miR-141 调控 KLRC 和 Ceacam 基因表达促进宫颈癌的增殖和转移。

Regulation of KLRC and Ceacam gene expression by miR-141 supports cell proliferation and metastasis in cervical cancer cells.

机构信息

Department of Molecular Biology, Genetic Engineering and Biotechnology Research Institute, University of Sadat City, P.O Box 79, Sadat City, Egypt.

Microbiology and Chemistry Department, Faculty of Science, Benha University, Benha, Egypt.

出版信息

BMC Cancer. 2024 Sep 3;24(1):1091. doi: 10.1186/s12885-024-12794-6.

DOI:10.1186/s12885-024-12794-6

PMID:39227808

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC11370040/

Abstract

INTRODUCTION

MicroRNAs (miRNAs) are single RNA molecules that act as global regulators of gene expression in mammalian cells and thus constitute attractive targets in treating cancer. Here we aimed to investigate the possible involvement of miRNA-141 (miR-141) in cervical cancer and to identify its potential targets in cervical cancer cell lines.

METHODS

The level of miR-141 in HeLa and C-33A cells has been assessed using the quantitative real-time PCR (qRT-PCR). A new miR-141 construct has been performed in a CMV promoter vector tagged with GFP. Using microarray analysis, we identified the potentially regulated genes by miR-141 in transfected HeLa cells. The protein profile of killer-like receptor C1 (KLRC1), KLRC3, carcinoembryonic antigen-related cell adhesion molecule 3 (CAM3), and CAM6 was investigated in HeLa cells transfected with either an inhibitor, antagonist miR-141, or miR-141 overexpression vector using immunoblotting and flow cytometry assay. Finally, ELISA assay has been used to monitor the produced cytokines from transfected HeLa cells.

RESULTS

The expression of miR-141 significantly increased in HeLa and C-33A cells compared to the normal cervical HCK1T cell line. Transfection of HeLa cells with an inhibitor, antagonist miR-141, showed a potent effect on cancer cell viability, unlike the transfection of miR-141 overexpression vector. The microarray data of HeLa cells overexpressed miR-141 provided a hundred of downregulated genes, including KLRC1, KLRC3, CAM3, and CAM6. KLRC1 and KLRC3 expression profiles markedly depleted in HeLa cells transfected with miR-141 overexpression accompanied by decreasing interleukin 8 (IL-8), indicating the role of miR-141 in avoiding programmed cells death in HeLa cells. Likewise, CAM3 and CAM6 expression reduced markedly in miR-141 transduced cells accompanied by an increasing level of transforming growth factor beta (TGF-β), indicating the impact of miR-141 in cancer cell migration. The IntaRNA program and miRWalk were used to check the direct interaction and potential binding sites between miR-141 and identified genes. Based on this, the seeding regions of each potential target was cloned upstream of the luciferase reporter gene in the pGL3 control vector. Interestingly, the luciferase activities of constructed vectors were significantly decreased in HeLa cells pre-transfected with miR-141 overexpression vector, while increasing enormously in cells pre-transfected with miR-141 specific inhibitor.

CONCLUSION

Together, these data uncover an efficient miR-141-based mechanism that supports cervical cancer progression and identifies miR-141 as a credible therapeutic target.

摘要

简介

microRNAs (miRNAs) 是一类单链 RNA 分子，可作为哺乳动物细胞中基因表达的全局调控因子，因此成为治疗癌症的有吸引力的靶点。本研究旨在探讨 miRNA-141 (miR-141) 与宫颈癌的可能相关性，并鉴定其在宫颈癌细胞系中的潜在靶基因。

方法

使用实时定量 PCR (qRT-PCR) 评估 HeLa 和 C-33A 细胞中 miR-141 的水平。在 CMV 启动子载体上构建带有 GFP 标签的新 miR-141 构建体。通过微阵列分析，我们鉴定了转染 miR-141 的 HeLa 细胞中可能受调控的基因。使用免疫印迹和流式细胞术检测转染 miR-141 抑制剂、拮抗剂或 miR-141 过表达载体的 HeLa 细胞中 killer-like receptor C1 (KLRC1)、KLRC3、癌胚抗原相关细胞黏附分子 3 (CAM3) 和 CAM6 的蛋白谱。最后，使用 ELISA 检测转染 HeLa 细胞产生的细胞因子。

结果

miR-141 在 HeLa 和 C-33A 细胞中的表达水平明显高于正常宫颈 HCK1T 细胞系。转染 miR-141 抑制剂的 HeLa 细胞显示出对癌细胞活力的显著影响，而转染 miR-141 过表达载体则不然。HeLa 细胞过表达 miR-141 的微阵列数据提供了上百个下调基因，包括 KLRC1、KLRC3、CAM3 和 CAM6。转染 miR-141 过表达载体的 HeLa 细胞中 KLRC1 和 KLRC3 的表达谱明显减少，同时白细胞介素 8 (IL-8) 减少，表明 miR-141 在 HeLa 细胞中避免程序性细胞死亡的作用。同样，CAM3 和 CAM6 的表达在 miR-141 转导的细胞中明显减少，同时转化生长因子 β (TGF-β) 水平升高，表明 miR-141 对癌细胞迁移的影响。使用 IntaRNA 程序和 miRWalk 检查 miR-141 与鉴定基因之间的直接相互作用和潜在结合位点。基于此，每个潜在靶基因的种子区域被克隆到 pGL3 对照载体的荧光素酶报告基因上游。有趣的是，在转染 miR-141 过表达载体的 HeLa 细胞中，构建载体的荧光素酶活性显著降低，而在转染 miR-141 特异性抑制剂的细胞中则显著增加。