HCMV-miR-US33-5p 通过靶向 EPAS1/SLC3A2 通路促进主动脉血管平滑肌细胞凋亡。
HCMV-miR-US33-5p promotes apoptosis of aortic vascular smooth muscle cells by targeting EPAS1/SLC3A2 pathway.
机构信息
Department of Vascular Surgery, Shanghai TCM-Integrated Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai, China.
Department of Vascular Surgery, Changhai Hospital, Navy Medical University, Shanghai, China.
出版信息
Cell Mol Biol Lett. 2022 May 20;27(1):40. doi: 10.1186/s11658-022-00340-w.
BACKGROUND
In patients with acute aortic dissection (AAD), increased vascular smooth muscle cell (VSMC) apoptosis has been found. Human cytomegalovirus (HCMV)-miR-US33-5p was significantly increased in the plasma of patients with AAD. However, the roles of miR-US33-5p in human aortic VSMC (HA-VSMC) apoptosis remain to be elucidated.
METHODS
In the current study, cell apoptosis was analyzed by flow cytometry, cell proliferation by CCK-8 assay, and differentially expressed genes by RNA sequencing. Luciferase reporter assay was used for binding analysis between miR-US33-5p and endothelial PAS domain protein 1 (EPAS1), and EPAS1 and amino acid transporter heavy chain, member 2 (SLC3A2). The enrichment degree of SLC3A2 promoter DNA was analyzed by chromatin immunoprecipitation assay. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and immunoblotting were performed for measuring messenger RNA (mRNA) and protein levels, respectively.
RESULTS
It was found that HCMV infection inhibited proliferation but promoted HA-VSMC apoptosis by upregulating HCMV-miR-US33-5p. Transfection of HCMV-miR-US33-5p mimics the significant effect on several signaling pathways including integrin signaling as shown in the RNA sequencing data. Western blotting analysis confirmed that HCMV-miR-US33-5p mimics suppression of the activity of key factors of the integrin signal pathway including FAK, AKT, CAS, and Rac. Mechanistic study showed that HCMV-miR-US33-5p bound to the 3'-untranslated region of EPAS1 to suppress its expression, leading to suppression of SLC3A2 expression, which ultimately promoted cell apoptosis and inhibited cell proliferation. This was confirmed by the findings that silencing EPAS1 significantly reduced the SLC3A2 expression and inhibited proliferation and key factors of integrin signal pathway.
CONCLUSIONS
HCMV-miR-US33-5p suppressed proliferation, key factors of integrin signal pathway, and EPAS1/SLC3A2 expression, but promoted HA-VSMC apoptosis. These findings highlighted the importance of HCMV-miR-US33-5p/EPAS1/SCL3A2 signaling and may provide new insights into therapeutic strategies for AAD.
背景
在急性主动脉夹层（AAD）患者中，已发现血管平滑肌细胞（VSMC）凋亡增加。人巨细胞病毒（HCMV）-miR-US33-5p 在 AAD 患者的血浆中显著增加。然而，miR-US33-5p 在人主动脉 VSMC（HA-VSMC）凋亡中的作用仍有待阐明。
方法
在本研究中，通过流式细胞术分析细胞凋亡，通过 CCK-8 测定法分析细胞增殖，通过 RNA 测序分析差异表达基因。荧光素酶报告分析用于分析 miR-US33-5p 与内皮 PAS 域蛋白 1（EPAS1）以及 EPAS1 和氨基酸转运蛋白重链成员 2（SLC3A2）之间的结合分析。通过染色质免疫沉淀测定法分析 SLC3A2 启动子 DNA 的富集程度。通过定量逆转录聚合酶链反应（qRT-PCR）和免疫印迹分别进行信使 RNA（mRNA）和蛋白质水平的测量。
结果
研究发现，HCMV 感染通过上调 HCMV-miR-US33-5p 抑制增殖但促进 HA-VSMC 凋亡。HCMV-miR-US33-5p 转染显示了几个信号通路的显著作用，包括整合素信号通路，如 RNA 测序数据所示。Western blot 分析证实，HCMV-miR-US33-5p 抑制了整合素信号通路的关键因子的活性，包括 FAK、AKT、CAS 和 Rac。机制研究表明，HCMV-miR-US33-5p 结合到 EPAS1 的 3'-非翻译区以抑制其表达，导致 SLC3A2 表达受到抑制，最终促进细胞凋亡并抑制细胞增殖。通过发现沉默 EPAS1 可显著降低 SLC3A2 的表达并抑制增殖和整合素信号通路的关键因子，证实了这一点。
结论
HCMV-miR-US33-5p 抑制增殖、整合素信号通路的关键因子和 EPAS1/SLC3A2 的表达，但促进 HA-VSMC 凋亡。这些发现强调了 HCMV-miR-US33-5p/EPAS1/SCL3A2 信号的重要性，并可能为 AAD 的治疗策略提供新的见解。
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