Xu Huiling, Huang Jinghong, Yao Lixia, Gu Wenyi, Ruzi Aynisahan, Ding Yufei, Li Ying, Liang Weihua, Jiang Jinfang, Pan Zemin, Cao Dongdong, Zhou Naiming, Li Dongmei, Zhang Jinli
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 City, Xinjiang 832002, China.
Department of Pathology, Central People's Hospital of Zhanjiang, Zhanjiang, Guangdong 52400, China.
Curr Mol Pharmacol. 2024 Jan 11. doi: 10.2174/0118761429264583231106104202.
This study aimed to investigate the influence of Notch1 on c-Fos and the effect of c-Fos on the proliferation of Kaposi's sarcoma-associated herpesvirus (KSHV)-infected neuronal cells.
Real-time PCR and western blotting were used to determine c-Fos expression levels in KSHV-infected (SK-RG) and uninfected SH-SY5Y cells. C-Fos levels were measured again in SK-RG cells with or without Notch1 knockdown. Next, we measured c-Fos and p-c-Fos concentrations after treatment with the Notch1 γ-secretase inhibitor LY-411575 and the Notch1 activator Jagged-1. MTT and Ki-67 staining were used to evaluate the proliferation ability of cells after c-Fos levels downregulation. CyclinD1, CDK6, and CDK4 expression levels and cell cycle were analyzed by western blotting and flow cytometry, respectively. After the c-Fos intervention, the KSHV copy number and gene expression of RTA, LANA and K8.1 were analyzed by real-time TaqMan PCR.
C-Fos was up-regulated in KSHV-infected SK-RG cells. However, the siRNA-mediated knockdown of Notch1 resulted in a significant decrease in the levels of c-Fos and p-c-Fos (P <0.01, P <0.001). Additionally, a decrease in Cyclin D1, CDK6, and CDK4 was also detected. The Notch1 inhibitor LY-411575 showed the potential to down-regulate the levels of c-Fos and p-c-Fos, which was consistent with Notch1 knockdown group (P <0.01), whereas the expression and phosphorylation of c-Fos were remarkably up-regulated by treatment of Notch1 activator Jagged-1 (P <0.05). In addition, our data obtained by MTT and Ki-67 staining revealed that the c-Fos down-regulation led to a significant reduction in cell viability and proliferation of the SK-RG cells (P <0.001). Moreover, FACS analysis showed that the cell cycle was arrested in the G0/G1 stage, and the expressions of Cyclin D1, CDK6, and CDK4 were down-regulated in the c-Fos-knockdown SK-RG cells (P <0.05). Reduction in total KSHV copy number and expressions of viral genes (RTA, LANA and K8.1) were also detected in c-Fos down-regulated SK-RG cells (P <0.05).
Our findings strongly indicate that c-Fos plays a crucial role in the promotion of cell proliferation through Notch1 signaling in KSHV-infected cells. Furthermore, our results suggest that the inhibition of expression of key viral pathogenic proteins is likely involved in this process.
本研究旨在探讨Notch1对c-Fos的影响以及c-Fos对卡波西肉瘤相关疱疹病毒（KSHV）感染的神经元细胞增殖的作用。
采用实时定量聚合酶链反应（Real-time PCR）和蛋白质免疫印迹法（western blotting）检测KSHV感染的（SK-RG）细胞和未感染的SH-SY5Y细胞中c-Fos的表达水平。在Notch1基因敲低或未敲低的SK-RG细胞中再次检测c-Fos水平。接下来，我们用Notch1γ-分泌酶抑制剂LY-411575和Notch1激活剂Jagged-1处理细胞后，检测c-Fos和磷酸化c-Fos（p-c-Fos）的浓度。采用噻唑蓝比色法（MTT）和Ki-67染色评估c-Fos水平下调后细胞的增殖能力。分别通过蛋白质免疫印迹法和流式细胞术分析细胞周期蛋白D1（CyclinD1）、细胞周期蛋白依赖性激酶6（CDK6）和细胞周期蛋白依赖性激酶4（CDK4）的表达水平及细胞周期。在c-Fos干预后，通过实时荧光定量TaqMan PCR分析KSHV拷贝数以及复制和转录激活因子（RTA）、潜伏相关核抗原（LANA）和K8.1的基因表达。
在KSHV感染的SK-RG细胞中c-Fos表达上调。然而，小干扰RNA（siRNA）介导的Notch1基因敲低导致c-Fos和p-c-Fos水平显著降低（P<0.01，P<0.001）。此外，还检测到细胞周期蛋白D1、CDK6和CDK4水平降低。Notch1抑制剂LY-411575显示出下调c-Fos和p-c-Fos水平的潜力，这与Notch1基因敲低组一致（P<0.01），而Notch1激活剂Jagged-1处理显著上调了c-Fos的表达和磷酸化水平（P<0.05）。此外，我们通过MTT和Ki-67染色获得的数据显示，c-Fos下调导致SK-RG细胞的活力和增殖显著降低（P<0.001）。此外，流式细胞术分析表明，细胞周期停滞在G0/G1期，在c-Fos基因敲低的SK-RG细胞中，细胞周期蛋白D1、CDK6和CDK4的表达下调（P<0.05）。在c-Fos下调的SK-RG细胞中还检测到总KSHV拷贝数以及病毒基因（RTA、LANA和K8.1）表达的降低（P<0.05）。
我们的研究结果有力地表明，在KSHV感染的细胞中，c-Fos通过Notch1信号通路在促进细胞增殖中起关键作用。此外，我们的结果表明，关键病毒致病蛋白表达的抑制可能参与了这一过程。
