男子生精功能发生过程中piRNA功能的初步研究及miR-96促进乳腺癌发生机制的研究

发布时间：2019-07-03 10:19

【摘要】：小分子非编码RNA(small non-coding RNA), 主要包括 tRNA.snRNA. snoRNA.microRNA(miRNA).siRNA和Piwi-interacting RNA(piRNA)等非编码蛋白质的RNA分子。随着对RNA研究的深入,小分子非编码RNA的研究也日益引起大家的注意和重视,现在已知道它们在基因的转录、转录后调控以及引导染色质修饰复合物中起到了重要的作用。piRNA是从哺乳动物生殖细胞中分离得到的一类长度约为26-31nt的小分子非编码RNA,并且与PIWI蛋白家族成员相结合。目前,尽管已经知道piRNA在精子形成中起着重要的作用,但在男性的精浆中的分布情况尚不完全清晰。在本研究第一节的第一部分中,我们系统地检测男性不育患者和正常对照的精浆中的piRNA差异表达谱,筛选出在不育男性患者中存在表达差异的piRNA.我们一共收集了91例正常对照和211例男性不育患者的精浆样本。选取正常对照17例、弱精症患者24例和无精症患者21例分别混合后提取RNA,然后通过高通量测序技术对RNA进行测序,初步筛选到有61个piRNA在不育患者组和正常对照组之间存在明显地表达差异,然后我们对每一个样本中piRNA进行实时荧光定量PCR(qRT-PCR)技术检测,先使用少量样本量(包括正常对照16例、弱精症患者20例和无精症患者20例)对初筛出61个piRNA进行复筛,复筛结果显示有4种piRNA(piR-31068,piR-31925,piR-43771和piR-43773)在弱精症患者精浆中显著降低；有5种piRNA(iR-31068,piR-31925,piR-43771,piR-43773和piR-30198)在无精组患者精浆中显著降低。然后使用大量样本(包括正常对照58例、弱精症患者74例和无精症患者52例)对复筛出的piRNA进行验证,结果和复筛结果变化趋势一致。在整个过程中我们使用对应piRN A的人工合成标准品构建标准曲线,所以计算出了piRNA的绝对浓度,同时我们使用ROC曲线分析及Risk_score分析显示该5种piRNA的确能够区分出正常对照与不育患者。在本研究第一节第二部分中,我们也通过高通量测序技术对正常对照和弱精症患者精子中的piRNA进行测序,结果显示在弱精症患者精子中piRNA的含量和种类明显减少。精子中蛋白检测发现MitoPLD在弱精症患者的表达量明显下降,这种差异极有可能影响精子中piRNA的形成。通过电镜检测发现精浆中存在大量的Exosome,并发现精浆piRNA大部分存在于Exosome中。miRNA是一类长度约为21 nt的小分子非编码RNA,大量研究表明,miRNA参与调控～30%人类基因,涉及到很多生理过程,包括器官发育、细胞分化、细胞周期和细胞凋亡等,还有1miRNA在癌症的发生和进程中扮演了一个重要的调控者,其中也包括乳腺癌。本研究第二节主要研究miR-96在乳腺癌中的表达情况及在乳腺癌发生过程中所起的生物学功能。本实验通过临床样本发现miR-96在乳腺癌样本中表达量上升,体外实验显示miR-96促进细胞的增殖、迁移和侵袭,动物实验发现miR-96促进肿瘤的生长。我们通过生物信息学预测及实验验证miR-96是通过靶向PTPN9蛋白来促进乳腺癌症的发生及发展,然后我们在乳腺癌细胞中通过下调或上调PTPN9蛋白的表达量,结果显示PTPN9的生物学功能与miR-96的生物学功能成负相关,这也说明miR-96是通过抑制PTPN9蛋白来行使生物学功能。本实验显示了miR-96在乳腺癌的发生发展中扮演了重要角色及通过靶向PTPN9蛋白来发挥生物学功能,同时也为乳腺癌的发生提供了新的分子生物学机制。
[Abstract]:Small-molecule non-coding RNA, mainly including tRNA. snRNA. SnRNA. microRNA (miRNA). RNA molecules of non-coding proteins such as siRNA and Piwi-intting RNA (piRNA). As the research of RNA is in-depth, the research of small-molecule non-coding RNA has also attracted attention and attention, and it is now known that they play an important role in the transcription, post-transcriptional regulation and the guiding of chromatin modification complexes. The piRNA is a class of small-molecular, non-coding RNAs, isolated from the mammalian germ cells, of a length of about 26-31nt, and is combined with the PIWI protein family member. At present, although it is known that the piRNA plays an important role in the formation of spermatogenesis, the distribution of piRNA in the seminal plasma of the male is not completely clear. In the first part of the first section of this study, we systematically examined the difference expression profile of the piRNA in the seminal plasma of the male sterile patient and the normal control, and screened a piece of piRNA expressing the difference in the male sterile male. We collected a total of 91 normal controls and 211 male sterile patients with seminal plasma samples. The RNA was extracted from 21 patients with normal control and 21 of the patients with azoospermia, and then the RNA was sequenced by high-throughput sequencing, and there were 61 pieces of piRNA clearly expressed in the sterile patient group and the normal control group. Then we used the real-time fluorescence quantitative PCR (qRT-PCR) technology to detect the piRNA in each sample, using a small amount of sample size (including 16 normal controls,20 patients with hypospermia and 20 patients with azoospermia) to screen out 61 pieces of piRNA from the primary screen, and the result of the