Fig. 3

MiR-190a-3p suppressed the expression of CSMD1 by targeting the 3′-UTR of CSMD1 mRNA. (A) Two candidate microRNA families (miR-10 and miR-190) that target the CSMD1 gene were identified in four databases (miRDB, TargetScan, miRanda and miRTarBase) and are listed in Table 1. (B) Luciferase reporter assays demonstrated that miR-190a-3p mimics could significantly reduce the luciferase activity of CSMD1 in the wild type (WT) group, whereas the CSMD1 mutational type (MT) group was not affected, indicating that the direct binding of miR-190a-3p to the CSMD1–3′-UTR. ns, no significance, ***p < 0.001. (C) qRT-PCR confirmed comparatively higher expression of miR-190a-3p in HTS derived fibroblasts (n = 4) than in NS derived fibroblasts (n = 3), *p < 0.05. (D, H) qRT-PCR was performed to confirm successful overexpression/knockdown of miR-190a-3p using miR-190a-3p mimics/inhibitors respectively. Mimics NC/inhibitors NC were served as negative controls. All experiments were performed in triplicate and the data were shown as mean ± SD. **p < 0.01, ***p < 0.001. (E, I) qRT-PCR was performed to detect the CSMD1 mRNA level in fibroblasts treated with miR-190a-3p mimics/inhibitors and their negative controls. All experiments were performed in triplicate and the data were shown as mean ± SD. *p < 0.05, **p < 0.01. (F, J) Immunofluorescence was performed to confirm successful down-regulation/up-regulation of CSMD1 protein expression in fibroblasts treated with miR-190a-3p mimics/inhibitors. Scale bar: 200 μm. (G) Histogram showing fluorescence intensity of CSMD1 in the mimics NC and miR-190a-3p mimics/inhibitors-treated fibroblasts from the IF photos taken and analyzed using the NIS-Elements D software