Circular RNA hsa_circ_0007367 promotes the progression of pancreatic ductal adenocarcinoma by sponging miR-6820-3p and upregulating YAP1 expression

Abstract

Circular RNAs (circRNAs) play critical regulatory roles in cancer biological processes. Nevertheless, the contributions and underlying mechanisms of circRNAs to pancreatic ductal adenocarcinoma (PDAC) remain largely unexplored. Dysregulated circRNAs between cancerous tissues and matched adjacent normal tissues were identified by circRNA microarray in PDAC. The biological effect of hsa_circ_007367 both in vitro and in vivo was demonstrated by gain- and loss-of-function experiments. Further, dual-luciferase reporter and RNA pull-down assays were performed to confirm the interaction among hsa_circ_007367, miR-6820-3p, and Yes-associated protein 1 (YAP1). The expression of hsa_circ_007367 and YAP1 were detected by in situ hybridization (ISH) and immunohistochemistry (IHC) using tissue microarray (TMA) in 128 PDAC samples. We first identified that a novel circRNA, hsa_circ_0007367, was markedly upregulated in PDAC tissues and cells. Functionally, in vivo and in vitro data indicated that hsa_circ_0007367 promotes the proliferation and metastasis of PDAC. Mechanistically, we confirmed that hsa_circ_0007367 could facilitate the expression of YAP1, a well-known oncogene, by sponging miR-6820-3p, which function as a tumor suppresser in PDAC cells. The results of ISH and IHC demonstrated that hsa_circ_0007367 and YAP1 were upregulated in PDAC tissues. Furthermore, clinical data showed that higher hsa_circ_0007367 expression was correlated with advanced histological grade and lymph node metastasis in PDAC patients. In conclusion, our findings reveal that hsa_circ_0007367 acts as an oncogene via modulating miR-6820-3p/YAP1 axis to promote the progression of PDAC, and suggest that hsa_circ_0007367 may serve as a potential therapeutic target for treatment of PDAC.

Fig. 1. Identification and characterization of hsa_circ_0007367 in PDAC.

A Hierarchical clustering of differentially expressed circRNAs in 3 pairs of PDAC and their matched normal tissues. Red represents upregulated circRNAs, and green represents downregulated circRNAs. B Volcano plot of differentially expressed circRNAs, the −log10 (p-value) and the log2 (fold change) are plotted on the y and x axes, respectively. The red dots represent the differentially expressed circRNAs with statistical significance. C The scatter plot shows the dysregulated circRNAs in PDAC tissues compared with matched normal pancreas tissues. The space above the top green line and below the bottom green line indicated more than 1.5-fold change. D The heatmap for 15 differentially up- and downregulated circRNAs. E qRT-PCR analysis of the five dysregulated circRNAs in PANC-1 cells compared to HPDE cells (the normal pancreatic cell line). F, G qRT-PCR for the expression of hsa_circ_0007367 in PDAC cell lines and tissues. H Schematic illustration of the genomic location and splicing pattern of hsa_circ_0007367, with the splicing site identified by Sanger sequencing. I FISH assay showed the subcellular localization of hsa_circ_0007367 in PDAC cells. All data are shown as the mean ± SD of at least three independent experiments. *p < 0.05, **p < 0.01.

Fig. 2. Hsa_circ_0007367 promotes the proliferation of PDAC cells in vitro.

A–C The expression of hsa_circ_0007367 in PANC-1 and AsPC-1 cells was analyzed by qRT-PCR after treated with three siRNAs (A, B) and transfected with hsa_circ_0007367 plasmid (C). D–I Cell proliferation was detected by EdU (D, E) and CCK-8 (F, G) assays in PANC-1 and AsPC-1 cells by knockdown of hsa_circ_0007367, and forced expression hsa_circ_0007367 in PANC-1 cells (H, I). All data are shown as the mean ± SD of at least three independent experiments. *p < 0.05, **p < 0.01.

Fig. 3. Hsa_circ_0007367 promotes the migration and invasion of PDAC cells in vitro and knockdown of hsa_circ_0007367 inhibits PDAC cell growth in vivo.

A, B The migration was measured by the wound-healing assays in PANC-1 and AsPC-1 cells after knockdown of hsa_circ_0007367. C, D Transwell invasion assays were performed to verify the invasion capability after knocking down hsa_circ_0007367 in PANC-1 and AsPC-1 cells. E, F PANC-1 cells transfected with the hsa_circ_0007367 plasmid to detect the migration and invasion capability by wound-healing and transwell invasion assays. G Hsa_circ_0007367 knockdown efficiency was detected by qRT-PCR in PANC-1 after transfected with sh-hsa_circ_0007367 and sh-NC plasmid. H Representative image of subcutaneous xenograft tumors at day 28 after knockdown of hsa_circ_0007367 compared with sh-NC in PANC-1 cells. I, J Tumor volume and weight of xenograft after knockdown of hsa_circ_0007367 compared with sh-NC. All data are shown as the mean ± SD of at least three independent experiments. *p < 0.05, **p < 0.01.

