【文献解析-鲤鱼|性腺发育|lncRNA】igf3敲除后鲤鱼中lnc RNA和mRNA的表达谱

  • 2019-12-10 12:56:36
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关键词:Insulin-like growth factors,Cyprinus carpio,Long noncoding RNA and mRNA

英文题目:Long noncoding RNA and mRNA expression profiles following igf3 knockdown in common carp, Cyprinus carpio

  名:Scientific data         发表时间:2019.02        IF5.305

作者单位南京农业大学

测序产品:mRNAlncRNA             

样品来源:鲤鱼

文章类型:研究型

 

英文摘要

Insulin-like growth factors (IGFs) are key factors that regulate the growth and reproduction axis。 As a new ligand of IGFs, igf3 plays an important role in the development and maturation of the gonads in fish1–3, especially in the maturation of oocytes and the process of spermatogenesis4,5。 We reported previously that the igf3 mRNA of common carp (Cyprinus carpio) was highly expressed in the gonads and blood, and Igf3 protein was localized in the ovary granulosa cells and testis spermatogonium and spermatids3。 In addition, igf3 also serves as a mediator of luteinizing hormone (LH) action in zebrafish (Danio rerio) ovulation6, and stimulate spermatogonial differentiation by activating β-catenin signaling or through some estrogens7,8。 To better understand the regulatory pathway of igf3 in gonadal development of fish, we are interested in the transcriptional regulation of lncRNAs and mRNAs expression profiles following igf3 knockdown in common carp。

The molecular mechanisms of gonadal development are complex processes that involve in many germ cell-specific gene products in testis or ovary that undergo strict developmental regulations9,10. LncRNAs are a novel class of RNAs with the length over 200 bp and without the potential of protein-coding11. By now, there are tens of thousands of lncRNAs have been discovered across human, mouse, nematode, zebrafish etc.12. For human, 58,648 of genes were classified as lncRNAs13. The existing studies show that lncRNAs play a key role in epigenetic, transcriptional and post-transcriptional control of gene expression and maintenance of stem cell, signal transduction, cell proliferation and differentiation, metabolism, and individual development14–17. In testis, lncRNAs affect germ cell development and spermatogenesis, such as tsx, mrh1, dmr and hongES218–21. In ovary, many lncRNAs that regulate ovarian development have been identified22–25. LncRNA A-30-P01019163 may affect ovarian cell cycle and proliferation by regulating p2rx7 expression in the ovary26. All of these observations indicate that lncRNAs play important roles in gonadal development.

RNA interference technology is an effective method in studying the gene function of organisms。 In this study, we first established a living model of igf3 knockdown C。 carpio via double-stranded RNA (dsRNA) injection, and then a high-throughput RNA sequencing (RNA-seq) was employed to profile the primordial gonad transcriptome of igf3 knockdown and control C。 carpio。 The aims of the experiment were to discover and characterize lncRNAs in carp gonad tissue and identify key genes, lncRNAs, and pathways that are associated with igf3。 This is the first study to describe the expression profiles of lncRNAs and mRNA in carp gonad with igf3 knockdown。 Our data will provide a very useful resource for studying the functional role of igf3 in gonadal development。

 

研究背景

胰岛素样生长因子(insulin like growth factorIGF)是一组具有促生长作用的多肽类物质,其广泛分布在人体肝、肾、肺、心、脑和肠等组织中。它是一类多功能细胞调控因子,大部分IGF在肝脏内合成,主要存在于血液循环中。IGF介导生长激素的功能,对身体成分的维持及营养代谢的调节起着重要的作用;通过内分泌、旁分泌和自分泌方式,由靶细胞表面受体介导发挥其生物学效应,并受到IGF结合蛋白的调节。

自从胰岛素样生长因子3Igf3)在鱼类卵巢中被发现以后,其在性腺发育中的作用开始受到业界关注。目前,仅在斑马鱼、尼罗罗非鱼和斜带石斑鱼中报道过Igf3。本文以“福瑞鲤”为材料,开展了igf3基因及其功能的研究。研究团队利用dsRNA干扰技术建立igf3基因被干扰的鲤活体模型,通过60天的连续干扰实验,发现igf3基因被敲降后,对鲤幼鱼的生长性能无显著性影响,但是能显著抑制卵巢和精巢的发育,并显著影响促性腺激素和固醇类性激素的分泌。

 

实验设计


研究结果


1. 幼年鲤鱼igf3基因干扰效率的研究


qRT-PCR法检测注射igf3-dsRNA后,RNA 干扰igf3mRNA表达水平。图中给出了igf3 RNAiqRT-PCR结果,注射igf3 dsRNAigf3 mRNA的表达较对照组明显下调(P<0.05),24小时下降87.4%



2. RNA测序结果


本次测序663158182150bp的原始序列,633474428clean reads。超过80%clean reads 比对到鲤鱼参考基因组。大约34%clean reads 分别比对到基因组的正链和负链。


3. lncRNAsmRNAs的鉴别


RNA-seq的结果进行筛选,获得1,4199lncRNA10,6932mRNA,去除不具有lncRNAsmRNAs特征的转录本。这些转录本随机分布在鲤鱼的50条染色体上,其中以31号和38号染色体上的lncRNAsmRNAs最多,分别占1。79%1.65%lncRNAs的长度范围为200-5759bp,其中82。69%15.07%lncRNAs的长度分别为200-1000bp1000-2000bp,而mRNA转录物的长度主要大于1000bplncRNA 的平均长度为643bp,小于mRNA的长度。此外,lncRNA与编码蛋白质的基因有重叠,lncRNA和编码蛋白质基因的注释可在数据库中找到。


4. lncRNAmRNA的差异表达


研究了对照组和处理组间lncRNAsmRNAs的差异表达,发现了124lncRNA353mRNA的显著差异表达。在差异表达的lncRNA中,处理组有69lncRNA上调,55lncRNA下调。在mRNA中,有234mRNA上调,119mRNA下调。在这些差异表达的基因中,有一些是与性腺发育相关的基因,如成纤维细胞生长因子受体2FGFR2)、纤维连接蛋白、丝氨酸/苏氨酸蛋白磷酸酶4等等。

5. qRT-PCR验证测序结果


为了进一步评价RNA测序的可靠性,随机选择20个差异表达的lncrna10个上调和10个下调)和20个差异表达的mrna10个上调和10个下调),用qRT-PCR方法验证对照组和治疗组性腺的相对表达水平。如图所示,这40个随机选择的基因(lncRNAs,左图;mRNAs,右图)的表达趋势与我们的RNA测序数据一致,表明RNA测序结果是可靠的。


文章亮点


①LncRNA-mRNA共表达网络显示,igf3基因被干扰后,上调了lncRNA MST42108的表达表达水平,继而诱导了细胞周期蛋白依赖性激酶、TBX18FGFR2、丝氨酸/苏氨酸蛋白激酶基因家族等基因的表达,影响性腺细胞的信号转导途径、细胞周期、分化以及生殖细胞分化,并最终影响性腺发育。

igf3基因被干扰后,鲤幼鱼的生长无显著性影响,但显著抑制卵巢和精巢的发育,并显著影响促性腺激素和固醇类性激素的分泌。

发现lncRNA MST42108可能是igf3基因的负调控因子,在igf3基因被干扰后性腺的发育中起重要作用。




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