Figures & data
Table 1. Association of circ-PGC expression with clinicopathological characteristics of patients with NSCLC
Figure 1. The expression of circ-PGC and FOXR2 was elevated in NSCLC tissues and cells
(a) The architecture and pathological characteristics of NSCLC tumor tissues and normal tissues were distinguished using HE staining. (b) The expression of circ-PGC in tumor tissues and normal tissues were detected by RT-qPCR. (c) The association between circ-PGC expression and overall survival of patients. (d) The expression of FOXR2 mRNA in tumor tissues and normal tissues were detected by RT-qPCR. (e) The correlation between circ-PGC expression and FOXR2 expression in tumor tissues was analyzed using Pearson analysis. (f and g) The expression of circ-PGC and FOXR2 mRNA in NSCLC cells (H460 and PC9) and BEAS-2B cells was detected by RT-qPCR. (h and i) The expression of FOXR2 protein in tissues (tumor and normal) and cell lines (H460, PC9 and BEAS-2B) was detected by western blot. (j and k) The effect of RNase R on the expression of circ-PGC (circular) and PGC (linear) was checked using RT-qPCR. (l) The distribution of circ-PGC in cytoplasmic fraction and nuclear fraction was determined. *P < 0.05, **P < 0.01.
Figure 2. Circ-PGC knockdown suppressed H460 and PC9 cell malignant phenotypes
(a) The efficiency of si-circ-PGC#1 and si-circ-PGC#2 was checked using RT-qPCR. (b) The viability of H460 and PC9 cells after circ-PGC knockdown was examined by CCK-8 assay. (c) The ability of colony formation in H460 and PC9 cells after circ-PGC knockdown was examined by colony formation assay. (d) The apoptotic effect of H460 and PC9 cells after circ-PGC knockdown was examined by flow cytometry assay. (e) Cell migration was monitored in H460 and PC9 cells after circ-PGC knockdown by wound healing assay. (f) Cell invasion was monitored in H460 and PC9 cells after circ-PGC knockdown by transwell assay. (g) The levels of glucose consumption, lactate production and ATP production were measured to assess glycolysis using commercial kits. (h) The protein levels of HK-2, MMP9 and E-cad were quantified by western blot. *P < 0.05, **P < 0.01.
Figure 3. FOXR2 knockdown inhibited H460 and PC9 cell malignant phenotypes
(a and b) The efficiency of si-FOXR2 was checked using RT-qPCR and western blot. In si-FOXR2-transfected H460 and PC9 cells, (c) cell viability, (d) colony formation ability, (e) cell apoptosis, (f) cell migration and (g) cell invasion were assessed using CCK-8 assay, colony formation assay, flow cytometry assay, wound healing assay and transwell assay, respectively. (h-j) Glycolysis metabolism was monitored according to the levels of glucose consumption, lactate production and ATP production. (k) The expression levels of HK-2, MMP9 and E-cad were measured using western blot. *P < 0.05, **P< 0.01.
Figure 4. FOXR2 overexpression rescued the inhibitory effects on H460 and PC9 cell malignant phenotypes caused by circ-PGC knockdown
(a and b) The efficiency of pcDNA-FOXR2 was checked by RT-qPCR and western blot. In H460 and PC9 cells transfected with si-circ-PGC, si-NC, si-circ-PGC+pcDNA-FOXR2 or si-circ-PGC+pcDNA, (c) cell viability, (d) colony formation ability, (e) cell apoptosis, (f) cell migration and (g) cell invasion were monitored using CCK-8 assay, colony formation assay, flow cytometry assay, wound healing assay and transwell assay, respectively. (h-j) Glycolysis metabolism was evaluated by the levels of glucose consumption, lactate production and ATP production. (k) The protein levels of HK-2, MMP9 and E-cad in these transfected cells were quantified by western blot. *P < 0.05, **P < 0.01.
Figure 5. MiR-532-3p was a target of circ-PGC, and miR-532-3p bound to FOXR2
(a) The potential relationship between circ-PGC and miR-532-3p was predicted using Circular RNA interactome. (b) The relationship between miR-532-3p and FOXR2 was predicted using Target Scan Human7.2. (c and d) The predicted relationship between miR-532-3p and circ-PGC or FOXR2 was verified by dual-luciferase reporter assay. (e and f) The relationship between miR-532-3p and circ-PGC or FOXR2 was also verified by RIP assay. (g) The expression of miR-532-3p in tissues (tumor and normal; n = 33) and cell lines (H460, PC9 and BEAS-2B) was detected by RT-qPCR. (h) The correlation between miR-532-3p expression and circ-PGC expression or FOXR2 expression in tumor tissues was analyzed using Pearson analysis. *P < 0.05, **P< 0.01.
Figure 6. Circ-PGC positively regulated FOXR2 expression by targeting miR-532-3p
(a and b) The expression of FOXR2 at the protein level in H460 and PC9 cells transfected with si-circ-PGC, si-NC, si-circ-PGC+anti-miR-532-3p or si-circ-PGC+anti-miR-NC was measured using western blot. *P < 0.05, **P < 0.01.
Figure 7. Wnt/β-catenin signaling pathway might be involved in the circ-PGC/miR-532-3p/FOXR2 regulatory network
(a and b) The expression levels of β-catenin and c-Myc in H460 and PC9 cells with the transfection of si-circ-PGC, si-NC, si-circ-PGC+anti-miR-532-3p, si-circ-PGC+anti-miR-NC, si-circ-PGC+pcDNA-FOXR2 or si-circ-PGC+pcDNA were determined using western blot. **P < 0.01.
Figure 8. Circ-PGC inhibited tumor growth in vivo.
(a) The representative tumor images from the sh-circ-PGC group and sh-NC group. (b) Tumor volume, and (c) tumor weight were measured to monitor tumor growth. (d) The expression of circ-PGC, miR-532-3p and FOXR2 mRNA in excised tumor tissues was detected by RT-qPCR. (e) The expression of FOXR2 protein was detected by western blot. (f) The protein levels of MMP9, c-Myc, β-catenin and E-cad in tumor tissues were detected by western blot. *P < 0.05, **P < 0.01.
Availability of data and materials
The analyzed data sets generated during the present study are available from the corresponding author on reasonable request.