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The piRNAs bounding to Piwil2 in the CA1 region of the transient Global Cerebral Ischemia rats hippocampus

ABSTRACT: Purpose：To identify piRNAs bound to Piwil2 in Sham,transient Global Cerebral Ischemia(tGCI) and transient Global Cerebral Ischemia(tGCI) with Hypoxic Postconditioning (HPC). Methods: Samples from CA1 subregion were lysed in 500 μl lysis buffer and centrifuged. After centrifugation, supernatant was incubated with monoclonal mouse antibody against Piwil2 (Santa Cruz, California, CA, USA), preincubated with Protein A/G Agarose. The beads were washed twice with lysis buffer. After quality check by Western blot, the RNA was extracted from the magnetic beads using Trizol reagent (Invitrogen). Then, the immunoprecipitated RNA was analyzed with piRNAs sequencing. Results: After tGCI, a total of 423 piRNAs interacted with Piwil2 in CA1 was upregulated after 26 h of reperfusion. Of the 423 piRNAs, 317 were significantly upregulated by more than 1.5-fold. Of the 317 piRNAs, 258 showed ≥2-fold change. Comparison between tGCI and HPC groups revealed that 401 piRNAs interacted with Piwil2 in CA1 were significantly downregulated after HPC, whereas only 7 piRNAs were found to be altered in all 3 groups studied (≥1.5-fold change). Conclusions: In our study, we identified a cohort of piRNAs in CA1 of rats. We demonstrated directly contrast changes of piRNAs interacted with Piwil2 in CA1 after tGCI, as compared to those in HPC rats. The results of the prediction showed that DNMT3A may be a potential common target of the 7 differentially expressed piRNAs interacted with Piwil2. Therefore, we focused on the expression of DNMT3A in CA1 after tGCI with or without HPC.

ORGANISM(S): Rattus norvegicus

PROVIDER: GSE163299 | GEO | 2023/01/18

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.

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The piRNAs bounding to Piwil2 in the CA1 region of the transient Global Cerebral Ischemia rats hippocampus

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