Exploration of the mechanism of NORAD activation of TGF-β1/Smad3 through miR-136-5p and promotion of tacrolimus-induced renal fibrosis

Background: Tacrolimus is a potent immunosuppressant, but has various side effects, with nephrotoxicity being the most common. Renal fibrosis is an important process of tacrolimus nephrotoxicity. Therefore, it is important to identify the factors that contribute to renal fibrosis after tacrolimus nephrotoxicity, and control its development.Methods: The present study aims to determine whether tacrolimus may speed up the course of renal fibrosis by upregulating noncoding RNA activated by DNA damage (NORAD) to compete with miR-136-5p, and activating the TGF-beta 1/Smad3 pathway. Furthermore, in vivo rat models and in vitro cell models were established. Then, the expression levels of NORAD and miR-136-5p were determined by RT-qPCR, while the expression of the TGF-beta 1/Smad3 pathway was determined by western blot and RT-qPCR. In order to investigate the interaction between NORAD and miR-136-5p, as well as miR-136-5p and SYK, two luciferase reporters were employed. The renal fibrosis of mice was observed using Masson and PAS staining. The expression of inflammatory factors IL-1, IL-6, MCP-1 and TNF-alpha was detected by ELISA.Results: In the in vitro experiments, NORAD was upregulated, while miR-136-5p was downregulated after tacrolimus induction. The expression of the TGF-beta 1/Smad3 pathway correspondingly changed after the induction by tacrolimus. In the in vivo experiments, the expression of NORAD and miR-136-5p, and the trend for renal fibrosis were consistent with the results in the in vitro experiments. Furthermore, the inflammatory factors correspondingly changed with the severity of renal fibrosis. Moreover, the expression trend of the TGF-beta 1/Smad3 pathway in tacrolimus-induced rats was consistent with that in the in vitro experiments.Conclusion: Through in vitro and in vivo experiments, the present study was able to successfully prove that tacrolimus upregulates NORAD to compete with miR-136-5p, resulting in a decrease in miR-136-5p expression, which in turn activates the TGF-beta 1/smad3 pathway, and finally induces the aggravation of renal fibrosis.

Automated summary

This study confirms that tacrolimus upregulates NORAD to compete with miR-136-5p, resulting in the downregulation of miR-136-5p expression, which activates the TGF-beta 1/Smad3 pathway and ultimately aggravates renal fibrosis.