re-screening showed that there were 4 pieces of piRNA (piR-31068, piR-31925, Pir-43771 and pir-43773) were significantly reduced in the seminal plasma of patients with asthenospermia;5 pieces of piRNA (iR-31068, piR-31925, piR-43771, piR-43773, and piR-30198) were significantly reduced in the seminal plasma of the unrefined group. The double-screened piRNA was then validated using a large number of samples (including 58 normal controls,74 patients with hypospermia and 52 patients with azoospermia) and the results were consistent with the results of the re-screening. In the whole process, we used the synthetic standard of piRNA to construct the standard curve, so the absolute concentration of piRNA was calculated. At the same time, we used the ROC curve analysis and the Risk _ score analysis to show that the five pieces of piRNA do distinguish between the normal control and the sterile patient. In the second part of the first part of this study, we also sequenced the pieces of piRNA in the sperm of the normal control and the asthenospermia by the high-throughput sequencing technique, and the results showed that the content and type of piRNA in the sperm of the patients with asthenospermia were significantly reduced. The detection of the protein in the sperm showed that the expression of the MitePLD in the patients with asthenospermia was significantly reduced, and the difference was highly likely to affect the formation of piRNA in the sperm. A large number of Exosomes were found in the seminal plasma by electron microscopy, and most of the piRNA was found to be present in the Exosome. MiRNA is a small molecule non-coding RNA with a length of about 21 nt, and a large number of studies have shown that the miRNA is involved in regulating the human gene of -30%, and relates to many physiological processes, including organ development, cell differentiation, cell cycle and cell apoptosis. There are also 1 miRNAs that play an important regulator in the occurrence and progression of cancer, including breast cancer. The second section of this study mainly studies the expression of miR-96 in breast cancer and its biological function in the process of breast cancer. In this experiment, the expression of miR-96 in the sample of breast cancer was increased by clinical samples, and the in vitro experiment showed that miR-96 promoted the proliferation, migration and invasion of the cells, and the animal experiment found that the miR-96 promoted the growth of the tumor. The biological function of the PTPN9 is negatively correlated with the biological function of the miR-96 by means of the bioinformatics prediction and the experiment to verify the occurrence and development of the breast cancer by targeting the PTPN9 protein, and then, the expression level of the PTPN9 protein is reduced or increased in the breast cancer cell, and the result shows that the biological function of the PTPN9 is inversely related to the biological function of the miR-96, This also indicates that miR-96 is a biological function by inhibiting the PTPN9 protein. The experiment shows that miR-96 plays an important role in the development of breast cancer and plays a biological function by targeting the PTPN9 protein, and also provides a new molecular biological mechanism for the occurrence of breast cancer.
【学位授予单位】：南京大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R737.9;R698.2

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