三磷酸鸟苷环化水解酶1调控的小胶质细胞环状RNA的表达

Guanosine-5′-triphosphate cyclohydrolase 1 regulates the expression of circular RNA in microglia

目的分析三磷酸鸟苷环化水解酶1(GCH1)在体外调控小鼠小胶质细胞(BV2)中环状RNA的相关表达。方法将BV2培养传代后转染病毒,分为低表达GCH1病毒载体组(Ad-shGCH1,n=3)与对照病毒载体组(Ad-NC,n=3)两组样本。孵育48 h观察转染效率并利用TRIzol试剂提取总RNA。使用第二代环状RNA(circRNA)高通量测序分析Ad-shGCH1与Ad-NC两组样本,筛选差异表达的环状circRNA。circRNA测序数据使用R包edge R计算基于负二项分布模型的fisher精确检验来确定差异显著性。应用编码-非编码基因共表达(CNC)网络分析对表达差异显著的circRNAs进行生物信息学分析,最后通过反转录-聚合酶链反应(RT-PCR)验证。结果与对照组比较,Ad-shGCH1中mmucirc0001322(实验组CPM值:10.40,对照组CPM值:7.63,P<0.05)、mmucirc0000454(实验组CPM值:10.84,对照组CPM值:8.20,P<0.05)、mmucirc0001383(实验组CPM值:10.76,对照组CPM值:8.53,P<0.05)、mmucirc0000898(实验组CPM值:11.22,对照组CPM值:9.32,P<0.05)发生高于对照组,差异有统计学意义,mmucirc0000652低于对照组(实验组CPM值:7.94,对照组CPM值:10.83,P<0.05)(倍数变化>1.2倍),差异有统计学意义。CNC分析表明mmucirc0000652可能通过参与特定的网络或生物学过程参与调控丝裂原活化蛋白激酶14(MAPK14)而发挥作用。结论GCH1调控的circRNA可能参与体外小胶质细胞的炎症过程。

Objective To analyze the relative expression of guanosine-5′-triphosphate cyclohydrolase 1(GCH1)in the regulation of circular RNA in mouse microglia(BV2)in vitro.Methods BV2 cells were cultured and passaged.The BV2 cells were transfected with viruses and divided into a low expression GCH1 adenovirus vector group(Ad-shGCH1,n=3)and a control adenovirus vector group(Ad-NC,n=3).Two sets of samples.Incubate for 48 h to observe the transformation efficiency and extract total RNA with TRIzol reagent.The second-generation circular RNA(circRNA)high-throughput sequencing was used to analyze the Ad-shGCH1 and Ad-NC samples to screen for differentially expressed circular circRNA.CircRNA sequencing data uses the R package edge R to calculate the fisher exact test based on the negative binomial distribution model to determine the significance of the difference.Finally,the coding-non-coding gene co-expression(CNC)network analysis was used to conduct bioinformatics analysis on circRNAs with significant expression differences,and finally verified by reverse transcription-polymerase chain reaction(RT-PCR).Results Compared with the control group,we found that mmu_circ_0001322(P<0.05),mmu_circ_0000454(P<0.05),mmu_circ_0001383(P<0.05),mmu_circ_0000898(P<0.05)were up-regulated and mmu_circ_0000652 was down-regulated(Multiple change>1.2 times)in Ad-shGCH1.The CNC analyzed that mmu_circ_0000652 may play a role by participating in the regulation of mitogen-activated protein kinase14(MAPK14)by participating in specific network or biological processes.Conclusion CircRNA regulated by GCH1 may be involved in the inflammatory process of microglia in vitro,and may provide new information for the treatment of neuropathic pain.