| 敲低RACK1基因抑制C2C12细胞中MyoG和MHC基因表达 |
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| 作者单位： | ;1.武汉轻工大学动物营养与饲料科学湖北省重点实验室;2.中南民族大学生命科学学院 |
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| 摘 要： | 本实验旨在研究RNA干扰(RNAi)RACK1基因对C2C12成肌细胞中肌分化标志基因MHC和MyoG表达的影响。将人工合成的靶向RAKC1基因的3条si RNA(1,2,3)转染C2C12细胞,用RT-qPCR方法检测并筛选干扰效率最高的1条siRNA。将筛选出的si RNA转染C2C12成肌细胞并诱导细胞分化,通过RT-qPCR、Western blot和免疫荧光方法在mRNA及蛋白水平检测RACK1基因沉默后对MHC和MyoG表达的影响。此外,Western blot方法检测RACK1基因沉默后对PI3K/Akt和Erk/MAPK通路激活的影响。结果表明:RACK1-si RNA-2干扰效率最高,转染48 h后抑制率可达70%。随后,C2C12细胞转染si RNA-2,诱导分化48h后RTqPCR表明,MHC和MyoG的m RNA表达均显著性下调(P<0.05);诱导分化72 h后,Western blot和免疫荧光结果表明MHC和MyoG的蛋白表达明显低于对照组,但AKT和Erk磷酸化水平未见明显变化。上述结果表明,干扰RACK1能显著抑制MHC和MyoG的表达,提示RACK1正向调控C2C12成肌细胞的分化。
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| 关 键 词： | 成肌细胞 激活性蛋白激酶C受体1 肌细胞生成素 肌球蛋白重链 RNA干扰 |
Knock-down of RACK1 Inhibits the Expressions of Myog and MHC Genes in C2C12 Myoblast |
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| Institution: | ,Hubei Key laboratory of Animal Nutrition and Feedscience, Wuhan Polytechnic University,College of lifesciences,South-Central University for Nationa lities |
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| Abstract: | The purpose of the study was to investigate the influence of RACK1 genesilencing by RNA interference(RNAi)on the expressions of two myogenic differentiation markgenes(MHC and Myo G) in C2C12 myoblast. We transfected three chemica lly synthesized RACK1 si RNA(si RNA-1,2,3) into C2C12 myoblasts, and detected the interference efficiency with RT-q PCR in order toselect thesi RNA with the highest interference efficiency. Then, we transfected the selected si RNA into C2C12 cells and induced C2C12 differentiation. In order to study the effect of RACK1 gene silencing on the expressions of MHC and Myo G, we examined MHC and Myo G m RNA and protein expressions by using RT-q PCR, Western blot and immunofluorescence methods. In addition, the effect of the RACK1 silencing on the activation of p I3K/Akt and Erk/MAp K pathway was detected by Western blot ana lysis. The result showed that RACK1-si RNA-2 had the highest interference efficiency, and its interference efficiency was approximately 70% at 48 h after the transfection. C2C12 myoblasts were transfected with si RNA-2 and induced into differentiation for 48 h and 72 h respectively. RT-q PCR showed that MHC and Myo G m RNA were significantly decreased in si RNA-2 transfected cells after 48 h of differentiation; Western blot and immunofluorescence showed that MHC and Myo G protein were decreased after 72 h of differentiation. However, no significant changes in phosphorylation levels of Akt and Erk were observed. We conclude that downregulation of RACK1 by RNAi significantly inhibits the expressions of MHC and Myo G, indicating that RACK1 is involved in positive regulation of C2C12 differentiation. |
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| Keywords: | Myoblast RACK1 Myogenin Myosin heavy chain RAN interference |
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