Dataset Information

MiR-497 Is Implicated in the Process of Chondrogenesis and Inhibits IHH Gene Expression in Human Chondrocytes.

ABSTRACT:

Objective

The aim of this study was to examine differences in microRNA-497 (miR-497) expression during cartilage tissue formation and to test whether miR-497 directly interferes with Indian hedgehog (IHH) gene and inhibits IHH expression in human chondrocytes.

Design

At different cartilage development stages and different time points in bone matrix gelatin-induced endochondral ossification (BMG-ECO) rat models, the expression of miR-497 and the Ihh gene was monitored at the mRNA level. Bioinformatic analysis, gene mutation, dual luciferase reporter gene assays and gene expression assays at both the mRNA and protein levels in human chondrocytes were subsequently performed to validate the interaction between miR-497 and the IHH gene.

Results

The mRNA expression of miR-497 or the Ihh gene in BMG-ECO rats showed significant differences between the cartilage development stages and between different time points, and the trends in the expression of miR-497 and Ihh were reversed. Bioinformatic and dual luciferase reporter gene assays demonstrated a direct interaction between miR-497 and the IHH gene. Differential mRNA and protein expression profiles of the IHH gene in human chondrocytes after 48 hours of transfection with miR-497 mimics and a negative control indicated that miR-497 inhibited IHH expression.

Conclusion

Our study provided new clues for further functional and molecular mechanism studies of miR-497 in chondrogenesis and demonstrated a potential target for clinical therapy for cartilage degenerative disease.

SUBMITTER: Zhang R

PROVIDER: S-EPMC7488943 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Assessing the quality of tissue engineered (TE) cartilage has historically been performed by endpoint measurements including marker gene expression. Until the adoption of promoter-driven reporter constructs capable of quantitative and real time non-destructive expression analysis, temporal gene expression assessments along a timeline could not be performed on TE constructs. We further exploit this technique to utilize microRNA (miRNA or miR) through the use of firefly luciferase reporter (Luc) containing a 3' UTR perfect complementary target sequence to the mature miR-145-5p. We report the development and testing of a firefly luciferase (Luc) reporter responsive to miR-145-5p for longitudinal tracking of miR-145-5p expression throughout MSC chondrogenic differentiation. Plasmid reporter vectors containing a miR-145-5p responsive reporter (Luc reporter with a perfect complementary target sequence to the mature miR-145-5p sequence in the 3'UTR), a Luc reporter driven by a truncated Sox9 (one of the targets of miR-145-5p) promoter, or the Luc backbone (control) vector without a specific miRNA target were transfected into MSCs by electroporation. Transfected MSCs were mixed with untransfected MSC to generate chondrogenic pellets. Pellets were imaged by bioluminescent imaging (BLI) and harvested along a preset time line. The imaging signals from miR-145-5p responsive reporter and Sox9 promoter-driven reporter showed correlated time-courses (measured by BLI and normalized to Luc-control reporter; Spearman r=0.93, p=0.0002) during MSC chondrogenic differentiation. Expression analysis by qRT-PCR suggests an inverse relationship between miR-145-5p and Sox9 gene expression during MSC chondrogenic differentiation. Non-destructive cell-pellet imaging is capable of supplementing histological analyses to characterize TE cartilage. The miR-145-5p responsive reporter is relatively simple to construct and generates a consistent imaging signal responsive to miR-145-5p during MSC chondrogenesis in parallel to certain molecular and cellular events.