The miR-155-5p/ FBXO11 axis inhibits the progression of gastric cancer via the mTOR pathway

Abstract

Background: Gastric cancer (GC) is a leading cause of cancer-related death. MicroRNAs (miRNAs or miRs) play a crucial role in the pathology of GC, including cell proliferation, invasion, and metastasis. In this study, genes targeted by miR-155-5p were predicted using bioinformatic tools. We found that the expression of miR-155-5p in GC cell lines differed relative to the expression of F-box protein 11 (FBXO11), which is involved in the regulation of cellular processes. This study sought to examine the function of miR-155-5p and the precise mechanism underlying its regulatory function in modulating proliferation and apoptosis in GC.

Methods: The luciferase reporter assay results showed that miR-155-5p bound directly to the three prime untranslated region (3'-UTR) of FBXO11, which further downregulated FBXO11 expression. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western-blot analyses confirmed that miR-155-5p negatively regulated the messenger RNA (mRNA) and protein expression of FBXO11. The effects of FBXO11 on cell proliferation and apoptosis in GC cell lines was further examined using Cell Counting Kit-8 (CCK-8) and flow cytometry.

Results: We found that FBXO11 promoted proliferation and decreased apoptosis in GC cells. Conversely, rescue experiments showed that the knockdown of FBXO11 limited the effects of miR-155-5p on the proliferation and apoptosis of GC cells, providing further evidence that FBXO11 is a functional target of miR-155-5p. Further, the overexpression of miR-155-5p inhibited cell growth via the targeted inhibition of FBXO11 that regulated mammalian target of rapamycin (mTOR) signaling pathway in the GC cells.

Conclusions: Overall, these results showed that miR-155-5p may serve as a tumor suppressor in GC and that the miR-155-5p/FBXO11 axis regulates tumor progression via the mTOR signaling pathway. Consequently, our findings may lead to the development a novel treatment strategy for GC.

Keywords: FBXO11; Gastric cancer (GC); mammalian target of rapamycin pathway (mTOR pathway); miR-155-5p; proliferation.

Figure 1

MiR-155-5p and FBXO11 expression in GC cells. (A) qRT-PCR showed miR-155-5p mRNA expression in GES-1 and GC. (B) qRT-PCR showed FBXO11 mRNA expression in GES-1 and GC. (C) Western blot showed the level of expression of FBXO11 in GC (HGC27, SGC7901, AGS, and MGC803) and GES-1. ***, P<0.001; ns, not significant. Every experiment was conducted at least three times. GC, gastric cancer; GES, gastric epithelial cells; qRT-PCR, quantitative reverse transcription-polymerase chain reaction.

Figure 2

Target genes of miR-155-5p GO and pathway enrichment analysis. (A) VennPainter intersected the target genes of miR-155-5p from four prediction databases (i.e., diana-microt, mirtarbase, mianda, and miranda). (B) R package cluster Profiler showed the top 10 GO terms enriched in this category based on the P values, Count: represents how many genes are enriched in each term. (C) R package cluster Profiler showed the top 10 KEGG terms enriched in this category based on the P values; Count: represents the quantity of enriched genes in each term. (D) String diagram for the KEGG analysis: KEGG pathway enrichment analysis of miR-155-5p target genes. BP, biological process; CC, cell component; MF, molecular function; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes.

Figure 3

MiR-155-5p directly targets FBXO11 in GC cells. (A) Seed matched outcomes of the 3'UTR of FBXO11 mRNA and miR-155-5p. FBXO11 mRNA (MUT-FBXO11) showed a 3'UTR mutation in the seed-matching sequence. (B) In the luciferase assay experiments, the HEK293T cells were co-transfected with 1.5 µg of recombinant plasmid (mutant or wild FBXO11-3'UTR) and 50 nM of miRNA mimics (miR-155-5p or NC) in each well. Luciferase activity was recorded 48 h post-transfection. (C) qRT-PCR showed the mRNA expression of FBXO11 after transfecting miR-155-5p mimics in the AGS and MGC803 cells. (D) Western blot was used to detect the protein expression of FBXO11 after the transfection of miR-155-5p mimics in the AGS and MGC803 cells. (E) qRT-PCR was performed to examine the mRNA expression of FBXO11 after the miR-155-5p inhibitor was transfected in the GES-1 cells. (F) Western blot was used to detect the expression of FBXO11 after the miR-155-5p inhibitor was transfected in the GES-1 cells. *, P<0.05; **, P<0.01. Every experiment was conducted at least three times. GC, gastric cancer; GES, gastric epithelial cells; MUT, mutant; NC, negative control; qRT-PCR, quantitative reverse transcription-polymerase chain reaction; WT, wild type.

Figure 4

The inhibitory effect of miR-155-5p on the proliferation and apoptosis of GC cells was reversed by FBXO11. (A) The protein expression of FBXO11 was decreased in the MGC803 cells after transfection with the miR-155-5p inhibitor. Conversely, an increase in the expression was observed after transfection with the si-FBXO11 and miR-155-5p inhibitor. (B) CCK-8 demonstrated that the cellular proliferation rate was decreased in the MGC803 cells after miR-155-5p inhibitor transfection, but increased after the transfection with the miR-155-5p inhibitor + si-FBXO11. (C) Flow cytometry analysis showed that transfection with the miR-155-5p inhibitor increased the rate of apoptosis in MGC803 cells. Conversely, transfection with si-FBXO11 and miR-155-5p inhibitor magnified this rate. *, P<0.05; **, P<0.01; ns, not significant. CCK-8, Cell Counting Kit-8; FITC, Fluorescein Isothiocyanate; GC, gastric cancer; NC, negative control; PI, Propidium Iodide.

Figure 5

miR-155-5p/FBXO11 axis affected proliferation and apoptosis via mTORC1 signaling pathways in GC cells. (A) Western blot was used to detect the related protein expression of the mTORC1 pathway after (respectively or common) transfected si-FBXO11 and MHY1485 in MGC803 cells. (B) CCK-8 assay revealed that the rate of cell proliferation after (respectively or common transfected) si-FBXO11 and MHY1485 in MGC803 cells. (C) Flow cytometry analysis revealed cell apoptosis after (respectively or common) transfected si-FBXO11 and MHY1485 in MGC803 cells. *, P<0.05; **, P<0.01; ns, not significant. Every experiment was conducted at least three times. CCK-8, Cell Counting Kit-8; FITC, Fluorescein Isothiocyanate; GC, gastric cancer; mTORC1, mammalian target of rapamycin complex 1; NC, negative control; PI, Propidium Iodide.