MiRNAs As Potential Regulators Of Enthesis Healing In A Rodent Injury Model

Jun 30, 2022, 12:00 PM
10m
Room: S3 B

Room: S3 B

Speaker

Peniche Silva, Carlos Julio (cBITE, MERLN Institute, Maastricht University)

Description

"Introduction: MicroRNAs are short, non-coding RNA sequences with the ability to inhibit the expression of a target mRNA at the transcriptional level. MiRNAs are involved in the regulation and modulation of both regenerative and degenerative processes, playing crucial regulatory roles in tissue healing and regeneration. This particular feature makes this family of molecules a very interesting niche to explore in pursuit of novel therapeutic tools in the fields of tissue engineering and regenerative medicine. Among the many tissues comprised in the musculoskeletal system, the tendon-to-bone enthesis is notoriously difficult to treat due to the heterogeneity of its composition. Upon injury, the fibrocartilaginous transition between the tendon and the bony ends of the enthesis usually doesn’t regenerate. Furthermore, the occurring fibrotic process typically yields a scar tissue with poor mechanical properties prone to recurrent rupture. With this study, we aimed to investigate the early expression patterns of fibrosis-related miRNAs in an injured enthesis to select the ultimate miRNA candidate(s) to be used as a therapeutic tool to aid in the treatment of entheses defects.
Methodology: A longitudinal defect was created at the patellar enthesis of adult rats. Explants were collected at one (n=6) and 10 days (n=6) after the injury. Tissue samples of the contralateral (healthy) side were used as control. MiRNA expression was assessed by a miScript qPCR array specific for fibrosis (Qiagen) containing a total of 86 miRNAs. Fourteen potentially enthesis-injury/regeneration-related miRNAs resulted de-regulated after the injury compared to the expression in the native tissue. The expression of these miRNAs was then validated in each separate sample. Target prediction was carried out by Ingenuity Pathway Analysis (IPA-Qiagen). The expression of the predicted mRNA targets was investigated by qPCR in each sample. Additionally, protein expression levels of the collagens type I, II, III, and X were investigated by western blot analysis.
Results: We observed that enthesis-injury/regeneration-related miRNAs showed a three- to five-fold down-regulation after one day of the injury and two- to 14-fold up-regulation after 10 days. Further IPA analysis predicted potential mRNA targets relevant for enthesis development and healing for seven of the fourteen de-regulated miRNAs. These were miR-16, -17, -100, -124, -133a, -155 and -182. Furthermore, the predicted mRNA targets included Col2a1, Runx2, Egfr1, Smad2, and Smad3. The mRNA expression pattern confirmed their regulation according to the up- or down-regulation of their respective targeting miRNA. Furthermore, at the protein level, the collagens type I and II resulted down-regulated directly after the injury and up-regulated after 10 days while collagens III and X showed the opposite pattern of expression.
Conclusions: These preliminary results bring new insights on the role of miRNAs in the early healing phases of the enthesis upon injury. Moreover, miRNA expression can be modulated by using mimics or antagomirs. Therefore, the mimics or antagomir of these de-regulated miRNAs could be used as time-sensitive, therapeutic tools to enhance or inhibit the miRNA modulatory effect over their mRNA target to aid in the regeneration process of the injured enthesis."

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