LINC01410-miR-532-NCF2-NF-kB feedback loop promotes gastric cancer angiogenesis and metastasis

Abstract

Dysregulation of non-coding RNAs, including miRNAs and lncRNAs has been reported to play vital roles in gastric cancer (GC) carcinogenesis, but the mechanism involved is largely unknown. Using the cancer genome atlas (TCGA) data set and bioinformatics analyses, we identified miR-532-5p as a potential tumor suppressor in GC, and found that lncRNA LINC01410 might be a negative regulator of miR-532-5p. We then conducted a series of in vivo and in vitro assays to explore the effect of LINC01410 on miR-532-5p-mediated GC malignancy and the underlying mechanism involved. MiR-532-5p overexpression inhibited GC metastasis and angiogenesis in vitro and in vivo, whereas miR-532-5p silencing had the opposite effect. Further study showed that miR-532-5p attenuated NF-κB signaling by directly inhibiting NCF2 expression, while miR-532-5p silencing in GC enhanced NF-κB activity. Furthermore, we demonstrated miR-532-5p down-regulation was caused by aberrantly high expression of LINC01410 in GC. Mechanistically, overexpression of LINC01410 promoted GC angiogenesis and metastasis by binding to and suppressing miR-532-5p, which resulted in up-regulation of NCF2 and sustained NF-κB pathway activation. Interestingly, NCF2 could in turn increase the promoter activity and expression of LINC01410 via NF-κB, thus forming a positive feedback loop that drives the malignant behavior of GC. Finally, high expression of LINC01410, along with low expression of miR-532-5p, was associated with poor survival outcome in GC patients. Our studies uncover a mechanism for constitutive LINC1410-miR-532-5p-NCF2-NF-κB feedback loop activation in GC, and consequently, as a potential therapeutic target in GC treatment.

Fig. 1

miR-532-5p suppresses gastric cancer metastasis in vitro and in vivo. a The wound healing rate in miR-532-5p-transfected SGC-7901 and MNK-45 cells was largely inhibited, while enhanced in miR-532-5p-silenced AGS cells. b The number of migrated cell was significantly decreased in miR-532-5p-overexpressing SGC-7901 and MNK-45, while increased in miR-532-5p-silenced AGS cells, as determined by transwell migration assay. c The number of invaded cell was decreased in miR-532-5p-overexpressing SGC-7901 and MNK-45, while increased in miR-532-5p-silenced AGS cells, as assessed by Matrigel invasion assay. d miR-532-5p inhibits tumor metastasis in vivo. Upper panel: (Left) Representative bright-field imaging of the lungs; (Right) hematoxylin and eosin (H&E) staining was performed on serial sections of metastatic tumors and normal lung. Arrows: lesions of lung. Lower panel: the number of nodules was qualified on lungs of SCID mice (n = 6 per group) 6 weeks after tail vein injection of SGC-7901/miR-532-5p or SGC-7901/control, and AGC/anti-miR-532-5p or AGC/shcontrol cells

Fig. 2

The inhibitory effects of miR-532-5p on tumor angiogenesis and NF-κB activity of GC cells. a GSEA plot showed miR-532-5p level was inversely correlated with angiogenesis gene signatures in the TCGA the stomach adenocarcinoma (STAD) data set. b and c The abilities of in vitro capillary tube formation (b) and migration (c) of HUVEC were significantly decreased after incubating with culture medium of miR-532-5p-transfected GC cell, while potentially strengthened after incubating with culture medium of miR-532-5p-silenced GC cell. d and e The VEGFA levels (d) and MVD (e) in tumor tissue of nude mice models with subcutaneous implantation of GC were noticeably reduced when miR-532-5p was up-regulated in SGC-7901 cells, while largely increased when miR-532-5p was down-regulated in AGS cells. f Luciferase-reported NF-κB activity was decreased in miR-532-5p-overexpressing, while increased in miR-532-5p-silenced GC cells. g EMSA indicated NF-κB activity dramatically decreased in miR-532-5p-transduced cells but increased in miR-532-5p-inhibited cells. OCT-1 DNA-binding complexes served as a control. h qRT-PCR analysis showed an apparent overlap between miR-532-5p-regulated gene expression and NF-κB-dependent gene expression. The pseudocolors represent the intensity scale of miR-532-5p versus control or anti-miR-532-5p versus shcontrol vector, which was generated by a log 2 transformation. i Western blotting assay showed that nuclear NF-κB/p65, p-IKK-β, p-IκBα, and c-FLP expressions were decreased in miR-532-5p-overexpressing SGC-7901 and MNK-45, while potentially increased in miR-532-5p-silenced AGS cells. p54 was used as a nuclear loading control

