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. 2022 Nov 24;22(1):443.
doi: 10.1186/s12890-022-02209-2.

CircRNA has_circ_0017109 promotes lung tumor progression via activation of Wnt/β-catenin signaling due to modulating miR-671-5p/FZD4 axis

Bo Yang #  1   2   3 , Bin Zhang #  1   2   3 , Qi Qi #  1   2   3 , Changli Wang  4   5   6
Affiliations

CircRNA has_circ_0017109 promotes lung tumor progression via activation of Wnt/β-catenin signaling due to modulating miR-671-5p/FZD4 axis

Bo Yang et al. BMC Pulm Med. .

Abstract

Introduction: Accumulating evidence highlights the critical roles of circular RNAs (circRNAs) in the malignant progression of cancers. In this study, we investigated the expression pattern of a newly identified circRNA (hsa_circ_0017109) in non-small cell lung cancer (NSCLC), and examined its downstream molecular targets.

Methods: Quantitative real-time PCR (qRT-PCR) and Western blotting (WB) were conducted to quantify gene and protein expression. In vitro functional assays such as colony formation assay, cell counting kit-8 (CCK-8) and flow cytometry were used to study cell proliferation and apoptosis. RNA pull-down assay, luciferase reporter assay and RNA immunoprecipitation were performed to validate molecular interaction. Mouse xenograft model of NSCLC cells was used to assess the role of circ_0017109 in tumorigenesis.

Results: Circ_0017109 was upregulated in NSCLC tumor samples and cells. Silencing circ_0017109 impaired cell proliferation and promoted apoptosis in NSCLC cells, and circ_0017109 knockdown suppressed in vivo tumorigenesis of NSCLC cells in mouse xenograft model. MiR-671-5p was identified as a target of circ_0017109, and circ_0017109 negatively impacted on miR-671-5p expression. MiR-671-5p downregulated FZD4 and dampened the activity of Wnt/β-catenin signaling pathway. Circ_0017109 modulated FZD4 expression by suppressing miR-671-5p activity.

Conclusions: Elevated circ_0017109 expression promotes tumor progression of NSCLC by modulating miR-671-5p/FZD4/β-catenin axis.

Keywords: FZD4; Lung cancer; NSCLC; Wnt/β-catenin; circRNA; miRNA.

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Conflict of interest statement

All authors report no conflicts of interest.

Figures

Fig. 1
Fig. 1
Circ_0017109 is upregulated in NSCLC and associated with poor prognosis in NSCLC patients. A Expression of circ_0017109 in normal lung tissues and NSCLC tumor samples of GSE101586 and GSE112214 (GEO). B Circ_0017109 levels within 50 pairs of NSCLC samples and matched non-carcinoma samples. C Circ_0017109 levels within NSCLC cells, (A549, CALU3, H1229 and CALU6) and human normal bronchial epithelial cells (HBE). D GAPDH and circ_0017109 levels in the RNA samples from H1229 and A549 cell lines with or without RNase R treatment. E RNA levels of circ_0017109, U6 (nuclear control), and GAPDH (cytoplasmic control) in the cytoplasmic and nuclear fraction of H1229 and A549 cells. F The association of NSCLC patient overall survival and circ_0017109 expression level. *, P < 0.05, and ***, P < 0.001
Fig. 2
Fig. 2
Circ_0017109 promotes cell growth and inhibits apoptosis of NSCLC cells. A Circ_0017109 expression within H1299 and A549 cells after the transfection of si-circ_0017109 or si-NC. B CCK-8 assay in H1299 and A549 cells after the transfection of si-circ_0017109 or si-NC. C Colony formation assay in H1299 and A549 cells after the transfection of si-circ_0017109 or si-NC. D Apoptosis detection in H1299 and A549 cells after the transfection of si-circ_0017109 or si-NC. E Protein levels of cleaved-caspase-3 and Bcl-2 in H1299 and A549 cells after the transfection of si-circ_0017109 or si-NC. **, P < 0.01, and ***, P < 0.001
Fig. 3
Fig. 3
MiR-671-5p is regulated by circ_0017109. A Predicted miR-671-5p target sequences in circ_0017109 sequence based on CircInteractome database. Luciferase activity of wide-type or mutated circ_0017109 reporter was determined in H293K cells in the presence of miR-NC or miR-671-5p mimic. B RNA-pull down assay of circ_0017109 enrichment using biotinylated miR-671-5p or biotin-NC. C RNA-immunoprecipitation assay using anti-AGO2 antibody or nonspecific rabbit IgG. ***, P < 0.001
Fig. 4
Fig. 4
Circ_0017109 promotes FZD4 expression via sponging miR-671-5p. A miR-671-5p target sequence in the FZD4 3′-UTR was predicted using Targetscan database. B Luciferase activity of wild type or mutated FZD4 reporter in the presence of miR-NC or miR-671-5p mimic. C FZD4 mRNA and protein expression in H1229 and A549 cells after the transfection of miR-671-5p mimic. D The effect of circ_0017109 silencing and miR-671-5p inhibitor on FZD4 protein level. E Relative FZD4 expression in NSCLC tumor and normal lung tissue samples. F Relative miR-671-5p levels in NSCLC tumor and normal lung tissue samples. G Correlation of FZD4 expression and miR-671-5p in NSCLC tumor samples. H FZD4 level and circ_0017109 expression in NSCLC tumor samples. ***, P < 0.001 compared with si-NC group, and ^^^, P < 0.001 compared with si-circ_0017109 group
Fig. 5
Fig. 5
Circ_0017109 modulates miR-671-5p/FZD4 axis and Wnt/β-catenin pathway. A FZD4 overexpression in H1229 and A549 cells upon the transfection of FZD4 expression vector. B CCK-8 assay in A549 and H1299 cells transfected with si-circ_0017109, si-NC, si-circ_0017109 + miR-671-5p inhibitor, or si-circ_0017109 + FZD4. C Colony forming assays in A549 and H1299 cells transfected with si-circ_0017109, si-NC, si-circ_0017109 + miR-671-5p inhibitor, or si-circ_0017109 + FZD4. D Apoptosis in A549 and H1299 cells transfected with si-circ_0017109, si-NC, si-circ_0017109 + miR-671-5p inhibitor, or si-circ_0017109 + FZD4. E Cleaved-caspase-3 and Bcl-2 levels within H1229 and A549 cells post-transfection. F Protein levels of FZD4, Bcl-2, β-catenin, non-phospho β-catenin, c-myc, cyclin D1, and cleaved caspase-3 in A549 and H1299 cells transfected with si-circ_0017109, si-NC, si-circ_0017109 + miR-671-5p inhibitor, or si-circ_0017109 + FZD4. **, P < 0.01, ***, P < 0.001 compared with si-NC group, ^, P < 0.05, and ^^^, P < 0.001 compared with si-circ_0017109 group
Fig. 6
Fig. 6
Xenograft tumorigenesis assay in nude mice. A Subcutaneous xenograft volumes and B weights were measured in mice injected with A549 cells stably expressing sh-NC and shRNA targeting circ_0017109. C Protein levels of cleaved caspase-3, FZD4, β-catenin, non-phospho β-catenin, Bcl-2, c-myc and cyclin D1 in xenograft tumor tissues with sh-NC or shRNA targeting circ_0017109. ***, P < 0.001
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