Xue Jian, Xue Jingwen, Zhang Ji, Li Dan, Jiang Lei
College of Animal Husbandry & Veterinary, Jinzhou Medical University, No. 48, Section 5, Renmin Street, Guta District, Jinzhou City, 121001, Liaoning Province, People's Republic of China.
College of Animal Science and Veterinary Medicine, Shandong Agricultural University, Tai'an, 271018, People's Republic of China.
Biotechnol Lett. 2017 Nov;39(11):1611-1619. doi: 10.1007/s10529-017-2400-8. Epub 2017 Jul 18.
To explore the roles of miR-130b-3p and miR-301b-3p which may regulate Rb1-inducible coiled-coil 1 (Rb1cc1) expression during myogenic differentiation of chicken primary myoblasts.
After 4 days of myogenic differentiation, myotubes appeared and after 6 days the cells fused to each other and expression of MyHC could be detected by immunofluorescence staining. TargetScan and RNAhybrid 2.2 showed miR-130b-3p and miR-301b-3p were well complementary with the target site of Rb1cc1 3'-untranslated region (3'-UTR). Using the dual-luciferase assay, we found miR-130b-3p and miR-301b-3p could inhibit Rb1cc1 expression by binding to its 3'-UTR. Real-time PCR showed Rb1cc1 mRNA expression level was almost reciprocal to that of miR-130b-3p or miR-301b-3p during myogenic differentiation. Furthermore, over-expression of miR-130b-3p or miR-301b-3p down-regulated the expression levels of Rb1cc1, myoblast determination protein, myogenin and myosin heavy chain.
miR-130b-3p or miR-301b-3p negatively regulate Rb1cc1 expression to affect myogenic differentiation.
探讨miR-130b-3p和miR-301b-3p在鸡原代成肌细胞成肌分化过程中可能对Rb1诱导卷曲螺旋蛋白1（Rb1cc1）表达的调控作用。
成肌分化4天后出现肌管，6天后细胞相互融合，免疫荧光染色可检测到肌球蛋白重链（MyHC）的表达。TargetScan和RNAhybrid 2.2显示miR-130b-3p和miR-301b-3p与Rb1cc1 3'非翻译区（3'-UTR）的靶位点具有良好的互补性。通过双荧光素酶报告基因检测，我们发现miR-130b-3p和miR-301b-3p可通过与Rb1cc1的3'-UTR结合来抑制其表达。实时定量PCR显示，在成肌分化过程中，Rb1cc1 mRNA表达水平与miR-130b-3p或miR-301b-3p的表达水平几乎呈负相关。此外，miR-130b-3p或miR-301b-3p的过表达下调了Rb1cc1、成肌决定蛋白、生肌调节因子和肌球蛋白重链的表达水平。
miR-130b-3p或miR-301b-3p通过负调控Rb1cc1表达影响成肌分化。
