GSE135934
Long-term Y-27632 treatment promotes senescence of primary human dermal fibroblasts by increasing IGFBP-5
Purpose: Y-27632, a well-characterized Rho-associated kinase (ROCK) inhibitor, has been shown to play an important role in regulation of cell growth in multiple types of cells, the regulation appeared to be cell specific.  To further understand its molecular mechanisms, we performed RNA-seq analysis of HDF at 12 hours and 48 hours after Y-27632 treatment. Methods: Total RNAs were isolated from dermal cells with/without Y-27632 treatment for 12 hours or 48 hours respectively. Then mRNAs were purified from above total RNA using poly-T oligo-attached magnetic beads. After sequencing libraries were prepared using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA), the library preparations were sequenced on an Illumina Hiseq platform and 125 bp/150 bp paired-end reads were generated. Results: We sequenced 12 of 229 differential expressed genes 48 hours after Y-27632 treatment and found genes which by GO analysis are negative regulators of cell growth ). Of these 12 genes, the expression level of 4 genes, IGFBP1, IGFBP-5, HNF4A and NKD1, changed significantly at both 12 hours and 48 hours of Y-27632 treatment. This results, validated by RT-qPCR analysis  showed that the expression of IGFBP-5 (Insulin Growth Factor Binding Protein 5) increased by 1.5 fold after 12 hours of treatment, and increased more than 10-fold after 48 hours. Expression of the other 3 genes didn't change significantly after Y-27632 treatment Conclusions:RNA-seq analysis of gene expression profile in HDFs collected from 12 hours and 48 hours treatment of Y-27632. From these analyses we found the IGFBP-5, a negative regulation of cell proliferation, whose expression increased with the duration of Y-27632 treatment (7-fold increase after the first 24 hours Overall design: RNA-seq analysis of gene expression profile in HDFs collected from 12 hours and 48 hours treatment of Y-27632 vs untreated controls.