Figure 3.

circ_0020123 positively regulates PDZD8 expression in LUAD tissues and cells. (a) circ_0020123 expression was upregulated in LUAD tissues as detected by RT-qPCR. **p < 0.01. (b) circ_0020123 showed high expression in LUAD cells as shown by RT-qPCR. ***p < 0.001 vs BEAS-2B group. (c) A positive expression correlation between circ_0020123 and PDZD8 in LUAD tissues was identified using Spearman correlation coefficient. R ² = 0.405, *p < 0.05. (d) circ_0020123 expression was successfully knocked down in LUAD cells as shown by RT-qPCR. ***p < 0.001 vs sh-NC group. (e) circ_0020123 knockdown reduced PDZD8 expression at both the mRNA and protein levels. ***p < 0.001 vs sh-NC group. (f) RT-qPCR was performed to detect the efficiency of circ_0020123 overexpression. ***p < 0.001 vs pcDNA3.1 group. (g) The influence of overexpressed circ_0020123 on PDZD8 mRNA expression and protein levels in LUAD cells was quantified by RT-qPCR and western blot analyses. ***p < 0.001 vs pcDNA3.1 group. (h) The effect of PDZD8 knockdown on circ_0020123 expression in LUAD cells were examined by RT-qPCR. (i) Luciferase reporter assays were conducted to explore the effect of circ_0020123 on the transcription level of PDZD8. (j) RIP assays implied that circ_0020123 and PDZD8 were enriched in RNA-induced silencing complex. ***p < 0.001 vs Anti-IgG group. (k) The expression levels of circ_0020123 and linear PDZD8 in A549 and H1975 cells after RNase R treatment were determined by RT-qPCR analysis. ***p < 0.001 vs Mock group. (l) Agarose gel electrophoresis showed that circ_0020123 can be amplified by divergent primers using total cDNA, but not genomic DNA (gDNA). Pair triangles indicate the directionality of primers. The data are presented as the mean value ± standard deviation. Student’s t test was used to compare differences between LUAD tissues and normal tissues. One-way analysis of variance followed by Tukey’s post hoc analysis was used to compare differences among groups.