Abstract
Purpose
The abnormal regulation of lncRNA CARMN has been proved to be a tumor suppressor gene of cervical cancer (CC). However, its role in CC is still elusive. The regulation of CARMN post-transcriptional level by m6A modification and miRNA has not been studied. This study aims to analyze the molecular mechanism of m6A modification and miRNA on the abnormal expression of CARMN in CC cells, so as to provide a new theoretical basis for the diagnosis and treatment of CC.
Methods
MeRIP-seq was used to identify the differential m6A-modified genes between tumor and normal cervical tissues. RT-qPCR assay was used to detect gene expression levels in tissues or cells. The m6A modification sites of CARMN was predicted by bioinformatics, and the modification of m6A and its regulatory effect on CARMN were analyzed by MeRIP-qPCR, Actinomycin D assay and RIP assay. RIP-microarray combined with bioinformatics methods to screen miRNAs that may target CARMN. The regulation mechanism between miRNA and CARMN was verified by RT-qPCR, nucleo-plasmic separation assay, mRNA stability assay, dual-luciferase reporter assay, and in vivo experiments.
Results
MeRIP-seq found that CARMN is a significant different gene in the abundance of m6A in CC, and the modification level of m6A in CC tissues was higher than that in normal cervical tissues. Further, this study verified that m6A reader YTHDF2 could recognize m6A-modified CARMN and promote its degradation in CC cells. miR-21-5p was proved to be the downstream target gene of CARMN, and miR-21-5p could negatively regulate the expression of CARMN. Further experiments showed that miR-21-5p could directly bind to CARMN and lead to the degradation of CARMN. The in vivo experimental results indicated that the level of miR-21-5p in the overexpressed CARMN group was significantly lower than that in the control group.
Conclusion
m6A modification and miR-21-5p play important roles in promoting the occurrence and development of tumors by regulating CARMN, provide new potential targets for the treatment of CC.
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Data availability
The datasets analysed during the current study are available in the TCGA GDC repository (https://portal.gdc.cancer.gov/).
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Funding
This study was supported by Natural Science Foundation of China (81872684), the Fundamental Research Funds for the Central Universities (2242023K40023), Southeast University “Zhongying Young Scholars” Project, Opening Foundation of Jiangsu Provincial Health Development Research Center (JSHD2022056), Natural Science Foundation for Colleges and Universities of Jiangsu Province (22KJB330004), Major Project of Jiangsu Health Vocational College, Scientific Research Project of Jiangsu Provincial Health Commission (Z2019009), Major Project of Jiangsu Health Vocational College (JKA202202), Foundation for Shihezi University School of Medicine (ZZZC2022016), and Postgraduate Research & Practice Innovation Program of Jiangsu Province (KYCX21_0162 and KYCX22_0301).
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Bingjia Yu conceived the study, performed data analysis and wrote the article. Xiuting Li and Wenjing Yan downloaded gene expression data of CC. Bo Ding, Xing Zhang, Siyuan Shen, Shuqian Xie, Jing Hu, Haohan Liuundertook data analysis and interpretation. Xue Chen, Yamei Nie, Fengying Liu performed manuscript review. Yan Zhang and Shizhi Wang critically revised the article for research content and administrative support. All authors reviewed the manuscript.
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Yu, B., Li, X., Yan, W. et al. Post-transcriptional regulation of tumor suppressor gene lncRNA CARMN via m6A modification and miRNA regulation in cervical cancer. J Cancer Res Clin Oncol 149, 10307–10318 (2023). https://doi.org/10.1007/s00432-023-04893-x
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DOI: https://doi.org/10.1007/s00432-023-04893-x