Abstract
Purpose :
Various diseases and age-dependent processes, such as AMD and glaucoma, are accompanied by alterations in stiffness of the surrounding tissue and concomitant changes in cell differentiation and function. The aim of the project was to characterize the impact of substrate stiffness on the transcriptome of RPE, with a distinct focus on the expression of miRNAs as part of the gene regulatory network.
Methods :
ARPE-19 cells were plated on polyacrylamide gels of defined substrate stiffness (30-80 kPa). Small RNAs and mRNAs were isolated after three weeks of confluent cultivation. RNA sample quality was assessed by a lab-on-a-chip system, total miRNA content was determined with a fluorescence-based assay. In a pilot experiment, small RNAs were studied by Next-Generation Sequencing. qPCR was performed to verify smallRNA Sequencing data. Changes in expression of miRNA targets were confirmed in qPCR and Western Blot.
Results :
755 different miRNAs were detected by RNA sequencing, 569 of them on all substrates. The miRNA fraction of small RNAs increased with substrate stiffness. Among others, hsa-miR-204-5p with a known relevance in RPE differentiation and hsa-miR-155-5p were reconfirmed by qPCR as examples of mechanosensitve miRNAs being more highly expressed on stiff or soft substrates, respectively. Comparative in silico gene expression analysis revealed targeting of different transcripts in the same functional pathways by significantly up- and downregulated miRNAs. Furthermore, upregulated miRNAs targeted some unique pathways including Wnt-signaling. A stiffness-dependent effect on the expression level and activity of MITF, a transcription factor downstream of Wnt, was observed.
Conclusions :
Substrate stiffness-dependent mechanotransduction modulates the expression patterns of small non-coding RNAs in retinal pigment epithelial cells. The miRNA fraction of small non-coding RNAs is enhanced on stiff substrates and distinct miRNAs are differentially expressed in a substrate-dependent manner. A strong overlap of targeted functional pathways by both up- and downregulated miRNAs supports a recently postulated homoeostatic function of miRNAs. Distinct factors in Wnt-signaling with a known impact on RPE show a stiffness-dependent expression pattern. Changes in tissue stiffness with age or disease may thus alter RPE differentiation and function in a miRNA mediated manner.
This is a 2020 ARVO Annual Meeting abstract.