Fig. 3

NCF2 is the direct target of miR-532-5p and affects the in vitro function of miR-532-5p in GC cells. a The predicted target sequence of miR-532-5p in 3′UTR of NCF2 (NCF2-3′UTR) and mutant containing three altered nucleotides in the seed sequence of miR-532-5p (miR-532-5p-mut). b Luciferase assay of pGL3-NCF2-3′-UTR reporters in the presence of increasing amounts (10, 20, 50 nM) of miR-532-5p mimic and mutant oligonucleotides, or increasing amounts (20, 50, 100 nM) of miR-532-5p inhibitor oligonucleotides in GC cell line. c and d qRT-PCR analysis (c) and western blot analysis (d) showed miR-532-5p tranfection decreased NCF2 mRNA and protein level in SGC-7901 and MNK-45 cells, while anti-miR-532-5p dramatically increased NCF2 mRNA and protein level in AGS cell. e Transwell migration assay (Left) and Matrigel invasion assay (Right) showed the migratory capacity and invasion ability of miR-532-5p-overexpressing SGC-7901 and MNK-45 cells was strengthened when transfected with full-length NCF2. f Restoration of NCF2 compromised the inhibitory effects of miR-532-5p on the abilities of capillary tube formation (Left) and in vitro migration (Right) of HUVECs. g Expression of NF-κB luciferase reporter activities were determined in the indicated cells. h EMSA showed that restoration of NCF2 counteracted the inhibitory effect of miR-532-5p on endogenous NF-κB activity. i and j qRT-PCR (i) and Western blotting (j) assay indicated that overexpression of NCF2 compromised the suppression effect of miR-532-5p on numerous NF-κB-targeted genes and proteins

Fig. 4

LINC01410 targets miR-532-5p by directly binding to miRNA response element. a Left panel: LINC01410 is abundant in cytoplasm of SGC-7901 and MNK-45 cells. U2 and ACTIN were used as positive controls in nucleus and cytoplasm, respectively. Right panel: Cytoplasm enrichment (cytoplasm/total expression ratio) of LINC01410 in GC and adjuvant normal tissues. b Schematic representation of the predicted binding sites for miR-532-5p, and the site mutagenesis design for the reporter assay. c The relative luciferase activities were inhibited in the HEK-293T cells transfected with the reporter vector LINC01410-WT, not with the reporter vector LINC01410-Mut. d Ectopic LINC01410 expression decreased miR-532-5p expression while inhibition of LINC01410 increased miR-532-5p expression in SGC-7901 and MNK-45 cells. e Ectopic miR-532-5p expression decreased LINC01410 expression, and inhibition of miR-532-5p enhanced LINC01410 expression. f miR-532-5p was identified in LINC01410-RISC complex. Control and anti-miR-532-5p cell lysates were used for RNA-IP with anti-Ago2 antibody. LINC01410 and miR-532-5p expression levels were detected using qRT-PCR

Fig. 5

LINC01410 promotes GC metastasis and angiogenesis in vitro and in vivo. a and b Enforced overexpression of LINC01410 largely promoted, while inhibition of LINC01410 potentially compromised the migratory (a) and invasion (b) ability of GC cells. c and d The abilities of in vitro capillary tube formation (c) and migration (d) of HUVEC were largely compromised after incubating with culture medium of LINC01410-transfected AGS cell, while potentially strengthened after incubating with culture medium of LINC01410-silenced SGC-7901 and MNK-45 cell. e Overexpression of LINC01410 increased, while silencing LINC01410 inhibited in vivo metastatic ability of GC cell. f The VEGFA levels and MVD in tumor tissue of nude mice models with subcutaneous implantation of GC were significantly decreased when LINC01410 was down-regulated in SGC-7901, while largely increased when LINC01410 was up-regulated in AGS cells

Fig. 6

LINC01410 promotes GC metastasis and angiogenesis through miR-532-5p. a and b Transwell assay (a) and Matrigel invasion assay (b) to examine the effect of LINC01410 and miR-532-5p on cell migration and invasion of GC cells. c and d The effect of LINC01410 and miR-532-5p on the capillary tube formation ability (c) and migration ability (d) of HUVEC. e Luciferase-reported NF-kB activity to evaluate the effect of LINC01410 and miR-532-5p on the NF-κB signaling in GC cells. f Western blot analysis for NCF2 in GC cells with the expression of LINC01410 and miR-532-5p changed

Fig. 7

NCF2 activates LINC01410 expression through NF-κB. a Schematic diagram showed the putative NF-κB/p65-binding sites in the LINC01410 promoter. b RT-qPCR illustrating ectopic NF-κB/p65 expression upregulates LINC01410 expression in SGC-7901 and MNK-45 cells. c RT-qPCR demonstrated overexpression of NCF2 enhanced LINC01410 expression, while IκBα-mut transfection or IDM0354 treatment diminished this effect. d ChIP assay of SGC-7901 and MNK-45 cells infected with NF-κB/p65 expression vector or control, and NCF2 expression vector or control. e ChIP assay of SGC-7901 and MNK-45 cells treatment with IDM0354 or control. The following PCR primers for ChIP assays were used: 5′-agaccatgcgcgagacag-3′, 5′-gagcacaccccgtcctaag-3′. f Luciferase reporter assays confirming NF-κB activation of the LINC01410 promoter through the NF-κB/p65-binding sites in SGC-7901 and MNK-45 cells. Expression of firefly luciferase (Fluc) was driven by LINC01410 promoter sequences containing either wild-type (Wt) or mutated (Mut) NF-κB-binding sites. g Schematic illustration of the positive feedback loop. LINC01410 upregulated NCF2 and NF-κB signaling by binding to and suppressing miR-532-5p. NCF2 in turn upregulated LINC01410 through NF-κB