miR-26a及其靶基因Tnrc6a参与调控鸡卵泡膜细胞增殖的分子机制

Follicle development in animal is a complex and finely regulated process involved a lot of biological regulatory networks.Studies have shown that miRNAs are involved in the regulation of ovarian functions, but their precise mechanisms have not yet been determined. Our previous studies have found that miR-26a was differentially expressed in chicken ovarian before and after sexual maturity, and has targeted regulation relationship with Tnrc6a, the overexpression of miR-26a could promote the proliferation of theca cells in vitro. Tnrc6a play a central role in miRNA-mediated gene silencing.We speculate that miR-26a might directly be involved in follicular development processes through regulating its target genes, or indirectly involved in these processes through regulating other miRNAs mediated by Tnrc6a. To clarify the molecular mechanism of miR-26a in regulating theca cell proliferation, in this study, we intend to overexpress miR-26a in co-culture model of chichen theca cells and granulosa cells,using transcriptome sequencing(Illumina RNA-seq), two-dimensional electrophoresis-mass spectrometry, and bioinformatics and screen the target genes mediated by miR-26a ,and further to clarify its signaling pathways. RNAi, qRT-PCR, Western blot technology researches will also be used to study the molecular mechanism regulating theca cell proliferation mediated by miR-26a and Tnrc6a. The results could provide a new clue to the analysis of follicular development mechanism in chicken from miRNAs , it also has great significance for further improving the regulatory networks in follicle development.

动物卵泡的发育是一个复杂而又被精细调控的过程，目前研究表明，miRNAs参与卵巢功能的调节，但其作用机制尚不清楚。申请人前期研究发现，miR-26a在鸡的卵泡发育过程中能靶向调控Tnrc6a 并能促进卵泡膜细胞的增殖，而Tnrc6a 又参与miRNA介导的基因沉默功能。因此miR-26a调控卵泡发育的机制是通过直接调控其靶基因参与卵泡发育过程，还是通过调控Tnrc6a介导的其它miRNA来间接参与此过程，需要进一步研究。本研究拟利用鸡卵泡膜细胞和颗粒细胞共培养模型过表达miR-26a，采用转录组测序和二维电泳-质谱技术结合生物信息分析，揭示受miR-26a介导调控的目标基因及其所在的信号通路；利用RNAi、qRT-PCR、Western blot等技术研究miR-26a及其靶基因Tnrc6a调控卵泡膜细胞增殖的分子作用机制。为从miRNA层面解析鸡的卵泡发育机制提供新的理论线索。

卵泡膜细胞对于卵母细胞成熟、卵泡颗粒细胞的分化以及甾体激素的合成分泌等起着重要的协同调控作用。本研究目标是明确miR-26a靶向调控TNRC6A基因在鸡卵泡细胞中的作用机制。研究主要结果如下：（1）通过在体外培养的卵泡细胞中过表达miR-26a后，利用转录组高通量测序技术筛选过表达前后差异表达基因，结果筛选到2249个差异表达基因，其中上调814个，下调1435个。这些基因的功能19.3%为预测，12.28%为未知，22.81%为信号转导机制，并初步确定miR-26a可能参与MAPK、Wnt等信号通路。并对可能的目标基因NLK、FOXL2、GnRH-1、VEGFA等在鸡卵泡细胞中的表达进行了定量分析。（2）通过过表达及RNAi等技术发现，miR-26a能通过直接调控靶基因TNRC6A表达影响膜细胞的增殖，过表达miR-26a及干扰TNRC6A均能上调抗凋亡基因BCL-2的表达。说明miR-26a促进鸡卵泡膜细胞增殖可能是通过抑制细胞凋亡来实现。（3）在鸡卵泡颗粒细胞和膜细胞中，miR-26a均能直接靶向调控NLK基因，但其在颗粒细胞中对NLK基因的表达调控存在剂量差异。生物信息学发现在，利用双荧光素酶报告基因检测系统确定了NLK 基因3′ UTR区miR-26a的3个识别靶点中只有第二个识别靶点起重要作用。NLK基因是Wnt信号的负调控因子，本研究发现在颗粒细胞中NLK能下调SMAD4基因的表达。（4）对不同鸡品种中miR-26a基因上游调控区的多态及其与宿主基因CTDSPL的表达进行分析。结果发现，两者在各时期卵巢中的表达趋势并不一致，在miR-26a基因上游调控区发现了C/T和 A/G两个SNP位点这种突变改变了miR-26a前体序列二级结构的自由能。本项目为miR-26a在鸡卵泡中的作用及其自身调控的研究积累了重要的资料，对于从miRNA层面揭示鸡卵巢卵泡发育的分子调控机制具有很好的借鉴意义。