The pro-α1(V) collagen gene (Col5a1) is coordinately regulated by miR-29b with core promoter in cultured cells

ABSTRACT

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 3ʹ-untranslated region (3ʹUTR) 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 3ʹUTR activity of the Col5a1 gene, chimeric plasmids constructs containing the core promoter and 3ʹUTR 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 3ʹUTR 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 3ʹUTR, which might play an important role in the pathogenesis of disease related to bone metabolism and fibrogenic reactions.

KEYWORDS:

• CRISPR/Cas9 system
• extracellular matrix
• miR-29b
• posttranscriptional regulation
• type V collagen

Acknowledgments

We thank Mr. K. Kai, Ms. H. Sato and the staff members of Department of Matrix Medicine, Faculty of Medicine, Oita University. J-J Z and H Yano designed and performed the experiments, analyzed the data, and wrote the manuscript. TS and NM interpreted the results. HY designed the experiments and wrote the manuscript. All authors reviewed the results and approved the final version of the manuscript.

Declaration of interest

The authors declare no conflicts of interest. The authors alone are responsible for the content and writing of the article.

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Funding

This work was supported by Grant-in-Aid (Nos. 25462375 and 17K11848 to H.Y.) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.