CircATP13A1 (hsa_circ_0000919) promotes cell proliferation and metastasis and inhibits cell apoptosis in pancreatic ductal adenocarcinoma via the miR-186/miR-326/HMGA2 axis: implications for novel therapeutic targets

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is a notoriously aggressive malignancy with a survival rate of merely 9%. The prognosis in patients with PDAC is relatively poor, particularly in patients with advanced distant metastases. However, the mechanisms of PDAC progression remain elusive. Circular RNAs (circRNAs) have been implicated in the development of various malignancies, including PDAC. Therefore, this study aimed to investigate how a novel circRNA, circATP13A1, regulates PDAC progression. We used the GEO database to determine circATP13A1 expression levels in cancer and adjacent cells and employed the limma package of R software to identify differentially expressed circRNAs. We detected the expression of circATP13A1, miR-186, and miR-326 using qRT-PCR and investigated the effect of circATP13A1 on cell proliferation, migration, invasion, and apoptosis in vitro using the Cell Counting Kit-8 (CCK-8), the transwell migration assay, and the flow cytometry assay. We then performed RNA pull-down assay, RNA immunoprecipitation (RIP), and Western blot to verify the interaction between circATP13A1, miR-186, miR-326, and HMGA2. Moreover, we used a naked mice model to determine how circATP13A1 affects tumor growth and progression in vivo. Loss and gain of function analyses revealed that circATP13A1 upregulation promotes cell proliferation, migration, invasion and tumor growth both in vitro and in vivo, which results in PDAC progression and poor prognosis in patients. CircATP13A1 knockdown significantly impaired cell proliferation and migration of PDAC cell lines. Additionally, circATP13A1 knockdown significantly increased the expression of miR-186 and miR-326, while reducing the expression of HMGA2 (P < 0.05), indicating that miR-186 and miR-326 are downstream targets of circATP13A1. Rescue experiments support the interactions between circATP13A1, miR-186, miR-326, and HMGA2. In conclusion, we demonstrated that circATP13A1 sponges the miR-186/miR-326/HMGA2/axis, acting as an oncogene to promote PDAC development.

Keywords: CircATP13A1; HMGA2; PDAC; miR-186; miR-326.

Figure 1

CircATP13A1 is highly expressed in PDAC tissues and cell lines. A. The volcano plot shows the differential expression of circRNAs in PDAC tissues of cancer patients. B. The Limma R package reveals the differential expression of circRNAs in normal and tumor tissues. Circ-ATP13A1 was significantly upregulated in PDAC cancer tissues relative to the adjacent normal tissues of 70 PDAC cancer patients downloaded from the GEO database. C. The schematic diagram displays the pre-mRNA structure of ATP13A1. D. QRT-PCR analysis indicates the significant upregulation of circATP13A1 in PDAC tissues compared to adjacent normal tissues of PDAC. E. Seventy patients with PDAC were grouped based on circATP13A1 expression levels (high vs low). High circATP13A1 expression was generally associated with poor survival prognosis. F. Expression of circATP13A1 in different PDAC cell lines, including HPDE6c7, AsPC-1, CFPAC-1, BxPC-3, SW1990, and Panc-1. G. Gel electrophoresis shows the amplification of circATP13A1 and ATP13A1 by divergent and convergent primers in SW1990 cells. H. RNase R treatment effectively degrades linear ATP13A1, while RNase is ineffective in degrading circATP13A1 in the SW1990 cell line. I. Actinomycin D significantly reduces the expression of linear ATP13A1 in a time-dependent manner but cannot affect circATP13A1 expression in the SW1990 cell line.

Figure 2

CircATP13A1 promotes proliferation, metastasis, and anti-apoptosis of PDAC cell lines in vitro. A. QRT-PCR shows that si-circ1 and si-circ2 effectively knock down circATP13A1 expression in the SW1990 cell line and oe-circ increases circATP13A1 expression in the BxPC-3 cell line. B. Knockdown of circATP13A1 by the two siRNAs (si-circ1 and si-circ2) significantly reduces cell viability in the SW1990 cell line compared with si-NC, and overexpressing circATP13A1 significantly increases cell viability in the BxPC-3 cell line. C. Knockdown of circATP13A1 by si-circ1 and si-circ2 reduces colony formation in SW1990, while overexpression circATP13A1 by oe-circ significantly increases the colony-forming ability of PDAC. D. Knockdown of circATP13A1 effectively reduces the invasion ability of SW1990 cells compared with si-NC, and the invasion ability of BxPC-3 cells was significantly increased. E. Flow cytometry reveals that cell apoptosis is significantly enhanced in SW1990 cell lines transfected with the two siRNAs, whereas cell apoptosis is significantly inhibited in BxPC-3 cells overexpressing circATP13A1 (oe-circ).

