Blocking circ-CNST suppresses malignant behaviors of osteosarcoma cells and inhibits glycolysis through circ-CNST-miR-578-LDHA/PDK1 ceRNA networks

Abstract

Background CircRNA CNST (circ-CNST) is a newly identified biomarker for prognosis of osteosarcoma (OS). However, its role in OS progression remains to be well documented. Methods Expression of circ-CNST, microRNA (miR)-578, lactate dehydrogenase A (LDHA), and pyruvate dehydrogenase kinase 1 (PDK1) was detected by quantitative real-time polymerase chain reaction and Western blotting. The physical interaction was confirmed by dual-luciferase reporter assay. Cell behaviors and glycolysis were measured by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide assay, colony formation assay, flow cytometry, transwell assays, xenograft experiment, and commercial kits. Results Circ-CNST was upregulated in human OS tissues and cells, accompanied with downregulation of miR-578 and upregulation of LDHA and PDK1. There were negative correlations between miR-578 expression and circ-CNST or LDHA/PDK1 in OS tissues. Moreover, high circ-CNST/LDHA/PDK1 or low miR-578 might predict shorter overall survival, advanced TNM stages, and lymph node metastasis. Physically, miR-578 was targeted by circ-CNST, and miR-578 could target LDHA/PDK1. Functionally, blocking circ-CNST and restoring miR-578 enhanced apoptosis rate and suppressed cell proliferation, colony formation, migration, and invasion in 143B and U2OS cells, accompanied with decreased glucose consumption, lactate production, and adenosine triphosphate (ATP)/adenosine diphosphate (ADP) ratio. Furthermore, in vivo growth of U2OS cells was retarded by silencing circ-CNST. Depletion of miR-578 could counteract the suppressive role of circ-CNST deficiency in 143B and U2OS cells, and restoring LDHA or PDK1 partially reversed the role of miR-578 inhibition as well. Conclusion Circ-CNST knockdown could antagonize malignant behaviors and glycolysis of OS cells by regulating miR-578-LDHA/PDK1 axes.

LDHA: The Obstacle to T cell responses against tumor

“…A study unveiled that circSLC25A16 interacted with miR-488-3p/HIF-1a to activate LDHA by promoting its transcription in non-small-cell lung cancer (60). Likewise, circ-CNST/miR-578 and circATRNL1/miR-409-3P regulated LHDA in osteosarcoma (61,62). In pancreatic ductal adenocarcinoma (PDAC), circSLIT2 functioned as a miRNA sponge to target the miR-510-5p/c-Myc axis to activate the transcription by binding to the promoter region of LDHA (63).…”

Metabolic reprogramming in osteosarcoma

“…A recent study has found that KDM6B promotes OS cell metastasis by regulating LDHA expression 61 . The knockdown of Circ‐CNST can antagonize the malignant behavior and glycolysis of OS cells by regulating the miR‐578‐LDHA/PDK1 axis 62 . Zhang et al.…”

“…61 The knockdown of Circ-CNST can antagonize the malignant behavior and glycolysis of OS cells by regulating the miR-578-LDHA/PDK1 axis. 62 Zhang et al found that CircATRNL1 enhanced glycolysis via the cir-cATRNL1/miR-409-3p/LDHA axis and promoted OS progression. 63 Other studies have found that non-coding RNAs: miR-323a-3p and miR-329-3p inhibit OS cell proliferation by targeting LDHA to repress OS glycolysis.…”

Circular RNAs Regulate Glucose Metabolism in Cancer Cells

“… 70 The isozymes bind phosphates to specific serine residues present within the α-subunits of the E1 of the complex. 71 , 72 Hu et al 73 reported that Circ-CNST regulates PDK1 expression by sponging miR-578 and glucose consumption. Moreover, lactate production and ATP/ADP ratios were found to be suppressed in Circ-CNST upregulated osteosarcoma cells.…”