Figures & data
Figure 1. The higher expression of circCTNNB1 was discovered in lung cancer.
(a) The mRNA expression of circCTNNB1 was examined in lung cancer tissues (n = 40) and normal tissues (n = 40) through RT-qPCR. (b) The prognosis of lung cancer patients with higher (n = 20) or lower (n = 20) circCTNNB1 expression was verified. (c) The mRNA expression of circCTNNB1 was determined in human bronchial epithelial cell line (BEAS-2B) and lung cancer cell lines (H1299, A549, PC9, and H1975) through RT-qPCR. (d) The relative abundance of circCTNNB1 and CTNNB1 was evaluated after RNase R treatment through RT-qPCR. (e) The relative abundance of circCTNNB1 and CTNNB1 was assessed after Actinomycin D treatment through RT-qPCR. (F) The location of circCTNNB1 in cytoplasm or nuclear was verified through RT-qPCR. ***p < 0.001.
Figure 2. CircCTNNB1 facilitated lung cancer cell proliferation, migration, invasion, and suppressed cell senescence.
(a) The knockdown efficiency of circCTNNB1 was confirmed through RT-qPCR in the sh-NC, sh-circCTNNB1#1, sh-circCTNNB1#2, and sh-circCTNNB1#3 groups. (b) The cell proliferation was tested through CCK-8 assay in the sh-NC and sh-circCTNNB1 groups. (c-d) The cell migration and invasion were examined through Transwell assay in the sh-NC and sh-circCTNNB1 groups. (e) The cell senescence was evaluated through SA-β-gal assay in the sh-NC and sh-circCTNNB1 groups. ***p < 0.001.
Figure 3. Knockdown of circCTNNB1 retarded the Wnt pathway.
(a) The nuclear translocation of β-catenin was notarized through IF assay in the sh-NC and sh-circCTNNB1 groups. (b) The protein expressions of WNT3A, β-catenin, and c-myc were determined through western blot in the sh-NC and sh-circCTNNB1 groups. ***p < 0.001.
Figure 4. CircCTNNB1 combined with miR-186-5p to target YY1.
(a) The binding sites between circCTNNB1 and miR-186-5p (YY1) were displayed. (b-c) The binding ability between circCTNNB1 and miR-186-5p (YY1) was confirmed through luciferase reporter and RIP assays. (d) The expressions of miR-186-5p and YY1 were detected through RT-qPCR. (e) The protein expression of YY1 was assessed after circCTNNB1 knockdown or miR-186-5p mimic through western blot. (f) The expressions of miR-186-5p and YY1 were confirmed in lung cancer tissues and normal tissues through RT-qPCR. (g) The expressions of miR-186-5p and YY1 were tested in lung cancer cell lines and normal bronchial epithelial cell line (BEAS-2B) through RT-qPCR. ***p < 0.001.
Figure 5. YY1 overexpression can rescue the decreased cell proliferation, migration, invasion, and the increased cell senescence mediated by circCTNNB1 suppression.
Groups were divided into the sh-NC, sh-circCTNNB1, and sh-circCTNNB1+pcDNA3.1/YY1 groups. (a) The cell proliferation was measured through a CCK-8 assay. (b-c) The cell migration and invasion were assessed through Transwell assay. (d) The cell senescence was confirmed through SA-β-gal assay. ***p < 0.001 vs the sh-NC group; ###p < 0.001 vs the sh-circCTNNB1.
Figure 6. YY1 overexpression can rescue the retarded Wnt pathway mediated by circCTNNB1 knockdown.
Groups were divided into the sh-NC, sh-circCTNNB1, and sh-circCTNNB1+pcDNA3.1/YY1 groups. (a) The nuclear translocation of β-catenin was examined through IF assay. (b) The protein expressions of WNT3A, β-catenin, and c-myc were determined through western blot. **p < 0.01, ***p < 0.001 vs the sh-NC group; ##p < 0.01, ###p < 0.001 vs the sh-circCTNNB1.
Figure 7. YY1 can transcriptionally activate circCTNNB1 to form YY1/circCTNNB1/miR-186-5p/YY1 loop.
(a) The correlation between circCTNNB1 and YY1 was verified. (b) The expression of circCTNNB1 was tested after YY1 knockdown through RT-qPCR. (b) The binding site of YY1 was displayed. (d-e) The binding ability between YY1 and the promoter of circCTNNB1 was measured through RIP and luciferase reporter assays. ***p < 0.001.
Figure 8. CircCTNNB1 accelerated tumour growth in vivo.
(a-c) The tumour size, volume, and weight were evaluated in the sh-NC and sh-circCTNNB1 groups. (d) The Ki67 expression was examined through IHC assay in the sh-NC and sh-circCTNNB1 groups. ***p < 0.001.
Supplemental material
Supplemental Material
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The data that support the findings of this study are available from the corresponding author.