The Study Of MiR-486-3P Influencing On Cell Proliferation And Its Molecular Mechanisms In H520Lung Cancer Cells Through Directly Targeting HAMP

Objective:Lung cancer, with the main pathologic type of non-small-cell lung cancer (NSCLC), is one of the most common malignant tumors and has substantial morbidity and mortality, as well as an unfavorable outcome; which need better understandings of the progression mechanism and new therapeutic strategies for treatment. HAMP gene encodes the peptide of hepcidin playing a crucial in regulation process of iron metabolism. Abnormal HAMP expression has been found in broad range of cancer types. microRNA (miRNAs), a single non-coding small RNA, can regulate target gene expression in posttranscriptional level. The expression profile of miR-486is useful for improving the diagnosis and providing prognosis information in lung cancer. This study is designed to indicate the expressions of HAMP and miR-486-3p, the regulation of miR-486-3p through directly targeting HAMP; to observe the influence on cell proliferation in H520lung cancer cells; and to investigate the molecular mechanism. This study can provide new experimental evidences of carcinogenesis and progress mechanism, and new treatment ideas for non-small cell lung cancer.Method:We evaluated the expression of HAMP and miR-486-3p in NSCLC tissues and several NSCLC cell lines by RT-PCR and immunohistochemistry respectively. We predicted the binding site of miR-486-3p on HAMP3’UTR by using bioinformatics software. Luciferase reporter assay was used to verify the prediction; and transfection of miR-486-3p mimics or inhibitor was used to determine whether miR-486-3p can regulate HAMP expression. To assess the effect miR-486-3p on NSCLC biological behavior by WST-8and colony formation experiments, we established a lung cancer cell lines stably expressing miR-486-3p, named H520-miR-486-3p. We also detected the expression of HAMP and ferroportin, as well as the content of total cellular iron in H520-miR-486-3p, in order to make the mechanism clear. Also, we tested the concentration of serum iron between NSCLC patients and normal volunteers by inductively coupled plasma-atomic emission spectrometry (ICP-AES).Result:Over-expression of HAMP and reduced expression of miR-486-3p have been found in NSCLC tissues and several NSCLC cell lines. Bioinformatics software results reveal the two target site of miR-486-3p on HAMP3’UTR. Transfection of miR-486-3p mimics significant suppress the activity of the luciferase reporter containing HAMP3’UTR and decrease the expression of HAMP mRNA in H520cells; In HBE cells, miR-486-3p inhibitor can inhibit the function of miR-486-3p result in the evaluation of the activity of luciferase reporter containing HAMP3’UTR and up-regulated expression of HAMP mRNA. Enforced expression of miR-486-3p leads to reduced rate of H520cell proliferation, which can be re-promoted by HAMP. Under-expression of HAMP, over-expression of ferroportin, and less iron content has been found in H520-miR-486-3p cells. Furthermore, the low concentration of serum iron also has been detected in NSCLC patients compared with normal volunteers.Conclusion:miR-486-3p inhibits cell proliferation by repressing the target gene HAMP, as a result of the influence on iron metabolism through hepcidin-ferroportin-iron axis system. Therapeutic strategies to rescue miR-486-3p expression or silence HAMP may be beneficial to patients with NSCLC in future.