miR-508-3p concordantly silences NFKB1 and RELA to inactivate canonical NF-κB signaling in gastric carcinogenesis

Abstract

Background: NF-κB signaling pathway plays an important role in gastric carcinogenesis. The basic expression and functional role of NFKB1 and RELA (components of canonical NF-κB pathway) in gastric cancer (GC) have not been well elucidated. In this study, the role of NFKB1 and RELA in gastric tumorigenesis will be investigated and their regulation by microRNAs (miRNAs) will be deeply explored.

Methods: The mRNA and protein expression of NFKB1 and RELA were investigated by qRT-PCR and Western blot in GC cell lines and primary tumors. The functional roles of NFKB1 and RELA in GC were demonstrated by MTT proliferation assay, monolayer colony formation, cell invasion and migration, cell cycle analysis and in vivo study through siRNA mediated knockdown. Identification of NFKB1 as a direct target of tumor suppressor miRNA miR-508-3p was achieved by expression regulation assays together with dual luciferase activity experiments.

Results: NFKB1 and RELA were up-regulated in GC cell lines and primary tumors compared with normal gastric epithelium cells and their upregulation correlation with poor survival in GC. siRNA mediated knockdown of NFKB1 or RELA exhibited anti-oncogenic effect both in vitro and in vivo. NFKB1 was further revealed to be a direct target of miR-508-3p in gastric tumorigenesis and their expression showed negative correlation in primary GC samples. miR-508-3p was down-regulated in GC cells compared with normal gastric epithelium samples and its ectopic expression in GC cell lines also exerts tumor suppressor function. NFKB1 re-expression was found to partly abolish the tumor-suppressive effect of miR-508-3p in GC.

Conclusion: All these findings supports that canonical NF-κB signaling pathway is activated in GC at least by the inactivation of miR-508-3p and this might have therapeutic potential in GC treatment.

Fig. 1

NFKB1 and RELA are up-regulated in GC cell lines and primary gastric tumors. a The mRNA expression of NFKB1 and RELA in nine GC cell lines compared with GES-1 cells (immortalized gastric epithelium cell line). The standard deviations (SDs) were achieved by qRT-PCR (−Delta Delta Ct values) in triplicate wells. b Western blot analysis of NFKB1 and RELA in nine GC cell lines, GES-1 cells and three normal gastric tissues (Normal 1–3 protein samples are from normal gastric mucosa obtained from weight reduction gastric surgery). c NFKB1 and RELA mRNA expression in 28 paired primary GC samples (NFKB1, P = 0.124; RELA, P = 0.188). d NFKB1 and RELA showed increased protein expression in primary gastric tumors compared with paired non-tumours adjacent tissues (n = 28; NFKB1, P = 0.004; RELA, P = 0.012). e Representative immunohistochemistry images of positive NFKB1 and RELA expression in GC tissue microarray (original magnification × 100, insertion × 400). NFKB1 and RELA expression are mainly localized in the cytoplasm. f Kaplan-Meier plots of disease specific survival according to NFKB1 or RELA expression status. RELA accumulation in cytoplasm was associated with poor disease specific survival in patients with GC (P = 0.045), but NFKB1 overexpression only correlated with a non-significant trend of poor survival (P = 0.274). g The prognosis plots according to NFKB1 and RELA mRNA expression in GC (from KM plotter). Only high RELA mRNA expression significantly correlated with overall survival in 876 GC samples (P < 0.001)

