Expression Analysis of WDR36 and WRN Genes of Borrelia Burgdorferi in HMC3 Cell Line Infection Model
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摘要:
目的 为探索神经莱姆病的分子机制，特别通过转录组学分析识别与伯氏疏螺旋体感染相关的两个差异表达基因WDR36和WRN。其次验证它们在伯氏疏螺旋体感染的发病中的联系，以证明是潜在的治疗靶点。 方法 通过建立非人灵长类动物模型，利用高通量测序技术获取转录组学数据。通过GO富集分析，识别出WDR36和WRN两个具有研究价值的差异表达基因。为了进一步验证这两个基因在Bb感染神经莱姆病中的作用，采用了人脑胶质细胞HMC3细胞株作为模型。实验组细胞接受了不同MOI（1和10）的Bb接种，并在感染后6 h、12 h和24 h收集细胞悬液。使用trizol法提取RNA，并通过qPCR法测定WDR36和WRN基因的mRNA相对表达水平。 结果 在Bb感染的HMC3细胞中，发现Bb MOI=1为适宜感染浓度。结果表明WDR36的表达量在24 h与12 h对比有显著的上调，且6 h的PBS组与试验组基因表达含量对比上升但不明显，12 h、24 h上升明显有统计学差异（P < 0.01），WRN的表达量在24 h与12 h对比有显著上调，且6 h、12 h、24 h的实验组分别比其PBS组基因表达含量呈上升趋势且有统计学差异（P < 0.01）。 结论 WDR36和WRN的上调可能与LNB的神经病理过程相关，这些发现为进一步研究LNB的分子机制提供了新的视角，并可能为开发针对Bb感染的新型治疗方案提供了潜在的分子靶点。 Abstract:Objective The aim is to explore the molecular mechanism of neurological Lyme disease, particularly by identifying two differentially expressed genes WDR36 and WRN associated with Burkholderia infection through transcriptome analysis. Secondly, to verify their association in the pathogenesis of Borrelia burgdorferi infection and demonstrate their potential therapeutic targets. Method By establishing a non-human primate model and utilizing high-throughput sequencing technology to obtain transcriptomic data. Through GO enrichment analysis, two differentially expressed genes with research value, WDR36 and WRN, were identified. To further validate the role of these two genes in Bb infection of Lyme disease, the HMC3 cell line of human glial cells was used as a model. The experimental group cells received Bb inoculation with different MOI （1 and 10）, and cell suspensions were collected at 6 h, 12 h, and 24 h after infection. Extract RNA using the triazol method and determine the relative mRNA expression levels of WDR36 and WRN genes using qPCR. Result it was found that Bb MOI=1 was the appropriate infection concentration in HMC3 cells infected with Bb. The results showed that the expression level of WDR36 was significantly upregulated at 24 h and 12 h, and the gene expression content of the 6 h PBS group increased but not significantly compared to the experimental group. The increase was significant at 12 h and 24 h, and there was a significant statistical difference with P < 0.01. The expression level of WRN was significantly upregulated at 24 h and 12 h, and the gene expression content of the 6 h, 12 h, and 24 h experimental groups showed an upward trend compared to their PBS group, and there was a significant statistical difference with P < 0.01. Conclusion The upregulation of WDR36 and WRN may be associated with the neuropathological processes of LNB. These findings provide a new perspective for further studying the molecular mechanisms of LNB and may provide potential molecular targets for developing novel treatment strategies for Bb infection. -
Key words:
- Borrelia burgdorferi /
- WDR36 /
- WRN /
- HMC3 cell line
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图 1 23个差异表达基因（DEGs）的筛选
Figure 1. Screening of 23 differentially expressed genes （DEGs）
图 2 免疫反应相关DEGs的相互作用关系网络
Figure 2. The interaction network of immune response related DEGs
图 4 组内HMC3在不同时间点WDR36表达量的比较
*P < 0.05，**P < 0.01。
Figure 4. Comparison of WDR36 secretion of HMC3 at different time points in the group
图 5 组间HMC3在不同时间点WDR36表达量的比较。
**P < 0.01。
Figure 5. Comparison of WDR36 secretion of HMC3 at different time points between groups
图 6 组内HMC3在不同时间点WRN表达量的比较。
**P < 0.01。
Figure 6. Comparison of WRN secretion of HMC3 at different time points in the group
图 7 组间HMC3在不同时间点WRN表达量的比较
**P < 0.01。
Figure 7. Comparison of WRN secretion of HMC3 at different time points between groups
表 1 主要差异表达基因BP（TMEM87A、WRN、FOLR2）和MF（BTAF1、MFN1、WRN）
Table 1. BP（TMEM87A、WRN、FOLR2）and MF（BTAF1、MFN1、WRN）Selected genes
基因 GO富集BP GO富集MF CFH
OGN
BTAF1
WDR36
TMEM87A
ALCAM
MFN1
WRN
CENPC
SF3B1
CTNNAL1
ZBED5
FOLR2未选择
未选择
未选择
未选择
选中
未选择
未选择
选中
未选择
未选择
未选择
未选择
选中未选择
未选择
选中
未选择
未选择
未选择
选中
选中
未选择
未选择
未选择
未选择
未选择
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