During latent infection, the HSV-1 virus generates only a single transcript, LAT, which encodes six miRNAs. The GABAergic pathway signaling system is an essential cell signaling pathway influenced by various therapeutic targets and some brain disorders, such as epilepsy. This study found that miRNAs encoding LAT might target the STXBP1 and GABBR2 genes, which are among the significant genes in the GABAergic pathway. Bioinformatic analysis utilizing TargetScan version 5.2 and the RNA22 tools uncovered miRNAs encoding LAT that can influence STXBP1 and GABBR2 transcripts. To evaluate the targeting effect of candidate microRNAs encoding LAT, namely, miR-H3 and miR-H4, LAT constructs were transfected into HEK 293T cells. The expression levels of microRNAs encoding LAT, as well as STXBP1 and GABBR2, were assayed by real-time PCR. Finally, the targeting potential of STXBP1 and GABBR2 3′UTR by LAT-encoded microRNAs was evaluated by the luciferase assay. In the current study, the bioinformatic tool TargetScan demonstrated that miR-H3 has the potential to target the transcripts of the STXBP1 and GABBR2 genes, whereas miR-H4 solely targeted GABBR2. On the other hand, the bioinformatic tool RNA22 validated the potential targeting of STXBP1 and GABBR2 by miR-H3 and miR-H4. Our findings showed that overexpression of miR-H4, miR-H3, or LAT significantly decreased STXBP1 gene expression by an average of 0.0593-fold, 0.237-fold, and 0.84-fold, respectively. Similarly, overexpression of miR-H3 or miR-H4 decreased GABBR2 expression by an average of 0.055- or 0.687-fold, respectively. Notably, targeting the GABBR2 3′UTR with the LAT transcript had no detectable effect. The evaluation of the targeting potential of STXBP1 and GABBR2 3′UTR by microRNAs encoded by LAT was conducted with a luciferase assay. Our results showed that miR-H3 overexpression reduces Renilla expression in psiCHECK2 plasmids with STXBP1 or GABBR2 3′UTR genes by 0.62- and 0.55-fold, respectively. miR-H4 reduced Renilla gene expression regulated by GABBR2’s 3′UTR plasmid but had no effect on the Renilla gene expression regulated by STXBP1’s 3′UTR. When the LAT transcript was overexpressed, there was a decrease in Renilla expression by 0.44-fold because of the regulation of STXBP1’s 3′UTR. However, there was no significant effect observed through the control of GABBR2’s 3′UTR.

在潜伏感染期间，HSV-1病毒仅产生一个转录本 LAT，它编码 6 个 miRNA。GABAergic 通路信号系统是重要的细胞信号通路，受各种治疗靶点和一些脑部疾病（例如癫痫）的影响。这项研究发现，编码 LAT 的 miRNA 可能靶向 STXBP1 和 GABBR2 基因，这两个基因是 GABA 能途径中的重要基因。利用 TargetScan 5.2 版和 RNA22 工具进行生物信息分析，发现编码 LAT 的 miRNA 可以影响 STXBP1 和 GABBR2 转录本。为了评估编码 LAT 的候选 microRNA（即 miR-H3 和 miR-H4）的靶向效果，将 LAT 构建体转染至 HEK 293T 细胞中。通过实时 PCR 检测编码 LAT 以及 STXBP1 和 GABBR2 的 microRNA 的表达水平。最后，通过荧光素酶测定评估了 LAT 编码的 microRNA 对 STXBP1 和 GABBR2 3'UTR 的靶向潜力。在当前的研究中，生物信息学工具TargetScan证明miR-H3有潜力靶向STXBP1和GABBR2基因的转录本，而miR-H4仅靶向GABBR2。另一方面，生物信息学工具RNA22验证了miR-H3和miR-H4对STXBP1和GABBR2的潜在靶向作用。我们的研究结果表明，miR-H4、miR-H3 或 LAT 的过表达使 STXBP1 基因表达量分别显着降低，平均分别为 0.0593 倍、0.237 倍和 0.84 倍。类似地，miR-H3或miR-H4的过表达使GABBR2表达分别平均降低0.055或0.687倍。值得注意的是，用 LAT 转录物靶向 GABBR2 3'UTR 没有可检测到的效果。通过荧光素酶测定评估 LAT 编码的 microRNA 对 STXBP1 和 GABBR2 3'UTR 的靶向潜力。我们的结果表明，miR-H3 过表达使带有 STXBP1 或 GABBR2 3'UTR 基因的 psiCHECK2 质粒中的Renilla表达分别降低了 0.62 倍和 0.55 倍。miR-H4降低了GABBR2的3'UTR质粒调控的Renilla基因表达，但对STXBP1的3'UTR调控的Renilla基因表达没有影响。当LAT转录本过表达时，由于STXBP1的3'UTR的调节，Renilla的表达量减少了0.44倍。然而，通过控制GABBR2的3'UTR并没有观察到显着的效果。