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

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. Brain hippocampal piRNA profiles in CA1 region of 7-8 weeks-old adult male Wistar rats（Sham,tGCI and HPC group, three replicates for each group. ）

研究目的：鉴定假手术（Sham）组、暂时性全脑缺血（transient Global Cerebral Ischemia, tGCI）组以及伴缺氧后处理（Hypoxic Postconditioning, HPC）组样本中，与Piwil2结合的piRNAs（piwi-interacting RNAs）。
实验方法：从大鼠海马CA1亚区获取的样本置于500 μl裂解缓冲液中裂解并离心。离心完成后，将上清液与预先经Protein A/G琼脂糖预孵育的抗Piwil2单克隆小鼠抗体（Santa Cruz，美国加利福尼亚州圣克鲁斯）进行孵育。随后用裂解缓冲液洗涤磁珠两次。经蛋白质印迹（Western blot）质检后，使用Trizol试剂（Invitrogen）从磁珠中提取RNA。随后对免疫沉淀得到的RNA开展piRNAs测序分析。
实验结果：暂时性全脑缺血造模后，缺血再灌注26小时时，CA1区共有423种与Piwil2结合的piRNAs表达上调。其中317种piRNAs的表达上调幅度超过1.5倍；在这317种piRNAs中，258种的表达变化倍数≥2。对比tGCI组与HPC组发现，经HPC处理后，CA1区共有401种与Piwil2结合的piRNAs表达显著下调；而在全部3个实验组中，仅7种piRNAs的表达变化幅度≥1.5倍。
研究结论：本研究鉴定了大鼠CA1区的一类piRNAs。相较于HPC组大鼠，我们直接证实了tGCI造模后CA1区与Piwil2结合的piRNAs呈现完全相反的表达变化趋势。生物信息学预测结果显示，DNMT3A可能是这7种差异表达的Piwil2结合piRNAs的潜在共同靶标。因此，我们重点探究了有无HPC处理的tGCI造模后，大鼠CA1区DNMT3A的表达情况。本研究的样本为7-8周龄成年雄性Wistar大鼠的海马CA1区脑组织，分为Sham组、tGCI组与HPC组，每组设置3个生物学重复。