School of Medical Science and Laboratory Medicine, Jiangsu University, Zhenjiang 212013, Jiangsu, China.
Marshall International Center for Digestive Diseases, Shanghai East Hospital, Tongji University, Shanghai 200120, China.
Aging (Albany NY). 2020 Jul 16;12(14):14467-14479. doi: 10.18632/aging.103488.
Coxsackie B3 virus (CVB3) is a member of small RNA viruses that belongs to the genus Enterovirus of the family Picornaviridae and CVB3 is the main pathogen of acute and chronic viral myocarditis. In this study RT-qPCR was used to determine the expression of miR-107 in CVB3-infected and uninfected HeLa cells. The experimental results show that the level of miR-107 began to rise at 4 h after the infection, and significantly boosted at 6 h. Based on the results of this experiment, we consider that miR-107 expression is related to CVB3 infection. In order to further clarify the effect of miR-107 in the process of CVB3 infection, we studied the effect of miR-107 upstream and downstream target genes on CVB3 replication. Levels of the target RNAs were detected by RT-qPCR after CVB3 infection, and the expression of CVB3 capsid protein VP1 by western blot analysis. Then the virus in the supernatant was quantitated via a viral plaque assay, reflecting the release of the virus. The experimental results showed that miRNA-107 expression is associated with CVB3 replication and proliferation, while KLF4 and BACE1 as the downstream of miR-107 weakened CVB3 replication. Overexpressions of KLF4 and BACE1 negatively regulated CVB3 replication, this effect on CVB3 was completely opposite to that of miR-107. Further experiments revealed that the upstream lncRNA004787, a new lncRNA that had not been reported, was located on chromosome 5, strand - from 37073250 to 37070908 (genome assembly: hg19). We sequenced and studied lncRNA004787 and found that it partially inhibited CVB3 replication. This prompted us to speculate that lncRNA004787 probably impacted the replication by other means. In conclusion, miR-107 interfered with CVB3 replication and release.
柯萨奇 B3 病毒（CVB3）属于小 RNA 病毒，属于小 RNA 病毒科的肠道病毒属，CVB3 是急性和慢性病毒性心肌炎的主要病原体。在本研究中，使用 RT-qPCR 确定了感染和未感染 HeLa 细胞的 CVB3 中的 miR-107 的表达。实验结果表明，miR-107 的水平在感染后 4 小时开始上升，并在 6 小时显着增加。基于该实验的结果，我们认为 miR-107 的表达与 CVB3 感染有关。为了进一步阐明 miR-107 在 CVB3 感染过程中的作用，我们研究了 miR-107 上下游靶基因对 CVB3 复制的影响。通过 RT-qPCR 检测 CVB3 感染后靶 RNA 的水平，并通过 Western blot 分析检测 CVB3 衣壳蛋白 VP1 的表达。然后通过病毒斑测定定量测定上清液中的病毒，反映病毒的释放。实验结果表明，miRNA-107 的表达与 CVB3 的复制和增殖有关，而作为 miR-107 下游的 KLF4 和 BACE1 减弱了 CVB3 的复制。KLF4 和 BACE1 的过表达负调控 CVB3 复制，对 CVB3 的作用与 miR-107 完全相反。进一步的实验表明，上游 lncRNA004787，一种尚未报道的新 lncRNA，位于染色体 5 上，链-从 37073250 到 37070908（基因组组装：hg19）。我们对 lncRNA004787 进行了测序和研究，发现它部分抑制了 CVB3 的复制。这促使我们推测 lncRNA004787 可能通过其他方式影响复制。总之，miR-107 干扰了 CVB3 的复制和释放。
