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目的探讨长链非编码RNA(lncRNA)在βB2基因敲除小鼠睾丸中的表达及其影响睾丸发育的可能机制。方法采用lncRNA芯片技术,筛选野生型(WT)和βB2基因敲除(KO)小鼠睾丸组织(均n=3)中lncRNA及信使RNA(mRNA)表达谱的变化;对差异表达lncRNA和mRNA进行GO数据库分析及KEGG数据库分析,建立调控网络图;采用实时荧光定量PCR(qRT-PCR)验证差异表达的lncRNA和mRNA,探讨调控通路。结果 (1)两组小鼠睾丸组织差异表达的lncRNA共140条,mRNA共477条;(2)通过GO数据库分析,筛选出差异表达lncRNA共12条,其中上调7条,下调5条;(3)经KEGG数据库进行路径分析,发现差异表达mRNA主要经由Ca2+信号、配体-受体相互作用等信号通路起作用;(4)用关联矩阵法建立了lncRNA和mRNA共表达网络图,共有17个节点,12条连接,包含9条lncRNA和8条mRNA;其中Rsl1由3条lncRNA调控,Lpo和Mpo各由2条lncRNA调控,Hdac1、Ephb4等各由1条lncRNA调控。(5)qRT-PCR分析结果表明,在βB2基因敲除小鼠睾丸组织中,lncRNA A-30-P01019163和P2rx7的表达均下调(P<0.05)。结论 lncRNA与βB2基因的晶状体外功能密切相关,其中lncRNA A-30-P01019163可能通过调控下游P2rx7mRNA的表达影响睾丸组织细胞周期及信号转导,进而调控睾丸发育。
Objective To investigate the expression of long chain non-coding RNA (lncRNA) in testes of βB2 knockout mice and its possible mechanism of affecting testicular development. Methods The expression of lncRNA and messenger RNA (mRNA) in wild-type (WT) and betaB2 knockout (KO) mice testis tissues (all n = 3) were screened by lncRNA microarray. The expression of differentially expressed lncRNA and mRNA GO database analysis and KEGG database analysis to establish regulatory network map; using real-time quantitative PCR (qRT-PCR) to verify differentially expressed lncRNA and mRNA, to explore regulatory pathways. Results (1) There were 140 lncRNAs differentially expressed in testes of the two groups, with a total of 477 mRNAs. (2) Twelve lncRNAs were screened by GO database, including 7 up-regulated and 5 down-regulated; 3) Path analysis by KEGG database showed that differentially expressed mRNA mainly via Ca2 + signal, ligand-receptor interaction and other signaling pathways; (4) established a co-expression network lncRNA and mRNA network with a correlation matrix method, a total of 17 Which contains 9 lncRNAs and 8 mRNAs. Among them, Rsl1 is regulated by 3 lncRNAs, Lpo and Mpo are regulated by 2 lncRNAs, and Hdac1 and Ephb4 are each regulated by 1 lncRNA. (5) The results of qRT-PCR analysis showed that the expression of lncRNA A-30-P01019163 and P2rx7 was down-regulated in betaB2 knockout mice testis (P <0.05). Conclusion lncRNA and βB2 gene are closely related to extra-corporeal function. LncRNA A-30-P01019163 may regulate testicular cell cycle and signal transduction by regulating the expression of downstream P2rx7 mRNA.