miR-34a-5p 通过靶向 c-fos 抑制 KSHV 感染的 SH-SY5Y 细胞的恶性进展。
miR-34a-5p inhibits the malignant progression of KSHV-infected SH-SY5Y cells by targeting c-fos.
机构信息
Key Laboratory of Xinjiang Endemic and Ethnic Diseases/NHC Key Laboratory of Prevention and Treatment of Central Asia High Incidence Diseases, School of Medicine, Shihezi University, Shihezi, Xinjiang, China.
Australian Institute for Bioengineering and Nanotechnology (AIBN), University of Queensland (UQ), St Lucia, Brisbane, Australia.
出版信息
PeerJ. 2022 Apr 15;10:e13233. doi: 10.7717/peerj.13233. eCollection 2022.
BACKGROUND
We aimed to investigate the effects of miR-34a-5p on c-fos regulation mediating the malignant behaviors of SH-SY5Y cells infected with Kaposi's sarcoma-associated herpesvirus (KSHV).
METHODS
The KSHV-infected (SK-RG) and uninfected SH-SY5Y parent cells were compared for differentially expressed miRNAs using transcriptome sequencing. Then miR-34a-5p was upregulated in SK-RG cells by the miRNA mimics transfection. Cell proliferation ability was determined by MTT and plate clone assays. The cell cycle was assessed by flow cytometry analysis, and CDK4, CDK6, cyclin D1 levels were determined by Western blot analysis. The migration behavior was detected by wound healing and transwell assays. The protein levels of MMP2 and MMP9 were measured by Western blot analysis. The regulation of c-fos by miR-34a-5p was detected by the dual-luciferase reporter gene assay. Rescue assays were carried out by upregulating c-fos in miR-34a-5p-overexpressing SK-RG cells. KSHV DNA copy numbers and relative virus gene expressions were detected. Xenograft tumor experiments and immunohistochemistry assays were further used to detect the effects of miR-34a-5p.
RESULTS
miR-34a-5p was lower in SK-RG cells. Restoration of miR-34a-5p decreased cell proliferation and migration, leading to a G1 cell cycle arrest and down-regulation of CDK4/6, cyclin D1, MMP2, MMP9. KSHV copy number and expression of virus gene including latency-associated nuclear antigen (LANA), replication and transcription activator (RTA), open reading frame (K8.1), and KSHV G protein-coupled receptor (v-GPCR) were also reduced. Furthermore, c-fos is the target of miR-34a-5p, while enhanced c-fos weakened cellular behaviors of miR-34a-5p-overexpressing cells. Xenograft experiments and immunohistochemistry assays showed that miR-34a-5p inhibited tumor growth and virus gene expression.
CONCLUSION
Upregulated miR-34a-5p in KSHV-infected SH-SY5Y cells suppressed cell proliferation and migration through down-regulating c-fos. miR-34a-5p was a candidate molecular drug for KSHV-infected neuronal cells.
背景
本研究旨在探讨 miR-34a-5p 对 c-fos 调控在感染卡波西肉瘤相关疱疹病毒（KSHV）的 SH-SY5Y 细胞恶性行为中的作用。
方法
采用转录组测序比较 KSHV 感染（SK-RG）和未感染的 SH-SY5Y 亲本细胞中的差异表达 miRNA。然后通过 miRNA 模拟物转染上调 SK-RG 细胞中的 miR-34a-5p。通过 MTT 和平板克隆测定评估细胞增殖能力。通过流式细胞术分析评估细胞周期，通过 Western blot 分析测定 CDK4、CDK6 和 cyclin D1 水平。通过划痕愈合和 Transwell 测定检测迁移行为。通过 Western blot 分析测定 MMP2 和 MMP9 的蛋白水平。通过双荧光素酶报告基因检测检测 miR-34a-5p 对 c-fos 的调控。通过上调 miR-34a-5p 过表达 SK-RG 细胞中的 c-fos 进行挽救实验。检测 KSHV DNA 拷贝数和相对病毒基因表达。进一步进行异种移植肿瘤实验和免疫组织化学检测，以检测 miR-34a-5p 的作用。
结果
miR-34a-5p 在 SK-RG 细胞中表达下调。恢复 miR-34a-5p 降低了细胞增殖和迁移，导致 G1 细胞周期停滞，并下调 CDK4/6、cyclin D1、MMP2 和 MMP9。KSHV 拷贝数和病毒基因表达包括潜伏相关核抗原（LANA）、复制和转录激活剂（RTA）、开放阅读框（K8.1）和 KSHV G 蛋白偶联受体（v-GPCR）也降低。此外，c-fos 是 miR-34a-5p 的靶标，而增强的 c-fos 削弱了 miR-34a-5p 过表达细胞的细胞行为。异种移植实验和免疫组织化学检测表明，miR-34a-5p 抑制肿瘤生长和病毒基因表达。
结论
在 KSHV 感染的 SH-SY5Y 细胞中上调 miR-34a-5p 通过下调 c-fos 抑制细胞增殖和迁移。miR-34a-5p 是 KSHV 感染神经元细胞的候选分子药物。
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