Figures & data
Figure 1. The circFAM53B level in glioma.
(a). qRT-PCR tested the circFAM53B profile in glioma tissues and adjacent non-tumor tissues, *** P< 0.001. (b). qRT-PCR examined circFAM53B expression in normal cell lines HEB and glioma cell lines (U251, A172, TJ861, TJ905, and LN18), NS represents P> 0.05, *** represents P< 0.001 vs. the HEB group. (c). The survival of glioma patients with high or low levels of circFAM53B was compared by Kaplan-Meier plotter assay. N = 3.
Figure 2. Up-regulation of circFAM53B intensified the malignant behaviors of glioma cells.
(a). CircFAM53B overexpression models were conducted in A172 and LN18 cells, and the circFAM53B profile was examined by qRT-PCR. (b). CCK-8 evaluated the influence of overexpressing circFAM53B on cell proliferation. (c). The colony formation assay verified cell colony formation ability. d. Western blot monitored the profiles of Bax and Bcl2 in A172 and LN18 cells. E. A172 and LN18 cell invasion was tested by Transwell assay. F. Western blot tested E-cadherin and Vimentin expression in A172 and LN18 cells. NS represents P> 0.05, ** represents P< 0.01, *** represents P< 0.001. N = 3.
Figure 3. Influence of CircFAM53B knockdown on cell proliferation, invasion, EMT and apoptosis.
(a): A circFAM53B knockdown model was established in A172 and LN18 cells. qRT-PCR gauged the circFAM53B expression. (b): CCK-8 assay evaluated the impact of circFAM53B knockdown on cell proliferation. (c): The colony formation assay checked cell colony formation. (d): Western blot tested the expression of apoptosis-related proteins (Bax and Bcl2) in A172 and LN18 cells. (e): Transwell assay was conducted for evaluating cell invasion. (f): Western blot was used for detecting EMT-related proteins (E-cadherin and Vimentin) in A172 and LN18 cells. N = 3, *P< 0.05, **P< 0.01, ***P< 0.001 (vs.si-NC group).
Figure 4. CircFAM53B boosted A172 cell proliferation and EMT in vivo.
A172 cells up-regulating circFAM53B were applied for in-vivo experiments. (a). Tumor volumes in nude mice were measured every five days. (b). Tumor images isolated from nude mice. (c). The tumor was weighed. (d). IHC was employed to examine Ki67 (a proliferation marker) in tumor tissues. E. The profiles of Bcl2 and Bax in tumor tissues were examined by Western blot. F. IHC gauged E-cadherin and Vimentin expression in tumor tissues. *** represents P< 0.001. N = 5.
Figure 5. miR-28-5p was a sponge RNA of circFAM53B.
a. Starbase and circInteractome searched potential target genes of circFAM53B. Venn’s diagram was applied to analyze common miRNAs. b. qRT-PCR determined miR-532-3p expression in glioma tissues and adjacent non-tumor tissues. ***P< 0.001 (vs.Normal group). c. Pearson linear regression was adopted to clarify the association between circFAM53B and miR-532-3p in glioma tissues. d. miR-532-3p expression in HEB, U251, A172, TJ861, TJ905, and LN18 was compared by Western blot. * represents P< 0.05, ** represents P< 0.01, *** represents P< 0.001 vs. HEB group. E. Dual-luciferase reporter assay affirmed the binding correlation between miR-532-3p and circFAM53B in A172 cells. F. Dual-luciferase reporter gene assay was conducted to confirm the binding association between miR-532-3p and circFAM53B. G. RIP assay verified the binding association between miR-532-3p and circFAM53B in A172 cells, and the enrichment of miR-532-3p and circFAM53B was determined by qRT-PCR. H. The miR-532-3p profiles in A172 and LN18 cells overexpressing circFAM53B were examined by qRT-PCR. I: qRT-PCR monitored the miR-532-3p expression after knocking down circFAM53B. *** represents P< 0.001. N = 3.
Figure 6. Impacts of circFAM53B/miR-532-3p on glioma cell proliferation, apoptosis and metastasis.
Figure 7. CircFAM53B/miR-532-3p modulated the MAPK/ERK pathway.
a. miRPath v.3 revealed that four pathways were regulated by miR-532-3p. B. Through miRanda and microT databases, the potential downstream targets of miR-532-3p were analyzed. qRT-PCR illustrated that MET and FZD6 were targets of miR-532-3p. NS represents P> 0.05, *** represents P< 0.001 vs.miR-NC group. N = 3. C. The binding sites between MET and miR-532-3p were exhibited. d. Dual-luciferase reporter gene assay was conducted to confirm the binding association between miR-532-3p and MET. NS represents P> 0.05, *** represents P< 0.001 vs.miR-NC group. N = 3. e-g: Western blot assessed the expression of the c-MET/PI3K/AKT pathway following up- or down-regulation of circFAM53B and miR-532-3p. H-J: Pearson’s correlation analyzed the correlations between circFAM53B and MET (H), circFAM53B and PIK3CA (I), circFAM53B and AKT1 (J) expressions in 40 glioma tissues. k-m: Pearson’s correlation analyzed the correlations between miR-532-3p and MET (K), miR-532-3p and PIK3CA (L), miR-532-3p and AKT1 (M) expression in 40 glioma tissues.
Figure 8. Impacts of inactivating the c-MET/PI3K/AKT pathway expression on the oncogenic effect of circFAM53B.
The MET inhibitor SGX523 (4 nM, Cat. No. S1112, Selleck), the PI3K inhibitor LY294002 (1 µM, Cat. No. S1105, Selleck), and the AKT inhibitor MK-2206 2HCL (5 µM, Cat. No. S1078, Selleck) into A172 cells stably transfected with circFAM53B overexpression plasmids. A: qRT-PCR detected circFAM53B expression. B: CCK8 tested cell viability. C: The colony formation assay was employed to measure the colony-forming ability of the cells. D: Transwell assay testified cell invasion. E-F: Western blot monitored the expression of apoptosis-related proteins (Bax and Bcl2) and EMT-related proteins (E-cadherin and Vimentin). G: Western blot evaluated MET, PI3K and AKT expression. ***P< 0.001 (vs.Vector); NSP>0.05, &&&P< 0.001 (vs. circFAM53B group). N = 3.
Figure 9. The mechanism diagram of circFAM53B in glioma.
circFAM53B boosts cell proliferation, invasion and EMT, and inhibits apoptosis. circFAM53B facilitates the activation of the c-MET/PI3K/AKT pathway through the inhibition of miR-532-3p.
Data availability statement
The data sets used and analyzed during the current study are available from the corresponding author on reasonable request.