组胎鼠舌体中Pax3表达均在E14.5达到峰值,Pax7表达均在E15.5达到峰值。RA诱导组Pax3的表达在E14.5显著高于正常组(P<0.05);Pax7的表达则在E13.5显著高于正常组(P<0.01)。结论:在舌肌分化过程以及RA诱导腭裂胎鼠的舌发育异常中,miR-1/miR-206与Pax3/Pax7及Myf5/Myo D的表达趋势具有相关性。RA可能通过下调miR-1/miR-206而靶向上调Pax3/Pax7,进而下调Myo D/Myf5表达,从而抑制舌肌分化,导致舌肌发育异常。

Objective：To research the mechanisms of the muscle-specific microRNAs in the tongue′s differentiation of the retinoic acid induced tongue dysplasia by measuring the expression of MyomiRs,MRFs and Pax3 /Pax7.Methods：A retinoic acid induced tongue dysplasia mouse model was established.The tongues were collected at E13.5,E14.5 and E15.5.The expression levels of MyomiRs, MRFs and Pax3 /Pax7 were detected by real-time quantitative PCR.Results：During the tongue development,the expression of miR-1 and miR-206 kept on rising both in the tongues of RA-treated mice and normal ones.While the expression of miR-1 and miR-206 in the RA-treated mice′s tongues was less than in the normal ones.The results of miR-1 at E14.5 and E15.5 had statistical significance （P〈0.01） and the results of miR-206 at E13.5 had statistical significance （P〈0.05）.The expression of MyoD and Myf5 in the normal and RA-treated mice′s tongues reached the peak at E14.5 then decreased.At E13.5 and E14.5,the expression of MyoD in the RA-treated mice′s tongues was less than the controls.But at E15.5,the expression of MyoD in the RA-treated mice′s tongues was higher （P〈0.01）.And at E14.5 and E15.5,the expression of Myf5 in the RA-treated mice′s tongues was lessthan the normal subjects.During the tongue myo-genesis of normal and RA-treated mice the expression of Pax3 in the tongue reached the peak at E14.5,and the expression of Pax7 in both of them reached the peak at E15.5.Compared to the normal mice,the expression of Pax3 was higher at E14.5 （P〈0.05）,and the ex-pression of Pax7 was higher at E13.5 （P〈0.01）in the RA-treated mice′s tongues.Conclusions：During the tongue myogenesis,miR1 /miR-206,Pax3 /Pax7 and Myf5 /MyoD were associated.In the retinoic acid induced tongue dysplasia,the correlation still existed.RA may down-regulate miR-1 /miR-206 which targeted on Pax3 /Pax7,following down-regulate the expression of MyoD /Myf5 to inhibit the myogen-esis in tongues of RA-treated mice,resulted in tongue dysplasia.