Cao Dongdong, Wu Shuyuan, Wang Xiaolu, Li Ying, Xu Huiling, Pan Zemin, Wu Zhaofu, Yang Lei, Tan Xiaohua, Li Dongmei
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, Beier Road, Shihezi, Xinjiang, China.
School of Medicine, Hangzhou Normal University, Hangzhou, Zhejiang, China.
Cancer Cell Int. 2021 Oct 30;21(1):577. doi: 10.1186/s12935-021-02269-0.
The cancer caused by Kaposi's sarcoma-associated herpesvirus (KSHV) infection is one of the major causes of death in AIDS patients. Some patients have neurological symptoms, which appear to be associated with KSHV infection, based on the neurotropic tendency of this virus in recent years. The objectives of this study were to investigate the effects of KSHV infection on neuronal SH-SY5Y cells and to identify differentially expressed genes.
KSHV was collected from islk.219 cells. Real-time PCR was used to quantify KSHV copy numbers. KSHV was used to infect SH-SY5Y cells. The KSHV copy number in the supernatants and mRNA levels of latency-associated nuclear antigen (LANA), ORF26, K8.1 A, and replication and transcriptional activator (RTA) were detected by real-time PCR. Proteins were detected by immunohistochemistry. The effect of KSHV infection on cell proliferation was detected by MTT and Ki-67 staining. Cell migration was evaluated by Transwell and wound healing assays. The cell cycle was analyzed by flow cytometry. The expression of CDK4, CDK5, CDK6, cyclin D1, and p27 were measured by western blotting. The levels of cell cycle proteins were re-examined in LANA-overexpressing SH-SY5Y cells. Transcriptome sequencing was used to identify differentially expressed genes in KSHV-infected cells. The levels of Notch signaling pathway proteins were measured by western blotting. RNA interference was used to silence Notch1 and proliferation were analyzed again.
SH-SY5Y cells were successfully infected with KSHV, and they maintained the ability to produce virions. KSHV-infected SH-SY5Y expressed LANA, ORF26, K8.1 A, and RTA. After KSHV infection, cell proliferation was enhanced, but cell migration was suppressed. KSHV infection accelerated the G0/G1 phase. CDK4, CDK5, CDK6, and cyclin D1 expression was increased, whereas p27 expression was decreased. After LANA overexpression, CDK4, CDK6 and cyclin D1 expression was increased. Transcriptome sequencing showed that 11,258 genes were upregulated and 1,967 genes were downregulated in KSHV-infected SH-SY5Y. The Notch signaling pathway played a role in KSHV infection in SH-SY5Y, and western blots confirmed that Notch1, NICD, RBP-Jĸ and Hes1 expression was increased. After silencing of Notch1, the related proteins and cell proliferation ability were decreased.
KSHV infected SH-SY5Y cells and promoted the cell proliferation. KSHV infection increased the expression of Notch signaling pathway proteins, which may have been associated with the enhanced cell proliferation.
卡波西肉瘤相关疱疹病毒（KSHV）感染所致癌症是艾滋病患者的主要死因之一。近年来，基于该病毒的嗜神经性倾向，一些患者出现了似乎与KSHV感染相关的神经症状。本研究的目的是调查KSHV感染对神经元SH-SY5Y细胞的影响，并鉴定差异表达基因。
从islk.219细胞中收集KSHV。采用实时荧光定量PCR（qPCR）对KSHV拷贝数进行定量。用KSHV感染SH-SY5Y细胞。通过实时荧光定量PCR检测上清液中的KSHV拷贝数以及潜伏相关核抗原（LANA）、ORF26、K8.1A和复制及转录激活因子（RTA）的mRNA水平。通过免疫组织化学检测蛋白质。采用MTT法和Ki-67染色检测KSHV感染对细胞增殖的影响。通过Transwell实验和伤口愈合实验评估细胞迁移。通过流式细胞术分析细胞周期。采用蛋白质免疫印迹法检测细胞周期蛋白依赖性激酶4（CDK4）、细胞周期蛋白依赖性激酶5（CDK5）、细胞周期蛋白依赖性激酶6（CDK6）、细胞周期蛋白D1（cyclin D1）和p27的表达。在过表达LANA的SH-SY5Y细胞中重新检测细胞周期蛋白水平。采用转录组测序鉴定KSHV感染细胞中的差异表达基因。采用蛋白质免疫印迹法检测Notch信号通路蛋白水平。利用RNA干扰沉默Notch1并再次分析细胞增殖情况。
SH-SY5Y细胞成功感染KSHV，并保持产生病毒颗粒的能力。感染KSHV的SH-SY5Y细胞表达LANA、ORF26、K8.1A和RTA。KSHV感染后，细胞增殖增强，但细胞迁移受到抑制。KSHV感染加速了G0/G1期。CDK4、CDK5、CDK6和cyclin D1表达增加，而p27表达降低。LANA过表达后，CDK4、CDK6和cyclin D1表达增加。转录组测序显示，在感染KSHV的SH-SY5Y细胞中，11258个基因上调，1967个基因下调。Notch信号通路在KSHV感染SH-SY5Y细胞中发挥作用，蛋白质免疫印迹证实Notch1、Notch胞内结构域（NICD）、重组信号结合蛋白Jκ（RBP-Jκ）和Hes1表达增加。Notch1沉默后，相关蛋白和细胞增殖能力降低。
KSHV感染SH-SY5Y细胞并促进细胞增殖。KSHV感染增加了Notch信号通路蛋白的表达，这可能与细胞增殖增强有关。
