小鼠精原上皮细胞系中受TGF-β1诱导的关键靶分子筛选

发布时间：2018-03-06 12:52

本文选题：TGF-β1　切入点：microRNA芯片分析　出处：《湖南大学》2014年硕士论文　论文类型：学位论文

【摘要】：男性不育一直是个世界性医学难题。男性不育与精子发生异常密切相关。TGF-β1(transforming growth factor β,TGF-β1)作为TGF-β超家族的主要成员之一,以其复杂的信号通路在睾丸发育不同阶段扮演不同角色,参与调节精子发生。因此,寻找并确定生殖细胞发育过程中参与TGF-β1信号通路的关键分子将有助于更好地诠释精子发生机制。基于此,本文以小鼠精原上皮细胞GC-1spg为实验材料,进行外源TGF-β1刺激后,开展了以下三个方面的工作：通过高通量的microRNA(miRNA)芯片分析和定量PCR验证,筛选出24种受TGF-β1调节的miRNA分子,其中重点验证了10个表达丰度较高的差异miRNAs,并通过生物信息学分析和定量PCR技术初步验证了这些miRNAs的可能靶基因：miR-5112的可能靶基因为Hbp1, miR-199a-3p的可能靶基因为Celsr2,miR-196a的可能靶基因为Nr6a1。通过高通量的2-DE(two-dimensional gel electrophoresis,2-DE)电泳结合MALDI-TOF-TOF-MS电泳结合质谱技术,鉴定得到了8个可能受TGF-β1调控的差异蛋白：Gelsolin(Gsn),Vinculin(Vcl),Ezrin(Ezr), NDKB(Nme2),PPIA(Ppia),Cofilin-1(Cfl1),TERA(Vcp)和IF5A1(Eif5a)。这8个差异蛋白在TGF-β1的作用下表达下调。通过GEO数据检索等生物信息学方法分析发现,上述预测的下游靶基因Nr6a1,Celsr2和Hbp1以及差异蛋白NDKB,COF1和PPIA等在畸形精子症和精原细胞瘤等病人组织中存在差异表达,提示它们可能在精子发生过程中扮演重要角色。值得关注的是,通过整合miRNA芯片结果以及蛋白质组学结果,同时结合生物信息学分析发现,有些差异miRNA与差异蛋白在TGF-β1的作用下,它们的表达趋势符合microRNA负性调控机制下的互反现象：如差异蛋白NDKB可能受miR-199a-3p调节,而差异蛋白PPIA可能受miR-196a或miR-149调节,从而响应TGF-β信号通路。结果暗示我们,在GC-1spg细胞中,这3个miRNA可能分别抑制N DKB和PPIA等蛋白的表达,从而参与精子发生过程。综上所述,我们在GC-1spg细胞系中筛选并重点验证了10个差异表达的miRNA和8个差异表达蛋白,并发现TGF-β1可能通过诱导miR-199a-3p,miR-196a和miR-149等的上调来抑制其各自的下游靶分子NDKB和PPIA等蛋白的表达,从而参与精子发生
[Abstract]:Male infertility has always been a worldwide medical problem. Male infertility is closely related to abnormal spermatogenesis. As one of the main members of the TGF- 尾 superfamily, male infertility plays different roles in different stages of testicular development with its complex signaling pathway. Therefore, finding and identifying the key molecules involved in TGF- 尾 1 signaling pathway during germ cell development will contribute to a better interpretation of the mechanism of spermatogenesis. Therefore, GC-1spg of mouse spermatogonial epithelial cells is used as the experimental material. After exogenous TGF- 尾 1 stimulation, the following three aspects were carried out:. Twenty-four miRNA molecules regulated by TGF- 尾 1 were screened by high-throughput microRNA-miRNA-chip analysis and quantitative PCR verification. Among them, 10 differentially expressed miRNAss with high abundance were verified, and the possible target gene of these miRNAs genes: 1 miR-5112 was confirmed by bioinformatics analysis and quantitative PCR technique. The possible target gene of these miRNAs genes was Hbp1, and the possible target gene of miR-199a-3p was Celsr2miR-196a because of Nr6a1. By means of high throughput 2-DEG gel electrophoresistics and MALDI-TOF-TOF-MS electrophoretic mass spectrometry, eight differentially regulated differential proteins (TGF- 尾 1) were identified and down-regulated by TGF- 尾 1. The eight differential proteins were down-regulated by TGF- 尾 _ 1. These proteins were down-regulated by TGF- 尾 _ 1. By GEO data retrieval and other bioinformatics methods, it was found that the predicted downstream target genes Nr6a1, Celsr2 and Hbp1, and the differential protein NDKB#en0# COF1 and PPIA were differentially expressed in the tissues of patients with malformation spermatozoa and seminoma. It suggests that they may play an important role in spermatogenesis. It is worth noting that by integrating the results of miRNA microarray and proteomics, and combining with bioinformatics analysis, some differential miRNA and differential proteins have been found under the action of TGF- 尾 1. Their expression trend is consistent with the reciprocal phenomenon under the negative regulatory mechanism of microRNA: for example, differential protein NDKB may be regulated by miR-199a-3p, while differential protein PPIA may be regulated by miR-196a or miR-149, thus responding to the TGF- 尾 signaling pathway. The results suggest that in GC-1spg cells, These three miRNA may inhibit the expression of N DKB and PPIA proteins, and thus participate in spermatogenesis. In conclusion, we screened 10 differentially expressed miRNA and 8 differentially expressed proteins in GC-1spg cell lines. It was also found that TGF- 尾 1 may inhibit the expression of NDKB and PPIA proteins in their downstream target molecules by inducing the up-regulation of miR-199a-3pnmiR-196a and miR-149, thus participating in spermatogenesis.
【学位授予单位】：湖南大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R698.2

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