Identification And Functions Of Long Noncoding RNAs In Mouse Spermatogonial Stem Cells

Spermatogonial stem cells have the ability to balance self-renewal and differentiation, which is essential for maintenance of the stem cell pool and spermatogenesis. Long noncoding RNAs are transcripts that structurally resemble message RNAs but without the potential for encoding proteins. Resent research indicate that long noncoding RNAs play essential roles in biological process, cellular component and molecular function. In this project, we analyzed the expression of AK005183in different organs and cell lines, and detected the responses of AK005183to GDNF and Retinoic Acid. The RNAi-plasmid that can down-regulate AK005183efficiently was constructed. Various genes were examined by quantitative real-time PCR in GC-1spg cell line using loss-of-function experiment. Specifically the results are as follows:1. We selected301ncRNAs from the transcriptome sequencing result of mouse embryonic stem cells and detected that19of them were also expressed in mouse testis and spermatogonial cell lines by RT-PCR. Interestingly, we found a long noncoding RNA AK005183was down regulated with GDNF and GFRal withdrawal and up regulated with GDNF and GFRal replacement. We also found that the expression level of AK.005183expression level decreased with the SSC differentiation induced by Retinoic Acid.2. We designed and synthesized complementary single-strand DNA oligonucleotides targeting AK005183and cloned into CD513B-U6vector. Then we transfected the RNAi-plasmid into NIH3T3cell line and found that AK005183can be down-regulated by real time PCR analysis. In addition, we packaged the lentivirus and obtained the viral titers.3. We obtained the GC-1spg cell strain using lentivirus-mediated RNAi in which the expression level of AK005183was down-regulated by64%. BrdU analysis showed that cell proliferation was significantly promoted when AK005183was interfered. We also detected that some functional genes, which are essential to SSC activities and associated with SSC self-renewal (eg. Etv5and Bcl6b) were down-regulated after interfering AK005183. Collectively, the results suggested that AK005183was associated with the self-renewal of spermatogonial stem cells.