摘要
[目的]探究长链非编码RNA(lncRNA)MSTRG.14200对猪骨骼肌卫星细胞(PSCs)成肌分化及不同类型肌纤维转化的影响.[方法]选择3头6月龄杜洛克猪,屠宰后采集心脏、肝脏、脾脏、肺脏、肾脏、胃、背最长肌、腿肌、比目鱼肌和趾长伸肌组织,利用实时荧光定量PCR检测MSTRG.14200在不同组织中的表达情况.构建MSTRG.14200过表达载体pcDNA3.1-14200并转染PSCs细胞,诱导分化3 d后收集细胞.通过实时荧光定量PCR和Western blotting分别检测肌球蛋白重链(MyHC)、肌细胞生成素(MyoG)和肌分化因子(MyoD)、MyHC Ⅰ、MyHCⅡa、MyHCⅡb和MyHCⅡxmRNA和蛋白表达情况;利用免疫荧光法检测MyHC、MyHC Ⅰ和MyHCⅡ b阳性细胞比率.[结果]MSTRG.14200在杜洛克猪不同组织中均有表达,且在背最长肌中表达量最高,极显著高于其他组织(P＜0.01),在趾长伸肌和比目鱼肌中的表达量存在显著差异(P＜0.05).过表达MSTRG.14200后,细胞分化相关基因MyoD和MyHC的mRNA水平极显著升高(P＜0.01),MyoD、MyoG和MyHC蛋白水平显著或极显著升高(P＜0.05;P＜0.01),MyHC阳性细胞比率极显著升高(P＜0.01);肌纤维类型转化相关基因MyHCⅠ的mRNA和蛋白表达量及阳性细胞比例率均显著或极显著降低(P＜0.05;P＜0.01),MyHCⅡ b基因mRNA和蛋白表达量以及阳性细胞比率均显著升高(P＜0.05).[结论]MSTRG.14200具有促进猪骨骼肌成肌分化以及促进慢肌纤维向快肌纤维转化的作用.研究结果为探究猪骨骼肌纤维类型转化的分子机制提供理论基础.
Abstract
[Objective]This experiment was conducted to investigate the effects of long non-coding RNA(lncRNA)MSTRG.14200 on myogenic differentiation of porcine skeletal muscle satellite cells and the transformation of different types of muscle fibers.[Method]Three 6-month-old Duroc pigs were slaughtered,and the heart,liver,spleen,lung,kidney,stomach,longissimus dorsi muscle,leg muscle,soleus and extensor digitorum longus were collected.Real-time quantitative PCR was used to detect the expression of MSTRG.14200 in different tissues.The MSTRG.14200 overexpression vector pcDNA3.1-14200 was constructed and transfected into PSCs cells,and the cells were collected after 3 days of induced differentiation.The mRNA and protein expressions of myosin heavy chain(MyHC),myogenin(MyoG),mydifferentiation factor(MyoD),MyHC Ⅰ,MyHC Ⅱ a,MyHC Ⅱ b and MyHC Ⅱ x were detected by Real-time quantitative PCR and Western blotting,respectively.The percentage of MyHC,MyHC Ⅰ and MyHC Ⅱ b positive cells was detected by immunofluorescence assay.[Result]MSTRG.14200 was expressed in different tissues of Duroc pigs,and the expression was the highest in longissimus dorsi muscle,which was extremely significantly higher than other tissues(P＜0.01),and there were significant differences in the expression of extensor digitorum longus and soleus(P＜0.05).After overexpression of MSTRG.14200,mRNA levels of MyoD and MyHC genes related to cell differentiation were extremely significantly increased(P＜0.01),and protein levels of MyoD,MyoG and MyHC were significantly or extremely significantly increased(P＜0.05 or P＜0.01),the percentage of MyHC positive cells was extremely significantly increased(P＜0.01).The mRNA and protein expressions of MyHC Ⅰ gene related to muscle fiber transformation and the proportion of positive cells were significantly or extremely significantly decreased(P＜0.05 or P＜0.01),the mRNA and protein expression of MyHC Ⅱ b gene and the percentage of positive cells were significantly increased(P＜0.05).[Conclusion]MSTRG.14200 could promote myogenic differentiation of porcine skeletal muscle and promote the transformation of slow muscle fibers into fast muscle fibers.The results provided a theoretical basis for exploring the molecular mechanism of transformation of porcine skeletal muscle fiber type.
基金项目
广东省重点研发计划项目(2022B0202090002)
维普
万方数据