摘要
目的研究在结核病(TB)患者体内血浆和血浆外泌体(Exo)中采用实时荧光定量聚合酶链式反应(qPCR)技术检测结核分枝杆菌(Mtb)编码的miRNAs水平时,U6、miR-16、miR-484作为候选内参基因的表达稳定性及适用性。方法收集健康人群(N组)和TB患者(T组)血浆,通过超速离心法分离外泌体(透射电镜和Nanosight验证),将健康人血浆外泌体组命名为N-Exo组(n=20),结核患者血浆外泌体组命名为T-Exo组(n=17),健康人血浆组命名为N-P组(n=20),结核患者血浆组命名为T-P组(n=20)。采用Trizol法提取总RNA并逆转录为cDNA。采用qPCR检测5种Mtb编码miRNA(MtbmiR-1、MtbmiR-2、MtbmiR-3、MtbmiR-5、MtbmiR-6)的表达,分别以U6、miR-16、miR-484为内参基因,通过GraphPad Prism 9.3软件判断血浆和血浆外泌体中目的基因表达量是否存在统计学差异。最后通过熔解曲线分析引物特异性、Cq值独立样本t检验及BestKeeper软件评估这3种内参基因的稳定性。结果以U6作为内参基因验证5种Mtb编码miRNAs的表达水平差异,发现在血浆中,T-P组5种Mtb-miRNA表达均显著低于N-P组,差异均有统计学意义(t=5.060、6.920、8.990、7.160、3.320,P均<0.05);而在血浆外泌体中仅MtbmiR-1、MtbmiR-2、MtbmiR-3、MtbmiR-5这4种基因的表达差异有统计学意义(t=5.538、4.529、9.354、7.346,P均<0.05),MtbmiR-6差异无统计学意义(P>0.05)。以miR-16为内参时,血浆及外泌体中,5种Mtb-miRNA均上调,但仅MtbmiR-2/6差异有统计学意义(t_(血浆)=15.150、10.580,t_(外泌体)=5.402、11.350,P均<0.05)。以miR-484为内参时,血浆中仅MtbmiR-2表达显著升高(t=3.603,P<0.05),外泌体中MtbmiR-1/2/6显著上调(t=3.046、8.596、3.281,P均<0.05)。这3个候选内参基因经Cq值独立样本t检验得到,U6在血浆中稳定(P>0.05),但在外泌体中表达显著波动(t=2.304,P=0.036);miR-16和miR-484在血浆及外泌体中均不稳定(P均<0.05);经Bestkeeper软件分析得到血浆中U6变异最小(SD=1.18,CV=3.55%),外泌体中miR-484稳定性最高(SD=1.07,CV=3.28%),但三者未达内参标准(SD>1)。结论传统内参基因(U6、miR-16、miR-484)在结核病感染模型中存在疾病依赖性表达波动;U6、miR-16、miR-484无论是在血浆样品还是外泌体样品中都不适宜作为TB患者基于qPCR技术检测miRANs表达水平的内参基因。
Objective To investigate the expression stability and applicability of U6,miR-16,and miR-484 as candidate reference genes in the detection of miRNAs encoded by Mycobacterium tuberculosis(Mtb)in plasma and plasma exosomes of tuberculosis(TB)patients by real-time fluorescence quantitative polymerase chain reaction(qPCR).Methods Plasma was collected from healthy people(N group)and TB patients(T group),and exosomes were separated by ultracentrifugation(transmission electron microscopy and Nanosight verification).The exosome group from healthy human plasma was named the N-Exo group(n=20),the exosome group from tuberculosis patients'plasma was named the T-Exo group(n=17),the plasma group from healthy human was named the N-P group(n=20),and the plasma group from tuberculosis patients was named the T-P group(n=20).Total RNA was extracted by Trizol method and reverse transcribed into cDNA.qPCR was used to detect the expression of five Mtb-encoded miRNAs(MtbmiR-1,MtbmiR-2,MtbmiR-3,MtbmiR-5,MtbmiR-6),with U6,miR-16,and miR-484 as reference genes,respectively.GraphPad Prism 9.3 software was used to determine whether there was a statistical difference in the expression of target genes in plasma and plasma exosomes.Finally,the stability of these three reference genes was evaluated by melting curve analysis of primer specificity,Cq value independent sample t test,and BestKeeper software.Results U6 was used as the internal reference gene to verify the expression level differences of the five Mtb-encoded miRNAs.It was found that in plasma,the expression levels of the five Mtb-miRNAs in the T-P group was significantly lower than those in the N-P group(t=5.060,6.920,8.990,7.160,3.320;all P<0.05);while in plasma exosomes,only the expression levels differences of MtbmiR-1,MtbmiR-2,MtbmiR-3,and MtbmiR-5 were statistically significant(t=5.538,4.529,9.354,7.346;all P<0.05),and there was no statistically significant dfference in MtbmiR-6(P>0.05).When miR-16 was used as an internal reference,the five+Mtb-miRNAs were upregulated in plasma and exosomes,but only MtbmiR-2/6 showed statistically significant differences(t_(plasma)=15.150,10.580,t_(exosome)=5.402,11.350;all P<0.05).When miR-484 was used as an internal reference,only MtbmiR-2 expression was significantly increased in plasma(t=3.603,P<0.05),and MtbmiR-1/2/6 was significantly upregulated in exosomes(t=3.046,8.596,3.281;all P<0.05).The three candidate internal reference genes were analyzed by independent sample t-test of Cq values.U6 was stable in plasma(P>0.05),but its expression level in exosomes fluctuated significantly(t=2.304,P=0.036);miR-16 and miR-484 were unstable in both plasma and exosomes(P<0.05);Bestkeeper software analysis showed that U6 had the smallest variation in plasma(SD=1.18,CV=3.55%),and miR-484 had the highest stability in exosomes(SD=1.07,CV=3.28%),but the three did not meet the internal reference standard(SD>1).Conclusion Traditional internal reference genes(U6,miR-16,miR-484)had disease-dependent expression fluctuations in the tuberculosis infection model;U6,miR-16,and miR-484 were not suitable as internal reference genes for detecting miRNAs expression levels in TB patients based on qPCR,whether in plasma samples or exosome samples.
作者
苏漫仪
刘宇恒
周萱
王华敏
祖好
杨金兰
徐君豪
廖瑶
王立富
SU Manyi;LIU Yuheng;ZHOU Xuan;WANG Huamin;ZU Hao;YANG Jinlan;XU Junhao;LIAO Yao;WANG Lifu(King Med School of Laboratory Medicine,Guangzhou Medical University,Guangzhou,Guangdong 510182,China)
出处
《热带医学杂志》
2025年第7期873-880,F0004,共9页
Journal of Tropical Medicine
基金
国家级大学生科技创新项目(202310570017)
广东省基础与应用基础研究基金委员会(省企联合基金-面上项目)(2023A1515220167)
广东省教育厅-广东省普通高校重点领域专项(生物医药与健康)(2023ZDZX2048)。
