Fig 1: COL22A1 is the target of miR-149-5p in HL-1 cells. (A) The predictive binding sequences between miR-149-5p and COL22A1. (B,C) The qRT-PCR and western blot assays showed that the overexpression of miR-149-5p significantly suppressed COL22A1 gene and protein expression, while the inhibition of miR-149-5p resulted in the upregulation of COL22A1 both in gene and protein as compared to the miRNA mimics and inhibitor NCs; U6 and GAPDH were used as the respective internal controls for each. (D) Luciferase assays were applied to confirm the binding between miR-149-5p and the 3'-UTR of COL22A1. Luciferase activity was measured. (E,F) qRT-PCR and western blot assays were performed to examine the mRNA and protein expression of COL22A1 in the mouse hearts induced by ISO or control; GAPDH was used as the internal control. ##, P<0.01; ###, P<0.001. NC, negative control; PBS, phosphate buffer saline; ISO, isoproterenol; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; qRT-PCR, quantitative real-time polymerase chain reaction.
Fig 2: HFRL and miR-149-5p synergistically regulated cardiac inflammation, oxidative damage, proliferation, and fibrosis. (A-G) The qRT-PCR, CCK-8, ROS, and western blot assays showed that the knockdown of HFRL by shRNA attenuated HL-1 cardiac function and pro-inflammatory related gene expression, enhanced HL-1 cell proliferation and viability, decreased ROS production, and prohibited pro-fibrotic genes and protein expression (i.e., COL22A1, collagen I, collagen III, a-SMA, and FN), and apoptosis marker expression (i.e., cleaved caspase3, and PARP). The inhibition effect of HFRL on HL-1 was partially rescued by the simultaneous silencing of miR-149-5p, indicating that HFRL and miR-149-5p synergistically regulated cardiac function, inflammation, proliferation, and fibrosis in HL-1 cells. ROS assays were performed to detect HL-1 cells oxidative damage after treatment. Then, the immunofluorescence was analyzed and captured using a laser scanning confocal microscope and the scale bar is 100 µm. *, P<0.05; **, P<0.01; ***, P<0.001. NC, negative control; HFRL, heart failure-relative lncRNA; ISO, isoproterenol; ANP, atrial natriuretic peptide; BNP, brain natriuretic peptide; ß-MHC, ß-myosin heavy chain; IL, Interleukin; TNF-a, tumor necrosis factor alpha; ROS, reactive oxygen species; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; qRT-PCR, quantitative real-time polymerase chain reaction; CCK-8, Cell Counting Kit-8; FN, fibronectin; PARP, poly (ADP-ribose) polymerase.
Supplier Page from Thermo Fisher Scientific for COL22A1 Antibody