miRNA-506-3p Directly Regulates rs10754339 (A/G) in the Immune Checkpoint Protein B7-H4 in Breast Cancer

Background: B7-H4 is a novel immune checkpoint protein that negatively regulates T cell activation and function. It is overexpressed in many malignant tumors, including Breast Cancer (BC). It was reported that the presence of the single nucleotide polymorphism rs10754339 (A/G) within the 3' UTR of the B7-H4 gene has a great influence on the risk and progression of BC as well as lymph node metastasis. On the other hand, mounting evidence demonstrated the potential of miR-506-3p to be employed in the diagnosis and treatment of a wide range of human malignancies. It is frequently down-regulated in BC despite its tumor suppressor role. Moreover, Myc, E2F and Rb proteins are key players in cell cycle regulation. In BC, the CDK-RB-E2F axis is extensively deregulated by several genetic mutations. Additionally, the potent proto-oncogene Myc is highly expressed in BC. Aim: The main aims of the study were to investigate the potential role of miR-506-3p in the regulation of B7-H4 SNP rs10754339 (A/G) in BC and to uncover the influence of miR-506-3p on cell cycle and tumor progression in BC cell lines. Methods: This study employed different BC cell lines, including MDA-MB-231 and MCF7 cells. Several bioinformatics analysis was performed to identify the miRNA that could potentially target B7-H4 SNP rs10754339 (A/G). To confirm the binding Relative luciferase activity was measured using Dual Luciferase Reporter Assay. Transfection experiments were performed using miR-506-3p oligonucleotides using lipofection technique. Furthermore, vital cell cycle regulatory proteins such as cMyc, Rb, and E2F were transfected into BC cells using superfect. Finally, several functional analysis experiments were performed, such as MTT and wound healing assays. Results: miR-506-3p down-regulates the disease-associated rs10754339 “G” allele in B7-H4 gene. It also inhibits cell cycle progression by simultaneously regulating important cell cycle proteins, including RB, E2F and c-Myc. Moreover, miR-506-3p decreased cellular viability and migration capacity of MDA-MB-231 TNBC cells and hormone receptor positive MCF-7 cells. Conclusion: miR-506-3p is a potential tumor suppressor miRNA in BC that has a potential role in regulating B7-H4 SNP rs10754339 (A/G).