Preliminary Study Of PiRNA In Man Spermatogenesis And Molecular Mechanism Of MiR-96 Promote Breast Cancer Progression

Small non-protein-coding RNA, including tRNA, snRNA, snoRNA, microRNA, siRNA, Piwi-interacting RNA (piRNA) and so on. With the researchs focusing on RNA, scientists begin to attach importance to study of small non-coding RNA. It is now clear that these RNAs play critical roles as transcriptional and post-transcriptional regulators and guides of chromatin-modifying complexes,piwi-interacting RNAs (piRNAs), as a type of small RNA, are expressed mainly in pachytene spermatocytes and spermatids in the testes of mammals> piRNAs are longer (26-31 nucleotides) than miRNAs (18-24 nucleotides) and bind to piwi-family proteins. Although piwi-interacting RNAs (piRNAs) play pivotal roles in spermatogenesis, little is known about piRNAs in the seminal plasma of infertile males. In first section of this study, we systematically investigated the profiles of seminal plasma piRNAs in infertile males to identify piRNAs that are altered during infertility. Seminal plasma samples were obtained from 211 infertile patients (asthenozoospermia and azoospermia) and 91 fertile controls. High-throughput sequencing technology was employed to screen piRNA profiles in seminal plasma samples pooled from healthy controls and infertile patients. The results identified 61 markedly altered piRNAs in the infertile patient groups compared with the control group. Next, a quantitative RT-PCR assay was conducted in the training and validation sets to measure and confirm the concentrations of altered piRNAs. The results identified a panel of 4 piRNAs (piR-31068, piR-31925, piR-43771 and piR-43773) that were significantly decreased in the seminal plasma of asthenozoospermia patients and a panel of 5 piRNAs (piR-31068, piR-31925, piR-43771, piR-43773 and piR-30198) that were significantly decreased in the seminal plasma of azoospermia patients compared with healthy controls. And each synthetic single-stranded piRNA was serially diluted and assessed using the qRT-PCR assay to generate a standard curve. The concentrations of piRNAs in seminal plasma were calculated based on the standard curve. ROC analysis and Risk score analysis based on the 5 piRNAs expression profile was used to distinguish seminal plasma samples of the infertile patients from those of the fertile control donors. High-throughput sequencing technology was employed to screen piRNA profiles in sperm samples pooled from healthy controls and infertile patients, the result showed types and reads significantly decreased in the sperm of asthenozoospermia patients compared with healthy controls. We found MitoPLD protein significantly decreased expression level in asthenozoospermia patients compared with healthy controls, these different levels may affect formation and secretion of piRNA from sperm. We found a large amounts of Exosome in seminal plasma by electron microscopy, and found most piRNAs exist in Exosome.microRNAs (miRNAs) are a short, about 21 nt in length, novel class of small noncoding RNAs that regulate about 30 percent gene of human, and play a critical role in many important biological processes and pathological development, including organ development, cell differentiation, cell cycle and cell apoptosis and so on. miRNAs also act as major regulators of the initiation and progression of human cancers, including breast cancer. The aim of the second section of this study is to determine the expression pattern of miR-96 in breast cancer and to investigate its biological role during tumorigenesis. We showed that miR-96 was significantly upregulated in breast cancer. We then investigated its function and found that miR-96 significantly promoted cell proliferation, migration and invasion in vitro and enhanced tumor growth in vivo. Furthermore, we explored the molecular mechanisms by which miR-96 contributes to breast cancer progression and identified PTPN9 (protein tyrosine phosphatase, non-receptor type 9) as a direct target gene of miR-96. Finally, we showed that PTPN9 had opposite effects to those of miR-96 on breast cancer cells, suggesting that miR-96 may promote breast tumorigenesis by silencing PTPN9. Taken together, this study highlights an important role for miR-96 in the regulation of PTPN9 in breast cancer cells and may provide insight into the molecular mechanisms of breast carcinogenesis.