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. 2022 Jun 16;12:10073. doi: 10.1038/s41598-022-13279-x

Figure 5.

Figure 5

MiR-628 targets JAG1 in PCa cells and affect JAG1 downstream Notch signaling in PCa cells. (A) Jagged-1 was identified as a potential miR-628 target using Targetscan. Wild type and mutant target sequences of miR-628 are shown. miR-628 and JAG1- 3′UTR interaction was assessed through luciferase assay in LNCaP and PC3 cells transfected with wild type miR-628 (WT) or mutant type miR-628 (MT) (**p < 0.01, ***p < 0.001). (B) JAG1 expression was assessed in RPWE-1 and PCa cells by western blotting. Membranes were cut before hybridization using the molecular weight marker as a reference and imaged using an Azure imaging system. All western blot full images are provided in supplementary information files. (C) JAG1 expression was assessed utilizing the GSE41969 dataset and analyzed using the GEO2R web tool in matched benign and PCa samples obtained from AA and CA men. Light blue triangles = benign AA (n = 80), dark blue diamonds = PCa AA (n = 270), light green circles = benign CA (n = 83), and dark green squares = PCa CA (n = 369), (p < 0.0001). (D) Effect of mir-628 on the expression of JAG1 and ZEB1 expression as measured by RT/qPCR in LNCaP and PC3 cells after transfection with 100 nM of miR-628 mimic or NC (*p < 0.05, **p < 0.01). (E) Protein expression of JAG1, Notch 1, Notch 2, Notch 2, and Hes1 is shown in LNCaP and PC3 cells after transfection with 100 nM of miR-628 mimic or NC. Membranes were cut before hybridization using the molecular weight marker as a reference and imaged using an Azure imaging system. All western blot full images are provided in supplementary information files. (F) Protein expression of JAG1, Notch intracellular domain (NICD), and Hes1 are shown in PC3 cells nuclear and cytosolic fractions after transfection with 100 nM of miR-628 mimic, miR-628 inhibitor, or NC. PARP and GAPDH were used as loading controls for nuclear and cytosolic protein fractions and imaged using film. (G) Protein expression of cleaved Notch1, cleaved-PARP are shown in PC3 cells after transfection with 100 nM of miR-628 mimic, miR-628 inhibitor, or NC and treatment with Scrambled control JAG1-peptide (SC) or JAG1-peptide for 30 min or 2 h and imaged using film.