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The changed piRNA profiles of GC-1 cells exposure to 500 nM MC-LR.

ABSTRACT: We treated GC-1 cells with 500 nM or 0 nM of MC-LR for 24 h.Next, total RNA from GC-1 cells of these two groups were extracted using TRIzol Reagent. The two small RNA libraries were prepared using the Illumina small RNA preparation kit and were deep-sequenced. After adapter sequences were trimmed, 10,824,057, and 11,566,902 small RNA reads that were smaller than 45 nucleotides were obtained from control group and MC-LR group, respectively. The sequences that correspond to known piRNAs were determined by perfect sequence matching to the NCBI database (http://www.ncbi.nlm.nih.gov). We identified 244066 reads of piRNAs and 220 unique piRNA sequences in control group and 359871 reads of piRNAs and 230 unique piRNA sequences in MC-LR group

ORGANISM(S): Mus musculus

PROVIDER: GSE100987 | GEO | 2018/07/09

REPOSITORIES: GEO

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Project description:Purpose:To evaluate the effect of transient Global Cerebral Ischemia(tGCI) with Hypoxic Postconditioning (HPC) on piRNA expression profiles, piRNAs sequencing was performed. Methods: Total RNA of each sample (n=3 in each group) was prepared using Trizol reagent (Invitrogen), and the RNA quality and integrity were confirmed. 5’- and 3’-Adaptors were ligated to the obtained small RNA. Reverse transcription followed by PCR was used to create cDNA constructs. Subsequently, an about 150 bp fraction corresponding to approximate the adaptor-ligated constructs derived from the 30 nts piRNA fragment was excised and purified. The purified libraries were sequenced on Illumina HiSeq 2000 apparatus. The raw data were refined using cutadapt software (v1.9.1). After filtering out low quality reads and short reads (<15 nts), trimmed reads from all samples were pooled, and piano software was used to predict novel piRNAs. The trimmed reads were aligned to the merged rat piRNA databases (known piRNA from piRNABank plus the newly predicted piRNAs) using Novoalign software (v3.02.12) with at most one mismatch. PiRNA-binding transposon and targets were predicted by miranda software (v3.3a). piRNA-targets networks were plotted by cytoscape software (v2.8.0). Results: After tGCI, a total of 5574 piRNAs in CA1 was detected after 26 h of reperfusion. Of those, 12 were significantly upregulated compared with Sham rats (≥1.5-fold change, p<0.05), including 6 novel piRNAs. Of the 12 piRNAs, 4 showed ≥2-fold change. In comparison to tGCI group, 6 piRNAs in CA1 were significantly downregulated after HPC. Interestingly, the piRNAs rno_piR_000618, rno_piR_017990 and rno_piR_014971 were significantly changed in both tGCI and HPC groups Conclusions: In our study, we identified a cohort of piRNAs in CA1 of rats. Furthermore, we demonstrated directly contrast changes of piRNAs interacted with Piwil2 in CA1 after tGCI, as compared to those in HPC rats. Therefore, this study revealed that PIWI/piRNAs respond to ischemic brain damage and neuroprotection mediated by HPC. To the best of our knowledge, this is the first study ever on piRNAs associated with Piwil2 in cerebral ischemic tolerance.

Project description:Small non-coding RNAs that associate with Piwi proteins, called piRNAs, serve as guides for repression of diverse transposable elements in germ cells of Metazoa. In Drosophila, the genomic regions that give rise to piRNAs, the so-called piRNA clusters, are transcribed to generate long precursor molecules that are processed into mature piRNAs. How genomic regions that give rise to piRNA precursor transcripts are differentiated from the rest of the genome and how these transcripts are specifically channeled into the piRNA biogenesis pathway are not known. We found that trans-generationally inherited piRNAs provide the critical trigger for piRNA production from homologous genomic regions in the next generation by two different mechanisms. First, inherited piRNAs enhance processing of homologous transcripts into mature piRNAs by initiating the ping-pong cycle in the cytoplasm. Second, inherited piRNAs induce installment of the H3K9me3 mark on genomic piRNA cluster sequences. The HP1 homolog Rhino binds to the H3K9me3 mark through its chromodomain and is enriched over piRNA clusters. Rhino recruits the piRNA biogenesis factor Cutoff to piRNA clusters and is required for efficient transcription of piRNA precursors. We propose that trans-generationally inherited piRNAs act as an epigenetic memory for identification of substrates for piRNA biogenesis on two levels, by inducing a permissive chromatin environment for piRNA precursor synthesis and by enhancing processing of these precursors. sequencing of argonaute-bound and total small RNAs from ovaries of different fly crosses: maternal deposition (MD) crosses, in which piRNAs from the P1152 locus are transmitted into the next generation through the mother. No maternak deposition (NMD) crosses, which are reciprocal to the MD crosses. Subsequently, no piRNAs from the P1152 locus are transmitted into the next generation. The strain P-1152, that carries insertion of P{lArB} construct in telomeric sequences of X chromosome (site 1A) is described in (Roche and Rio 1998). The strain BC69 that has insertion of P{A92} construct at a euchromatic location on chromosome 2L (site 35B10-35C1) is described in (Lemaitre et al. 1993).Both stocks were a generous gift from S. Ronsseray.

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The changed piRNA profiles of GC-1 cells exposure to 500 nM MC-LR.

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