Yang Kai, Pan Ying, Liu Ping, Yu Furong, Wei Xiaokang, Zhang Fasu, Wang Qin
Anhui Medical College, Hefei 230601, China. *Corresponding author, E-mail:
Anhui Medical College, Hefei 230601, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2024 Aug;40(8):704-709.
Objective To explore the effects of Myxovirus resistance protein A (MxA) on the Janus kinase/Signal transducer and activator of transcription (JAK/STAT) pathway in HepG2 cells. Methods HepG2 cells were transfected with the pcDNA3.1-Flag-MxA construct, and subsequent localization and expression of the MxA protein were detected through immunofluorescence cytochemistry. The presence of MxA protein was further confirmed by using Western blot analysis. Following transfection with MxA small interfering RNA (si-MxA) and subsequent treatment with alpha interferon (IFN-α), real-time fluorescent quantitative PCR was employed to measure the mRNA levels of myxovirus resistance protein A (MxA), protein kinase R (PKR), and oligoadenylate synthase (OAS). Western blot analysis was used to detect the protein expression of MxA, PKR, OAS, signal transducer and activator of transcription 1 (STAT1), phosphorylated STAT1 (pSTAT1), STAT2, phosphorylated STAT2 (p-STAT2) and interferon regulatory factor 9 (IRF9). Additionally, pcDNA3.1-Flag-MxA and pISRE-TA-luc were co-transfected into HepG2 and HepG2.2.15 cells, respectively, to assess the activity of the interferon-stimulated response element (ISRE) by using a luciferase activity assay. Results MxA protein was expressed in both the cytoplasm and nucleus of HepG2 cells, with higher expression levels in the cytoplasm than in the nucleus. Knocking down MxA expression in HepG2 cells did not affect the expression of STAT1, p-STAT1, STAT2, p-STAT2, and IRF9 proteins induced by IFN-α, but significantly reduced the expression of antiviral proteins PKR and OAS. Overexpression of MxA in HepG2 cells enhanced ISRE activity and increased the expression of PKR and OAS proteins, but this effect was inhibited in HepG2.2.15 cells. Conclusion MxA induces the expression of antiviral proteins by enhancing the activity of the JAK/STAT signaling pathway ISRE.
目的 探讨黏液病毒抗性蛋白A（MxA）对HepG2细胞中Janus激酶/信号转导子和转录激活子（JAK/STAT）信号通路的影响。方法 将pcDNA3.1-Flag-MxA构建体转染至HepG2细胞，通过免疫荧光细胞化学检测MxA蛋白的定位及表达情况。采用蛋白质免疫印迹法进一步确认MxA蛋白的存在。用MxA小干扰RNA（si-MxA）转染细胞后，再用α干扰素（IFN-α）处理，采用实时荧光定量PCR检测黏液病毒抗性蛋白A（MxA）、蛋白激酶R（PKR）和寡腺苷酸合成酶（OAS）的mRNA水平。用蛋白质免疫印迹法检测MxA、PKR、OAS、信号转导子和转录激活子1（STAT1）、磷酸化STAT1（pSTAT1）、STAT2、磷酸化STAT2（p-STAT2）和干扰素调节因子9（IRF9）的蛋白表达。此外，将pcDNA3.1-Flag-MxA和pISRE-TA-luc分别共转染至HepG2和HepG2.2.15细胞，通过荧光素酶活性测定评估干扰素刺激反应元件（ISRE）的活性。结果 MxA蛋白在HepG2细胞的细胞质和细胞核中均有表达，细胞质中的表达水平高于细胞核。敲低HepG2细胞中MxA的表达不影响IFN-α诱导的STAT1、p-STAT1、STAT2、p-STAT2和IRF9蛋白的表达，但显著降低抗病毒蛋白PKR和OAS的表达。HepG2细胞中MxA的过表达增强了ISRE活性并增加了PKR和OAS蛋白的表达，但在HepG2.2.15细胞中这种作用受到抑制。结论 MxA通过增强JAK/STAT信号通路ISRE的活性诱导抗病毒蛋白的表达。
