Circular RNA circVAPA knockdown suppresses colorectal cancer cell growth process by regulating miR-125a/CREB5 axis

Abstract

Background: Colorectal cancer (CRC) is a malignant tumor, and the overall prognosis of patients with advanced CRC is still unsatisfactory. Circular RNAs (circRNAs) vesicle-associated membrane protein-associated protein A (circVAPA) could act as an underlying biomarker in CRC. This study aimed to explore the mechanism of circVAPA in the regulation of CRC growth.

Methods: CircVAPA level was measured in CRC tumor tissues. The expression levels of circVAPA, VAPA mRNA, microRNA-125a (miR-125a), and cAMP response element binding 5 (CREB5) in CRC cells were detected by RT-qPCR. Cell cycle progression, migration and invasion, extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) were measured by flow cytometry, transwell assays and Seahorse XF96 Glycolysis Analyzer, severally. The levels of glucose uptake, lactate and ATP production were examined by Glucose Uptake Colorimetric Assay kit, Lactate Assay kit and ATP Colorimetric Assay kit, respectively. The interaction between miR-125a and circVAPA or CREB5 was predicted by Starbase or DIANA TOOL, and verified by the dual-luciferase reporter and RNA Immunoprecipitation (RIP) assays.

Results: CircVAPA level was up-regulated in CRC tumor tissues. Expression levels of circVAPA and CREB5 were increased, and miR-125a was decreased in CRC cells. CircVAPA knockdown repressed CRC cells cycle progression, migration, invasion and glycolysis. CircVAPA acted as a miR-125a sponge to regulate CREB5 expression. Rescue assay confirmed that miR-125a deletion or CREB5 overexpression weakened the inhibitory effect of circVAPA knockdown on CRC growth.

Conclusion: Our studies disclosed that circVAPA knockdown suppressed CRC cells cycle progression, migration, invasion and glycolysis partly by modulating miR-125a/CREB5 axis, suggesting a potential therapeutic strategy for CRC treatment.

Keywords: CREB5; CircVAPA; Colorectal cancer; miR-125a.

Fig. 1

CircVAPA was elevated in CRC tissues and cells. a RT-qPCR assay was applied to measure the expression level of circVAPA in 42 pairs of CRC tumor mucosal tissues and adjacent normal mucosal tissues. b CircVAPA expression level was examined in CRC cell lines (HCT116 and LOVO) and human normal colon mucosal epithelial cell line (NCM460). c, d Relative levels of circVAPA and VAPA mRNA were tested in HCT116 and LOVO cells treated with or without RNase R. *P < 0.05

Fig. 2

CircVAPA deficiency repressed cycle progression, migration and invasion of CRC cells. aTransfection efficiency of si-circVAPA in HCT116 and LOVO cells was detected by RT-qPCR assay. b, c Flow cytometry was applied to test cell cycle progression of G0/G1 phase in si-circVAPA-transfected HCT116 and LOVO cells. d, e Transwell assay was conduct to detect the abilities of migratory and invasive in si-circVAPA-transfected HCT116 and LOVO cells. *P < 0.05

Fig. 3

CircVAPA knockdown blocked aerobic glycolysis of CRC cells. a, b Extracellular acid ratio (ECAR) upon cell was examined by Seahorse XF Glycolysis Stress Test Kit in si-circVAPA-transfected HCT116 and LOVOcells. c, d Oxygen consumption rate (OCR) was measured by Seahorse XF Cell Mito Stress Test Kit in si-circVAPA-transfected HCT116 and LOVO cells. e–g The levels of glucose uptake, lactate production and ATP were detected in si-circVAPA-transfected HCT116 and LOVO cells. *P < 0.05

Fig. 4

CREB5 was positively regulated by circVAPA/miR-125a. a, b The binding sites betweenmiR-125a and circVAPA or CREB5 3′UTR were predicted by starBase or DIANA TOOL. c, d Dual-luciferase reporter assay was performed to confirm the bindings among circVAPA, miR-125a and CREB5. e, f The interacting between circVAPA, miR-125a and CREB5 was proved by RIP assay. g The effects of circVAPA upregulation and deletion on miR-125a expression were assessed in HCT116 and LOVO cells. h The effects of miR-125a overexpression and knockdown on CREB5 protein level were detected in HCT116 and LOVO cells. i The protein levels of CREB5 were examined in HCT116 and LOVO cells transfected with vector, circVAPA, circVAPA + miR-con and circVAPA + miR-125a. j CREB5 protein levels were measured in HCT116 and LOVO cells transfected with si-con, si-circVAPA, si-circVAPA + anti-miR-con and si-circVAPA + anti-miR-125a. *P < 0.05

Fig. 5

Regulation of circVAPA on cell cycle progression, migration and invasion was mediated by miR-125a/CREB5 axis. a, b The expression levels of miR-125a and CREB5 were detected in CRC cell lines (HCT116 and LOVO) and human normal colon mucosal epithelial cell line (NCM460). c, d Cell cycle in G0/G1 phase was examined in HCT116 and LOVOcells transfected with si-con, si-circVAPA, si-circVAPA + anti-miR-con, si-circVAPA + anti-miR-125a, si-circVAPA + vector and si-circVAPA + CREB5. e, f Migratory ability was assessed in transfected HCT116 and LOVO cells. g, h Invasive capacity was tested in transfected HCT116 and LOVO cells. *P < 0.05

Fig. 6

CircVAPA-mediated glycolysis was regulated by miR-125a/CREB5 axis. a, b The change of ECAR level was measured in HCT116 and LOVOcells transfected with si-con, si-circVAPA, si-circVAPA + anti-miR-con, si-circVAPA + anti-miR-125a, si-circVAPA + vector and si-circVAPA + CREB5. c, d The change of OCR was detected in transfected HCT116 and LOVO cells. e, f Glucose uptake was examined in transfected HCT116 and LOVO cells. g, h The production of lactate was detected in transfected HCT116 and LOVO cells. i, j ATP was determined in transfected HCT116 and LOVO cells. *P < 0.05