基于转录组数据分析环形泰勒虫对诱导宿主细胞转化相关基因的影响

doi:10.11843/j.issn.0366-6964.2022.12.025

摘要/Abstract

摘要： 本试验主要通过研究布帕伐醌(BW720)处理前后，环形泰勒虫转化牛单核细胞(TaNM Ⅰ)转录组数据的变化情况，为进一步研究环形泰勒虫诱导宿主细胞转化的分子机制奠定基础。以环形泰勒虫感染的牛单核细胞为试验材料，设置对照组(二甲基亚砜,DMSO)和试验组(BW720)，利用Illumina HiSeq4000测序平台进行高通量测序，将测序得到的原始数据(Raw reads)与GenBank和Rfam数据库进行比对过滤，通过质控获得最终数据(Clean read)。利用Venn分析软件筛选出DMSO组和BW720组中差异表达明显的基因，进行功能注释(GO)以及信号通路(KEGG)分析。随机选择10个基因，利用荧光定量PCR (qPCR)检测基因在不同处理条件下细胞中的表达量。在验证测序结果的准确性后，选出差异表达明显的基因，分析其在TaNM Ⅰ细胞的凋亡、增殖等信号通路方面的作用。结果显示：BW720组和DMSO组中分别得到6 854 019 704和6 627 265 854条Raw reads，过滤、质控后分别得到22 925 606和22 171 427条Clean reads。BW720组和DMSO组中筛选出差异表达明显的基因共计4 054个(P＜0.05)，其中，2 146个基因显著上调，1 908个基因显著下调；进一步筛选出共同差异表达的基因367个，其中，显著上调的196个，显著下调的171个。同时，qPCR验证随机选择10个基因的表达趋势与转录组测序结果一致，表明测序结果可靠。然后对差异表达基因进行GO功能注释，筛选到与细胞增殖相关的20个生物学过程、15个细胞组分以及11个分子功能条目。KEGG富集分析结果显示，在Top20的信号通路中筛选出与环形泰勒虫转化细胞有关的一些信号通路，如：癌症信号通路、PI3K-Akt信号通路、MAPK信号通路等。对这些信号通路进一步分析发现：PI3KR3、FOXO1、IL23A、FZD3、AKT、MMP9等基因在环形泰勒虫转化细胞的研究中也有相关文献的报道。本研究表明，在TaNM Ⅰ细胞中DAPK1、FZD3、FOXO3、PI3KR3等可能在感染细胞无限增殖的过程中发挥作用，为后续研究TaNM Ⅰ细胞无限增殖机制奠定了基础。

关键词: 环形泰勒虫, 牛单核细胞, 转录组, 信号通路

Abstract: This study was conducted to study the changes of transcriptome in Theileria annulata infected bovine monocytes (TaNM Ⅰ) before and after treatment with Bupavaquone (BW720) for laying a foundation for further study on the molecular mechanism of host cell transformation induced by T. annulata. Bovine TaNM Ⅰ cells infected by T. annulata were used as experimental materials, control group (DMSO) and experimental group (BW720) were set up. High-throughput sequencing was carried out by Illumina HiSeq4000 sequencing platform. The original reading segment (Raw reads) was compared with GenBank and Rfam database, and the clean value (Clean reads) was obtained by quality control. The differentially expressed genes in DMSO treatment group and BW720 treatment group were screened by Venn analysis software, and functional annotation (GO) and signal pathway (KEGG) analysis were performed. Ten genes were randomly selected and the expression of genes in cells under different treatment conditions was detected by fluorescence quantitative PCR (qPCR). After verifying the accuracy of the sequencing results, the differentially expressed genes were selected and their roles in apoptosis and proliferation of TaNM Ⅰ cells were analyzed. Results showed that 6 854 019 704 and 6 627 265 854 Raw reads were obtained in BW720 treatment group and DMSO treatment group, and 22 925 606 and 22 171 427 Clean read were obtained after filtration and quality control, respectively. A total of 4 054 genes were expressed in BW720 group and DMSO group, of which 2 146 genes were significantly up-regulated and 1 908 genes were significantly down-regulated. Three hundred and sixty-seven differentially expressed genes were further screened, of which 196 were significantly up-regulated and 171 were significantly down-regulated. At the same time, the expression trend verified by qPCR was consistent with that of transcriptome sequencing, indicating that the sequencing results were reliable. Then GO functional annotation was performed on the differentially expressed genes, and 20 biological processes, 15 cell components and 11 molecular functional items related to cell proliferation were screened. KEGG enrichment analysis showed that some signaling pathways related to T. annulata transformation cells were screened out in Top20 signaling pathway, such as cancer signaling pathway, PI3K-Akt signaling pathway, MAPK signaling pathway, etc. The progress analysis of these signaling pathways found that PI3KR3, FOXO1, IL23A, FZD3, AKT, MMP9 and other genes have also been reported in the study of transformation cells of T. annulata. This study shows that DAPK1,FZD3,FOXO3 and PI3KR3 may be the key candidate genes affecting the infinite proliferation of infected cells in TaNM Ⅰ cells. It lays a foundation for the follow-up study of the mechanism of infinite proliferation of TaNM Ⅰ cells.

Key words: T. annulata, bovine monocyte, transcriptome, signal pathways

中图分类号:

S852.723

赵保才, 赵帅阳, 关贵全, 罗建勋, 曹天行, 张志刚, 石苗, 刘军龙, 赵洪喜. 基于转录组数据分析环形泰勒虫对诱导宿主细胞转化相关基因的影响[J]. 畜牧兽医学报, 2022, 53(12): 4398-4409.

ZHAO Baocai, ZHAO Shuaiyang, GUAN Guiquan, LUO Jianxun, CAO Tianxing, ZHANG Zhigang, SHI Miao, LIU Junlong, ZHAO Hongxi. Transcriptome-based Analysis of the Effects of Theileria annulata on Host Cell Transformation-related Genes[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(12): 4398-4409.

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