Zheng Liming, Wang Shenghua, Ling Mengyu, Lv Zhengmei, Lin Shuai
Anhui Medical University, Hefei, 230032 People's Republic of China.
3 Biotech. 2020 Jul;10(7):301. doi: 10.1007/s13205-020-02296-0. Epub 2020 Jun 12.
Hemolysin-coregulated protein (Hcp) of is known to be a structural and effector protein of the T6SS, but little is known about the role of Hcp in host cells. In this study, Hcp was expressed by plasmid pEGFP-N1-hcp in BHK-21 cells and the results showed that the subcellular localization of Hcp was predominantly in the cytoplasm of BHK-21 cells. When Hcp was over-expressed by transfecting plasmid pCI-neo-hcp in BHK-21 cells and mRNA sequencing was performed to analyze differentially expressed genes, the results showed a change in the expression levels of 307 mRNAs (fold change > 2, and < 0.01). Amongst these, 125 mRNAs were up-regulated and 182 mRNAs were down-regulated. Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis showed that differentially expressed genes were enriched in tumor necrosis factor (TNF) signaling pathway, IL-17 signaling pathway, and cytokine-cytokine receptor interaction. Subsequently, we selected differentially expressed genes of TNF signaling pathway and verified the changes by real-time PCR. The results were consistent with the trend observed for the sequencing results. In conclusion, we demonstrated that Hcp of caused the change of mRNAs expression of TNF signaling pathway in the cytoplasm of BHK-21 cells.
已知嗜水气单胞菌的溶血素共调控蛋白（Hcp）是VI型分泌系统（T6SS）的一种结构和效应蛋白，但关于Hcp在宿主细胞中的作用知之甚少。在本研究中，通过质粒pEGFP-N1-hcp在BHK-21细胞中表达Hcp，结果表明Hcp的亚细胞定位主要在BHK-21细胞的细胞质中。当通过在BHK-21细胞中转染质粒pCI-neo-hcp过表达Hcp并进行mRNA测序以分析差异表达基因时，结果显示307个mRNA的表达水平发生了变化（倍数变化>2，且P<0.01）。其中，125个mRNA上调，182个mRNA下调。京都基因与基因组百科全书（KEGG）富集分析表明，差异表达基因富集于肿瘤坏死因子（TNF）信号通路、IL-17信号通路和细胞因子-细胞因子受体相互作用。随后，我们选择了TNF信号通路的差异表达基因并通过实时PCR验证了变化。结果与测序结果观察到的趋势一致。总之，我们证明了嗜水气单胞菌的Hcp导致了BHK-21细胞细胞质中TNF信号通路mRNA表达的变化。