Fig. 2

NFKB1 and RELA knockdown exerts tumor suppressor function in GC cells. a Transfection with siNFKB1 or siRELA decreased the mRNA expression of NFKB1 and RELA respectively in MKN28, MGC-803 and SGC-7901 cells. b 6-day MTT assays revealed NFKB1 or RELA knockdown significantly suppressed proliferation rate of GC cells (**, P < 0.001). c siNFKB1 or siRELA decreased monolayer colony formation in MKN28, MGC-803 and SGC-7901 cells (*, P < 0.05; **, P < 0.001). The experiments were performed in triplicate wells to get SDs. d NFKB1 or RELA knockdown inhibited GC cell invasion (*, P < 0.05; **, P < 0.001). Three random vision fields were selected for invaded cell counting to get SDs. e The GC cell migration abilities were suppressed by siNFKB1 or siRELA (*, P < 0.05; **, P < 0.001). The cells were counted in three random vision fields to get SDs. f Flow cytometry analysis of NFKB1 or RELA knockdown transfectants together with scramble siRNA transfectants as control. Two independent experiments were performed and the representative one was shown in the bar chart. g Effects of siNFKB1 and siRELA on the induction of early apoptosis (Annexin V-FITC and PI double-staining). The cell population with early apoptosis was shown in the lower right of each treatment. siNFKB1 or siRELA induced early apoptosis compared with siScramble control after 20-h transfection (*, P < 0.05, **, P < 0.001). h Upper, Western blot analysis of NFKB1, p21, p27, cleaved-PRAP and p-Rb after siNFKB1 transfection; Lower, the Western blot result of RELA, cleaved-PRAP and p-Rb in the transfectants of siRELA. i siNFKB1 and siRELA formed smaller xenografts than siScramble using MGC-803 cells in a 28-day inoculation and the elevated cleaved-PARP was detected in siNFKB1 and siRELA xenografts

Fig. 3

NFKB1 is a direct target of miR-508-3p in GC. a The binding site in the NFKB1 3'UTR for miR-508-3p as predicted by TargetScan (www.targetscan.org). b NFKB1 mRNA expression was down-regulated by ectopic miR-508-3p expression in MKN28, MGC-803 and SGC-7901 cells (**, P < 0.001). c Ectopic miR-508-3p expression decreased the RELA mRNA expression in GC cells (**, P < 0.001). d Both NFKB1 and RELA protein were down-regulated by miR-508-3p in three GC cell lines. e miR-508-3p overexpression inhibited the luciferase activity in the constructs containing wild type binding site, but the luciferase activity in the construct containing mutated binding site of NFKB1 3'UTR was not affected (Wild type, the construct containing the complementary sequence of seed region; Mutation, the binding site was deleted; **, P < 0.001). f ChIP-qPCR analysis on the promoter region of IL-1β and IL-6 after treating the cells with siNFKB1 or miR-508-3p. siNFKB1 or miR-508-3p decreased the binding affinity of NFKB1 on the promoter region of downstream targets IL-1β and IL-6 (**, P < 0.001). IP by IgG was as experimental negative control

Fig. 4

miR-508-3p is down-regulated in GC cell lines and has tumor suppressor potential. a The expression of miR-508-3p in ten GC cell lines compared with commercial normal gastric total RNA (AM7996, Ambion). b 6-day MTT proliferation assays revealed the growth-inhibition effect of miR-508-3p in MKN28, MGC-803 and SGC-7901 cells (**, P < 0.001). c Ectopic miR-508-3p expression decreased monolayer colony formation in all the three GC cell lines (**, P < 0.001). The experiments were performed in three wells to get SDs. d The GC cell invasion ability was significantly inhibited by ectopic expression of miR-508-3p (*, P < 0.05). The invaded cells from the matrigel were counted in three random vision fields for getting SDs. e The mRNA expression of CCND1 and MMP9 was down-regulated upon ectopic miR-508-3p expression in MKN28, MGC-803, and SGC-7901 cells (**, P <0.001)

Fig. 5

NFKB1 re-expression partly abrogated the inhibitory effect of miR-508-3p in GC. a Expression of miR-508-3p in paired primary GC samples (n = 28; P = 0.155). b miR-508-3p expression was negatively correlated with NFKB1 protein expression in primary gastric tumors (P = 0.033). c The Western blot analysis of NFKB1 in the rescue experiments. d NFKB1 re-expression promoted cell proliferation compared with miR-508-3p alone in MKN28 and SGC-7901 cells (*, P < 0.05; **, P < 0.001). The SDs were get by the 575 nm absorbance readings in 4 wells of each item. e NFKB1 re-expression counteracted proliferation-inhibition effect of miR-508-3p revealed by monolayer colony formation assays revealed (*, P < 0.05). The representative colony formation figures were shown in the bottom