AIMS: 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. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSION: 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.
AIMS: 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. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSION: 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.
Entities:
Keywords:
CRISPR/Cas9 system; extracellular matrix; miR-29b; posttranscriptional regulation; type V collagen