Department of Biotechnology, College of Science, University of Tehran, Tehran, Iran.
Department of Medical Genetics, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran.
Iran J Basic Med Sci. 2015 Feb;18(2):108-14.
An earlier meta-analysis on gene expression data derived from four microarray, two cDNA library, and one SAGE experiment had identified RGS5 as one of only ten non-housekeeping genes that were reported to be expressed in human trabecular meshwork (TM) cells by all studies. RGS5 encodes regulator of G-protein signaling-5. The TM tissue is the route of aqueous fluid outflow, and is relevant to the pathology of glaucoma. MicroRNAs constitute the most recently identified components of the cellular machinery for gene regulation in eukaryotic cells. Given our long standing interest in glaucoma, we aimed to identify miRNAs that may target RGS5.
Eight miRNAs were selected for study using bioinformatics tools and available data on miRNAs expressed in the eye. Their effects were assessed using the dual luciferase assay. 3'-UTR segments of RGS5 mRNA were cloned downstream of a luciferase coding gene in psiCHECK2 vectors, and these were co-transfected with each of the miRNAs into HEK293 cells.
The outcomes evidenced that one or more of the segments are in fact targeted by miR-7, miR-9, miR-96, miR-23a, miR-23b, miR-204, and miR-211. Down regulations by the miRNAs were statistically significant. The effect of miR-204 is considered particularly important as this miRNA is well known to regulate eye development and to affect multiple ocular functions.
Our results justify further studies on regulation of RGS5 expression and RGS5 downstream functions by these miRNAs.
之前对源自四个微阵列、两个 cDNA 文库和一个 SAGE 实验的基因表达数据进行的荟萃分析，已将 RGS5 鉴定为仅有的十个非管家基因之一，所有研究均报道其在人眼小梁网（TM）细胞中表达。RGS5 编码 G 蛋白信号调节因子-5。TM 组织是房水流出的途径，与青光眼的病理有关。microRNAs 构成真核细胞基因调控细胞机制中最近发现的组成部分。鉴于我们对青光眼的长期兴趣，我们旨在鉴定可能靶向 RGS5 的 microRNAs。
使用生物信息学工具和眼睛中表达的 microRNAs 的可用数据，选择了 8 种 microRNAs 进行研究。使用双荧光素酶测定法评估它们的作用。RGS5 mRNA 的 3'-UTR 片段被克隆到 psiCHECK2 载体下游的荧光素酶编码基因中，并将这些载体与每种 microRNA 共转染到 HEK293 细胞中。
结果表明，一个或多个片段实际上受到 miR-7、miR-9、miR-96、miR-23a、miR-23b、miR-204 和 miR-211 的靶向调控。microRNAs 的下调具有统计学意义。miR-204 的作用被认为尤为重要，因为这种 microRNA 众所周知可调节眼睛发育并影响多种眼部功能。
我们的结果证明，进一步研究这些 microRNAs 对 RGS5 表达和 RGS5 下游功能的调控是合理的。
