Zhang Juan Juan, Yano Hiroyuki, Sasaki Takako, Matsuo Noritaka, Yoshioka Hidekatsu
a Department of Matrix Medicine, Faculty of Medicine , Oita University , Oita , Japan.
b Research Promotion Institute , Oita University , Oita , Japan.
Connect Tissue Res. 2018 May;59(3):263-273. doi: 10.1080/03008207.2017.1370465. Epub 2018 Mar 1.
Col5a1 encodes the α1 chain of type V collagen, a quantitatively minor fibrillar collagen that is critical for the formation and function of the organs in the body. MicroRNAs (miRNAs) are small noncoding RNAs that posttranscriptionally regulate biological functions by binding to the 3'-untranslated region (3'UTR) of specific target mRNA. In this study, we investigated the posttranscriptional regulation of miRNAs on the Col5a1 gene expression.
We cultured osteoblasts and fibroblasts of cell lines. To examine the 3'UTR activity of the Col5a1 gene, chimeric plasmids constructs containing the core promoter and 3'UTR of Col5a1 were generated and luciferase assays were performed. We also evaluated the role of miRNA using constructs that were mutated at the putative binding sites of miRNA. In addition, we evaluated the endogenous mRNA and protein, and luciferase activity of the Col5a1 gene after miRNA overexpression/knockdown or CRISPR/Cas9-induced knockout.
The luciferase assay showed a decreased activity of the 3'UTR of Col5a1 gene. However, the expression of the mutant constructs of miRNA-binding sites was restored. The overexpression of miRNA inhibited the Col5a1 gene not only with regard to the luciferase activity and endogenous mRNA but also at the protein level. In contrast, the RNAi-mediated knockdown or CRISPR/Cas9 system increased the expression of the Col5a1 gene.
These results provided evidence that miR-29b regulates the Col5a1 gene expression through binding to the 3'UTR, which might play an important role in the pathogenesis of disease related to bone metabolism and fibrogenic reactions.
Col5a1编码V型胶原蛋白的α1链，这是一种数量上较少的纤维状胶原蛋白，对身体器官的形成和功能至关重要。微小RNA（miRNA）是小的非编码RNA，通过与特定靶mRNA的3'-非翻译区（3'UTR）结合在转录后调节生物学功能。在本研究中，我们研究了miRNA对Col5a1基因表达的转录后调控。
我们培养了细胞系的成骨细胞和成纤维细胞。为了检测Col5a1基因的3'UTR活性，构建了包含Col5a1核心启动子和3'UTR的嵌合质粒，并进行了荧光素酶测定。我们还使用在miRNA假定结合位点发生突变的构建体评估了miRNA的作用。此外，我们评估了miRNA过表达/敲低或CRISPR/Cas9诱导敲除后Col5a1基因的内源性mRNA和蛋白质以及荧光素酶活性。
荧光素酶测定显示Col5a1基因3'UTR的活性降低。然而，miRNA结合位点突变构建体的表达得以恢复。miRNA的过表达不仅在荧光素酶活性和内源性mRNA方面，而且在蛋白质水平上均抑制了Col5a1基因。相反，RNAi介导的敲低或CRISPR/Cas9系统增加了Col5a1基因的表达。
这些结果提供了证据，表明miR-29b通过与3'UTR结合来调节Col5a1基因表达，这可能在与骨代谢和纤维化反应相关的疾病发病机制中起重要作用。
