布鲁氏菌wbkc基因影响RAW264.7细胞自噬的分子机制研究

Brucellosis is a human zoonosis caused by Brucella, but its pathogenesis is still unclear. In the previous study, we found that the deletion of wbkc gene in Brucella resulted in differential expression of 13 mRNAs and 8 miRNAs related to autophagy pathway, suggesting that Brucella wbkc gene affects the process of RAW264.7 autophagy. To further reveal it’s molecular mechanism, this project through lncRNA sequencing analysis, mining Brucella wbkc gene affected the regulatory network of lncRNA-miRNA-target gene related to the autophagy pathway; overexpression or inhibition of lncRNA mediated by lentivirus, overexpression of miRNA or inhibition of miRNA mediated by transfection mimic or inhibitor, 3'UTR report gene and qRT-PCR were used to verify the regulatory network; Western-blot, transmission electron microscope and laser confocal detection were used to analyze the impact of Brucella wbkc gene on the level of autophagy. The molecular mechanism of the lncRNA-miRNA-target gene related to the RAW264.7 autophagy pathway affecting by Brucella wbkc gene is fully explained, not only pave the way for revealing the pathogenic mechanism of Brucella, but also provide a scientific basis for the prevention and treatment of brucellosis.

布鲁氏菌病是由布鲁氏菌引起的一种人兽共患传染病，但其致病机制仍不清楚。申请人在前期研究中发现，布鲁氏菌wbkc基因的缺失，导致自噬通路相关的13个mRNAs和8个miRNAs差异表达，提示布鲁氏菌wbkc基因影响RAW264.7自噬的过程。为了进一步揭示其分子机制，本课题拟用lncRNA测序，挖掘布鲁氏菌wbkc基因影响自噬通路相关的lncRNA-miRNA-靶基因调控网络；应用慢病毒介导lncRNA过表达或抑制表达、mimic过表达或inhibitor抑制表达miRNA、3'UTR报告基因和qRT-PCR验证调控网络；利用Western-blot、透射电镜和激光共聚焦的方法，分析布鲁氏菌wbkc基因对细胞自噬水平的影响。从而全面阐释布鲁氏菌wbkc基因影响RAW264.7自噬通路相关的lncRNA-miRNA-靶基因的分子机制，为布鲁氏菌致病机制的揭示做铺垫，还为布病的防治提供科学依据。

本课题通过成功制备布鲁氏菌B. melitensis的WbkC基因多克隆抗体和过表达腺病毒Ad-WbkC，并成功介导了B. melitensis的WbkC基因在RAW264.7细胞中的过表达，利用全转录测序，筛选出表达上调的48个长链非编码RNAs (lncRNAs)，表达下调的47个lncRNAs，利用GO、KEGG功能富集性分析和qRT-PCR验证，成功挖掘出布鲁氏菌WbkC基因影响RAW264.7细胞自噬通路相关的lncRNA 4933430A20Rik、lncRNA B930036N10Rik，为进一步揭示布鲁氏菌致病的分子机制和靶向防治布鲁氏菌病奠定了坚实的科学基础。