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miRNA-506-3p 直接调控乳腺癌免疫检查点蛋白 B7-H4 中的 rs10754339(A/G)。

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

机构信息

Department of Biochemistry, German University in Cairo, New Cairo City, Main Entrance Al Tagamoa Al Khames, 11835, Cairo, Egypt.

Pharmaceutical Biology Department, German University in Cairo, New Cairo City, Main Entrance Al Tagamoa Al Khames, 11835, Cairo, Egypt.

出版信息

Microrna. 2020;9(5):346-353. doi: 10.2174/2211536609666201209152949.

DOI:10.2174/2211536609666201209152949

PMID:33297930

Abstract

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).

摘要

背景

B7-H4 是一种新型免疫检查点蛋白，可负向调节 T 细胞激活和功能。它在许多恶性肿瘤中过度表达，包括乳腺癌（BC）。据报道，B7-H4 基因 3'UTR 内的单核苷酸多态性 rs10754339（A/G）的存在对 BC 的风险和进展以及淋巴结转移有很大影响。另一方面，越来越多的证据表明 miR-506-3p 具有用于诊断和治疗广泛的人类恶性肿瘤的潜力。尽管具有肿瘤抑制作用，但它在 BC 中经常下调。此外，Myc、E2F 和 Rb 蛋白是细胞周期调节的关键因子。在 BC 中，CDK-RB-E2F 轴被多种遗传突变广泛失调。此外，强大的原癌基因 Myc 在 BC 中高度表达。

目的

本研究的主要目的是探讨 miR-506-3p 在调节 BC 中 B7-H4 SNP rs10754339（A/G）中的潜在作用，并揭示 miR-506-3p 对 BC 细胞系中细胞周期和肿瘤进展的影响。

方法

本研究使用了不同的 BC 细胞系，包括 MDA-MB-231 和 MCF7 细胞。进行了几项生物信息学分析，以鉴定可能靶向 B7-H4 SNP rs10754339（A/G）的 miRNA。为了确认结合，使用双荧光素酶报告基因测定测量相对荧光素酶活性。通过脂质体转染技术使用 miR-506-3p 寡核苷酸进行转染实验。此外，使用 superfect 将重要的细胞周期调节蛋白（如 cMyc、Rb 和 E2F）转染到 BC 细胞中。最后，进行了几项功能分析实验，如 MTT 和划痕愈合实验。

结果

miR-506-3p 下调 B7-H4 基因中与疾病相关的 rs10754339“G”等位基因。它还通过同时调节重要的细胞周期蛋白，包括 RB、E2F 和 c-Myc，抑制细胞周期进程。此外，miR-506-3p 降低了 MDA-MB-231 TNBC 细胞和激素受体阳性 MCF-7 细胞的细胞活力和迁移能力。