Carcinoma-associated fibroblasts (CAFs) are one of the crucial parts of in the tumor microenvironment and contribute to
tumor progression.
Interleukin-33 (IL-33), a tissue-derived nuclear
cytokine from the
IL-1 family, has been found abnormally expressed in
tumor cells and Fibroblast. However, the role and mechanism of
IL-33 in the interaction between
gastric cancer (GC) cells and CAFs need investigation. Presently, we inquire into the function of
lncRNA NORAD-miR-496 axis-mediated
IL-33 in modulating the GC-CAFs interaction. Real-time reverse transcription-polymerase chain reaction (RT-PCR) was adopted to gauge the expression of NORAD, miR-496, and
IL-33 in GC tissues and cells, and gain- or loss-of-function assays were conducted to investigate the role of them in GC. A GC cell-CAFs co-culture model was established to explore the interaction between CAFs and GCs. As exhibited, NORAD was up-regulated in GC tissues and cells, while miR-496 was remarkably down-regulated. Overexpressing NORAD substantially promoted the proliferation, migration, invasion, and EMT of GC cells and repressed cell death, while overexpressing miR-496 had the opposite effects. Additionally, NORAD enhanced the
IL-33 expression and the release of
IL-33 from GC cells. The dual-
luciferase reporter assay confirmed that miR-496 was a target of NORAD and targeted
IL-33. CAFs aggravated the malignant behaviors of GC cells as indicated by both experiments. However, NORAD knockdown in CAFs reversed CAFs-mediated promotive effects on GC cells. In conclusion, NORAD enhanced the promotive effect of CAFs in GC cells by up-regulating
IL-33 and targeting miR-496, which provided new insights into the microenvironment of GC cells and CAFs.Abbreviation ANOVA: Analysis of Variance; BCA:
Bicinchoninic acid; CAFs:
carcinoma-associated fibroblasts; CCK-8: cell counting kit-8;
ceRNA:
competing endogenous RNA;
DAPI: 4',6-diamidino-2-phenylindole; DMEM: Dulbecco's minimal essential medium/Ham's; ECL: enhanced chemiluminiscent; ELISA:
Enzyme-Linked
Immunosorbent Assay; EMT: epithelial-mesenchymal transition; FBS:
fetal bovine serum; FISH:Fluorescence in situ hybridization;
FITC:
fluorescein isothiocyanate; FSP:fibroblast-specific
protein; GAPDH:
glyceraldehyde-3-phosphate dehydrogenase; GC:
gastric cancer; IHC: immunohistochemistry; IL:
Interleukin;
lncRNA:
long Noncoding RNA; miR-496:
microRNA-496;
MMP-14:
matrix metalloproteinase-14; MUT:mutant; MYH9:
myosin heavy chain 9; NFs: normal fibroblasts; NORAD:
Noncoding RNA activated by DNA damage; ORF: open reading frame; PBS:
phosphate-buffered saline; PMSF:
Phenylmethylsulfonyl fluoride;
PVDF:
polyvinylidene difluoride; RIPA: Radio-Immunoprecipitation Assay; RT-PCR: Real-time reverse transcription polymerase chain reaction; S100A4:
S100 calcium binding protein A4; SDS-PAGE:
sodium dodecyl sulfate-
polyacrylamide gel electrophoresis; sh-NC:
short-hairpin RNA negative control; sh-NORAD:
short-hairpin RNA of NORAD; α-SMA: α-smooth muscle actin; TBST: Tris-buffered saline with
Tween-20; TGF-β1:
Transforming growth factor β1; TUNEL: TdT-mediated dUTP Nick-End Labeling; TWIST1: the
twist-related protein 1;
VEGF-C:
vascular endothelial growth factor C; WT: Wildtype.