Long Non-Coding RNA LINC00518 Induces Radioresistance by Regulating Glycolysis Through An miR-33a-3p/HIF-1α Negative Feedback Loop in Melanoma

Abstract Background: The long non-coding RNA (lncRNA),LINC00518, is highly expressed in many human cancers and is involved in cancer progression. However, the potential function and regulatory mechanism of LINC00518 in cutaneous malignant melanoma (CMM) remain unclear. Methods:Short hairpin RNA (shRNA) was used to silence LINC00518 and HIF-1α, and real-time PCR was performed to determine mRNA expression. Then, cell proliferation, colony formation, flow cytometric, scratch, and transwell assays were to examine the influence of LINC00518 silencing on cellular radiosensitivity. Dual luciferase reporter system,CHIP and COIP was used to verify the target relationship between LINC00518,miR‐33a-5b and HIF-1α,.Glycolysis assays were conducted to exam cell glycolysis process. Western blotting was performed to explore the expression of HIF-1α and LDHA. Finally, animal experiments were performed to demonstrate the effect of LINC00518 silencing on the radiosensitivity of melanoma in vivo.Results: LINC00518 expression was significantly upregulated in CMM samples, and LINC00518 levels were associated with poor prognosis of patients with CMM. Knockdown of LINC00518 in CMM cells significantly inhibited cell invasion, migration, proliferation, and clonogenicity. LINC00518-mediated invasion, migration, proliferation, and clonogenicity were negatively regulated by the microRNA, miR-33a-3p, in vitro, which intensified sensitivity to radiotherapy via inhibition of the hypoxia-induced factor 1α (HIF-1α)/lactate dehydrogenase A (LDHA)-glycolysis axis. Additionally, HIF-1α recognized the miR-33a-3p promoter region and recruited histone deacetylase2 (HDAC2), which decreased the expression of miR-33a-3p and formed an LINC00518/miR-33a-3p/HIF-1α negative feedback loop. Furthermore, signalling initially activated glycolysis and radioresistance in CMM cells was recovered by Santacruzamate A (a histone deacetylase inhibitor) and 2-deoxy-D-glucose (a glycolytic inhibitor). Lastly, knockdown of LINC00518 expression sensitized CMM cancer cells to radiotherapy in an in vivo subcutaneously implanted tumour model. Conclusion: LINC00518 was confirmed to be an oncogene in CMM, which induces radioresistance by regulating glycolysis through an miR-33a-3p/HIF-1α negative feedback loop. Our research may provide a potential strategy to improve the treatment outcome of radiotherapy in CMM.