Fig. 4. Hsa_circ_0007367 functions as a sponge for miR-6820-3p in PDAC.

A Schematic drawing showing potential target miRNAs of hsa_circ_0007367 predicted by starbase, circbank, and circular RNA interactome. B The relative expression of seven potential target miRNAs in PANC-1 and AsPC-1 cells lysates were detected by qRT-PCR. MiR-6820-3p was stably pulled down by hsa_circ_0007367. C Schematic illustration of the wild-type (WT) and mutant (mut) hsa_circ_0007367 luciferase plasmid. D The luciferase activities of the hsa_circ_0007367 luciferase reporter vector (WT or mut) in HEK293T cells transfected with miR-6820-3p mimics or mimic NC. E, F qRT-PCR detected the relative expression of miR-6820-3p in PDAC tissues (E) and in HPDE and PANC-1 cell lines (F). G The relative expression of hsa_circ_0007367 in PANC-1 cells was detected by qRT-PCR after hsa_circ_0007367 knocking down. H Correlation analysis between hsa_circ_0007367 and miR-6820-3p expression in PDAC tissues. All data are shown as the mean ± SD of at least three independent experiments. *p < 0.05, **p < 0.01.

Fig. 5. MiR-6820-3p inhibits the proliferation, migration, and invasion of PDAC cells.

A–D EdU (A, C) and CCK-8 B, D assay were used to evaluate the proliferation ability of the cells transfected with miR-6820-3p inhibitor or mimics. E–H Cell migratory and invasive capabilities were determined by wound-healing and transwell invasion assays in PANC-1 cells transfected with miR-6820-3p inhibitor or mimics. All data are shown as the mean ± SD of at least three independent experiments. *p < 0.05, **p < 0.01.

Fig. 6. MiR-6820-3p inhibitor reverses the effects of si-hsa_circ_0007367 on proliferation, migration, and invasion in PDAC cells.

A–D EdU (A), wound healing (B), CCK-8 (C), and transwell invasion D assays demonstrated that co-transfection with the miR-6820-3p inhibitor could reverse the proliferation, migration and invasion ability of PANC-1 cells after treated with si-hsa_circ_0007367. All data are shown as the mean ± SD of at least three independent experiments. *p < 0.05, **p < 0.01.

Fig. 7. YAP1 is a downstream target of miR-6820-3p and is indirectly regulated by hsa_circ_0007367.

A Venn diagram showing the potential target genes of miR-6820-3p predicted by miRDB, mirDIP, miRWalk, and TargetScan databases. B qRT-PCR analysis of four putative target genes mRNA level in cells transfected with si-hsa_circ_0007367. C, D YAP1 expression in PDAC cells was detected by qRT-PCR and western blot assays after transfection with the miR-6820-3p inhibitor. E Schematic illustrations showed the complementary sequence of miR-6820-3p with the 3′ UTR of YAP1. F The luciferase activities of the YAP1 3′ UTR reporter vector (WT or mut) in HEK293T cells transfected with miR-6820-3p mimic. G qRT-PCR analysis the expression of YAP1 in PDAC and adjacent tissues. H, I Correlation analysis between YAP1, hsa_circ_0007367, and miR-6820-3p expression in PDAC tissues. J Western blot analysis was performed to measure the protein levels of YAP1. β-actin was used as the loading control. All data are shown as the mean ± SD of at least three independent experiments. *p < 0.05, **p < 0.01.

Fig. 8. Upregulated hsa_circ_0007367 related with poor prognosis of PDAC patients.

A, B The numbers of hsa_circ_0007367 (A) and YAP1 B expression in TMAs. C, D Representative images showed the expression of hsa_circ_0007367 detected by ISH (C) and YAP1 detected by IHC (D) in TMAs. E The correlation between hsa_circ_0007367 and YAP1 expression in TMAs. F Kaplan–Meier survival curve for the overall survival of PDAC patients according to the relative expression of YAP1 in TMAs.

Fig. 9. Hsa_circ_0007367/miR-6820-3p/YAP1 axis promoted the progression of PDAC.

Hsa_circ_0007367 upregulated YAP1 expression via sponging miR-6820-3p to facilitate the proliferation, migration and invasion of PDAC cells.