Zhao Yabo, Wang Zaiwei, Hou Yue, Zhang Kang, Peng Xiuli
Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction (Huazhong Agricultural University), Ministry of Education, College of Animal Science and Technology & College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China.
Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction (Huazhong Agricultural University), Ministry of Education, College of Animal Science and Technology & College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China.
Gene. 2017 Sep 5;627:239-247. doi: 10.1016/j.gene.2017.06.039. Epub 2017 Jun 23.
Mycoplasma gallisepticum (MG), one of the primary etiological agents of poultry chronic respiratory disease, has caused significant economic losses worldwide, and increasing evidence has recently indicated that miRNAs are involved in its microbial pathogenesis. gga-miR-99a, a member of the miR-99 family, plays an essential role in a variety of diseases. Through miRNA Solexa sequencing, we previously found that gga-miR-99a is significantly down-regulated in the lungs of MG-infected chicken embryos. In this study, we further verified that the expression of gga-miR-99 was significantly down-regulated in both MG-infected lungs and a chicken embryonic fibroblast cell line (DF-1) by qPCR. Moreover, we predicted and validated SMARCA5 as its target gene through a luciferase reporter assay, qPCR, and western blot analysis. The over-expression of gga-miR-99a significantly depressed SMARCA5 expression, whereas a gga-miR-99a inhibitor enhanced the expression of SMARCA5. Inversely, SMARCA5 was significantly up-regulated and gga-miR-99a was obviously down-regulated in MG-HS-infected chicken embryonic lungs and DF-1 cells. At 72h post-transfection, the over-expression of gga-miR-99a significantly repressed the proliferation of DF-1 cells by inhibiting the transition from the G1 phase to the S and G2 phases. This study reveals that gga-miR-99a plays a key role in MG infection through the regulation of SMARCA5 expression and provides new insights regarding the mechanisms of MG pathogenesis.
鸡毒支原体（MG）是家禽慢性呼吸道疾病的主要病原体之一，在全球范围内造成了重大经济损失，最近越来越多的证据表明，微小RNA（miRNA）参与了其微生物发病机制。gga-miR-99a是miR-99家族的成员之一，在多种疾病中起重要作用。通过miRNA Solexa测序，我们之前发现gga-miR-99a在MG感染的鸡胚肺中显著下调。在本研究中，我们通过定量聚合酶链反应（qPCR）进一步验证了gga-miR-99在MG感染的肺组织和鸡胚成纤维细胞系（DF-1）中表达均显著下调。此外，我们通过荧光素酶报告基因检测、qPCR和蛋白质免疫印迹分析预测并验证了SMARCA5是其靶基因。gga-miR-99a的过表达显著抑制了SMARCA5的表达，而gga-miR-99a抑制剂则增强了SMARCA5的表达。相反，在MG-HS感染的鸡胚肺和DF-1细胞中，SMARCA5显著上调，gga-miR-99a明显下调。转染后72小时，gga-miR-99a的过表达通过抑制从G1期到S期和G2期的转变，显著抑制了DF-1细胞的增殖。本研究揭示了gga-miR-99a通过调节SMARCA5的表达在MG感染中起关键作用，并为MG发病机制提供了新的见解。
