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

Abstract

<jats:title>Abstract</jats:title> <jats:p><jats:bold>Background</jats:bold>: The long non-coding RNA (lncRNA),<jats:italic>LINC00518,</jats:italic> is highly expressed in many human cancers and is involved in cancer progression. However, the potential function and regulatory mechanism of <jats:italic>LINC00518</jats:italic> in cutaneous malignant melanoma (CMM) remain unclear. <jats:bold>Methods</jats:bold>:Short hairpin RNA (shRNA) was used to silence <jats:italic>LINC00518 </jats:italic>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 <jats:italic>LINC00518</jats:italic> silencing on cellular radiosensitivity. Dual luciferase reporter system,CHIP and COIP was used to verify the target relationship between<jats:italic> LINC00518</jats:italic>,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 <jats:italic>LINC00518 </jats:italic>silencing on the radiosensitivity of melanoma in vivo.<jats:bold>Results</jats:bold>: <jats:italic>LINC00518</jats:italic> expression was significantly upregulated in CMM samples, and <jats:italic>LINC00518</jats:italic> levels were associated with poor prognosis of patients with CMM. Knockdown of <jats:italic>LINC00518</jats:italic> 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, <jats:italic>in vitro</jats:italic>, 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 <jats:italic>LINC00518</jats:italic>/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 <jats:italic>LINC00518</jats:italic> expression sensitized CMM cancer cells to radiotherapy in an <jats:italic>in vivo</jats:italic> subcutaneously implanted tumour model. <jats:bold>Conclusion</jats:bold>: <jats:italic>LINC00518</jats:italic> 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.</jats:p>