Figure 3

CircATP13A1 sponges miR-186 and miR-326 in PDAC cell lines. A. Circular interactome analyses predicted that circATP13A1 can target and bind to miR-186 and miR-326. B. RNA pull-down experiments in SW1990 and BxPC-3 cell lines demonstrated that circATP13A1 efficiently enriched miR-186 and miR-326 compared to other miRNAs and the control probe (ctrl). C. Constructed sequences of wild-type circATP13A1, mutant circATP13A1, miR-186, and miR-326. D. Luciferase reporter assay indicated a significant increase in the fluorescent intensity of the mutant circATP13A1 sequence compared with the wild-type circATP13A1 in the SW1990 and BxPC-3 cell lines. E. QRT-PCR showed that miR-186 and miR-326 were remarkably upregulated when circATP13A1 was knocked down compared with the vector and normal control (Si-NC) in the SW1990 and BxPC-3 cell lines. F. QRT-PCR showed the expression of miR-186 and miR-326 in cancer and adjacent tissues of the 70 PDAC patients.

Figure 4

HMGA2 is a common target of MiR-186 and MiR-326. A. The construction of miR-186, miR-326, the wild-type HMGA2 sequence, and the mutated HMGA2 sequence. B. Luciferase activity in the wild-type miR-186 and miR-326 sequences was significantly reduced compared to miR-NC mimics in the SW1990 and BxPC-3 cell lines but did not change in the mutated miR-186 and miR-326 sequences compared with the wild-type miR-186 and miR-326 sequences in SW1990 and BxPC-3 cells. C. The RIP experiment shows that circATP13A1, miR-186, miR-326, and HMGA2 were efficiently enriched on Ago2 compared to IgG. D. HMGA2 expression was remarkably inhibited by miR-186 and miR-326 in SW1990. E. Western blot confirmed that miR-186 and miR-326 inhibited HMGA2 expression. F. QRT-PCR showed that HMGA2 expression was remarkably upregulated in PDAC cancer tissues compared to the normal adjacent tissue. G. Western blot confirmed that HMGA2 was highly expressed in PDAC cancer tissues compared to normal adjacent tissues.

Figure 5

CircATP13A1 promotes proliferation and metastasis via the miR-186/miR-326/HMGA2 axis in PDAC cell lines. A. QRT-PCR analysis showed HMGA2 expression levels in different groups. Both si-circ+inh-miR-186 significantly reversed HMGA2 downregulation resulting from circATP13A1 knockdown in the SW1990 cell line. B. Western blot confirmed that HMGA2 expression was significantly restored in the si-circ1+inh-miR-186 and si-circ1+inh-miR-326 groups with circATP13A1 knockdown. C. CCK8 assay indicated that cell viability was significantly reduced in the si-circ1+si-circ1-inh-NC group compared with the normal control and other groups (si-circ1+si-circ1-inh-miR-186+si-circ1-inh-miR-326) in a time-dependent manner. D. A colony formation assay showed that the colony formation ability was significantly recovered when si-circ-inh-miR-186 and si-circ-inh-miR-326 were transfected in the SW1990 cell line. E. Transwell experiments revealed that si-circ-inh-miR-186 and si-circ-inh-miR-326 could restore the cell migration ability caused by circATP13A1 in the SW1990 cell line. F. A flow cytometry experiment showed that the rate of apoptosis was remarkably higher in the si-circ and si-circ+inh-NC groups compared to other groups in SW1990 cell lines.

Figure 6

CircATP13A1 promotes PDAC tumor progression in vivo. A. An in vivo experiment using nude mice revealed that the tumor size was significantly smaller in nude mice where circATP13A1 was silenced compared to the control mice. B. The tumor size in circATP13A1 knockdown mice was significantly reduced in a time-dependent manner compared to the control. C. Tumor weight in circATP13A1 knockdown mice was significantly reduced compared to the normal control. D. The mechanism underlying the regulation of PDAC by circATP13A1.