mRNA, lncRNA, circRNA, and miRNA sequencing to investigate silibinin's anti-fibrotic activity in TGF-beta2 treated human trabecular meshwork cells

Background and Objective: TGF-beta-induced fibrogenic changes in human trabecular meshwork (HTM) cells contribute to IOP elevation in glaucoma. Silibinin, a natural polyphenolic flavonoid from milk thistle seeds, has been shown in our previous work to prevent TGF-beta2 effects on HTM cells. The transcriptomic changes of non-coding RNAs contributing to the pro-fibrotic effect of TGF-beta2 and the anti-fibrotic effect of silibinin in HTM cells remain unknown. Methods: The mRNA, lncRNA, circRNA, and miRNA profiles in TGF-beta2-treated HTM cells with or without silibinin administration were detected by RNA sequencing, followed by pathway enrichment analysis and competing endogenous RNA network construction. Results: TGF-beta2 upregulated 1119 mRNAs, 319 lncRNAs, 383 circRNAs, and 30 miRNAs in HTM cells, of which 545 mRNAs, 183 lncRNAs, 265 circRNAs, and 22 miRNAs were reversed by silibinin. These 545 mRNAs, including COL1A1 and alpha-SMA, were enriched in pathways including cell migration, cell adhesion, extracellular matrix, PI3K-AKT, and TGF-beta signaling pathway. Furthermore, TGF-beta2 downregulated 1165 mRNAs, 212 lncRNAs, 449 circRNAs, and 15 miRNAs, of which 395 mRNAs, 54 lncRNAs, 104 circRNAs, and 7 miRNAs were reversed by silibinin. By predicting the binding of differentially expressed mRNAs, lncRNAs, and circRNAs to miRNAs using Miranda (v 3.3a) software, we constructed multiple lncRNA-miRNA-mRNA and circRNA-miRNA-mRNA networks that might be involved in the pro-fibrotic effect of TGF-beta2 and the anti-fibrotic activity of silibinin in HTM cells. Conclusions: Silibinin mitigated TGF-beta2-induced alterations in the coding and non-coding RNA profiles of HTM cells, presumably contributing to its anti-fibrotic activity.