Emad Dabous, Mai Alalem, Ahmed M Awad, Khaled A Elawdan, Ahmed M Tabl, Shorouk Elsaka, Walid Said, Adel A Guirgis, Hany Khalil
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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.</p><p><strong>Results: </strong>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.</p><p><strong>Conclusion: </strong>Together, these data uncover an efficient miR-141-based mechanism that supports cervical cancer progression and identifies miR-141 as a credible therapeutic target.</p>","PeriodicalId":9131,"journal":{"name":"BMC Cancer","volume":null,"pages":null},"PeriodicalIF":3.4000,"publicationDate":"2024-09-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11370040/pdf/","citationCount":"0","resultStr":"{\"title\":\"Regulation of KLRC and Ceacam gene expression by miR-141 supports cell proliferation and metastasis in cervical cancer cells.\",\"authors\":\"Emad Dabous, Mai Alalem, Ahmed M Awad, Khaled A Elawdan, Ahmed M Tabl, Shorouk Elsaka, Walid Said, Adel A Guirgis, Hany Khalil\",\"doi\":\"10.1186/s12885-024-12794-6\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><strong>Introduction: </strong>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. 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引用次数: 0
摘要
导言:微RNA(miRNA)是单个RNA分子,在哺乳动物细胞中作为基因表达的全局调控因子,因此是治疗癌症的诱人靶点。在此,我们旨在研究 miRNA-141 (miR-141)可能与宫颈癌的关系,并确定其在宫颈癌细胞系中的潜在靶点:方法:使用定量实时 PCR(qRT-PCR)技术评估了 HeLa 和 C-33A 细胞中 miR-141 的水平。在带有 GFP 标记的 CMV 启动子载体中构建了一种新的 miR-141。通过微阵列分析,我们确定了转染 HeLa 细胞中可能受 miR-141 调控的基因。在转染了抑制剂、拮抗剂 miR-141 或 miR-141 过表达载体的 HeLa 细胞中,我们使用免疫印迹法和流式细胞术检测了类杀伤性受体 C1 (KLRC1)、KLRC3、癌胚抗原相关细胞粘附分子 3 (CAM3) 和 CAM6 的蛋白质谱。最后,使用 ELISA 检测法监测转染 HeLa 细胞产生的细胞因子:结果:与正常宫颈 HCK1T 细胞系相比,miR-141 在 HeLa 和 C-33A 细胞中的表达明显增加。与转染 miR-141 过表达载体不同的是,用抑制剂--拮抗剂 miR-141 转染 HeLa 细胞对癌细胞的存活率有很强的抑制作用。过表达 miR-141 的 HeLa 细胞的芯片数据提供了上百个下调基因,包括 KLRC1、KLRC3、CAM3 和 CAM6。在转染了 miR-141 的 HeLa 细胞中,KLRC1 和 KLRC3 的表达明显减少,白细胞介素 8(IL-8)也随之减少,这表明 miR-141 在避免 HeLa 细胞程序性死亡方面发挥了作用。同样,在 miR-141 转导的细胞中,CAM3 和 CAM6 的表达明显减少,同时转化生长因子β(TGF-β)的水平上升,这表明 miR-141 对癌细胞迁移有影响。研究人员利用 IntaRNA 程序和 miRWalk 检查了 miR-141 与已识别基因之间的直接相互作用和潜在结合位点。在此基础上,在 pGL3 控制载体的荧光素酶报告基因上游克隆了每个潜在靶点的播种区域。有趣的是,在预转染了 miR-141 过表达载体的 HeLa 细胞中,所构建载体的荧光素酶活性明显降低,而在预转染了 miR-141 特异性抑制剂的细胞中,荧光素酶活性则大大提高:总之,这些数据揭示了基于 miR-141 的支持宫颈癌进展的有效机制,并确定 miR-141 为可靠的治疗靶点。
Regulation of KLRC and Ceacam gene expression by miR-141 supports cell proliferation and metastasis in cervical cancer cells.
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.
期刊介绍:
BMC Cancer is an open access, peer-reviewed journal that considers articles on all aspects of cancer research, including the pathophysiology, prevention, diagnosis and treatment of cancers. The journal welcomes submissions concerning molecular and cellular biology, genetics, epidemiology, and clinical trials.