微小RNA-7-5p在O-连接的N-乙酰葡糖胺糖基化及癌症代谢中的作用

MicroRNA-7-5p's role in the O-GlcNAcylation and cancer metabolism.

作者信息

Woo Sin Yung, Lee Su Yel, Yu Seong-Lan, Park Se Jin, Kang Daeun, Kim Jin Suk, Jeong In Beom, Kwon Sun Jung, Hwang Wan Jin, Park Chang Ryul, Son Ji Woong

机构信息

Department of Internal Medicine, Konyang University Hospital, South Korea.

Priority Research Center, Myunggok Research Institute, College of Medicine, Konyang University, South Korea.

出版信息

Noncoding RNA Res. 2020 Nov 10;5(4):201-207. doi: 10.1016/j.ncrna.2020.11.003. eCollection 2020 Dec.

DOI:10.1016/j.ncrna.2020.11.003

PMID:33251387

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC7677666/

Abstract

O-GlcNAc Transferase (OGT) is a complementary enzyme that regulates O-linked N-acetylglucosaminylation(O-GlcNAcylation) and plays a critical role in various cancer phenotypes, including invasion, migration, and metabolic reprogramming. In our previous study we found that miR-7-5p was downregulated at lung cancer cells with highly metastatic capacity. In the in-silico approach, OGT is the predicted target of miR-7-5p. To identify miR-7-5p's role in cell growth and metabolism, we transfected various lung cancer cell lines with miR-7-5p. The expression level of miR-7-5p was confirmed by qRT-PCR in lung cancer cell lines. Western blot assays and qRT-PCR were performed to demonstrate miR-7-5p's effect. Bioinformatic analysis indicated that OGT is a direct target of miR-7-5p. The binding sites of miR-7-5p in the OGT 3' UTR were verified by luciferase reporter assay. To investigate the role of miR-7-5p in the cancer metabolism of non-small cell lung cancer (NSCLC) cells, mimic of miR-7-5p was transfected into NSCLC cells, and the effect of miR-7-5p on cancer metabolism was analyzed by LDH assays, glucose uptake, and mitochondrial ATP synthase inhibitor assay. O-GlcNAcylated protein level was determined by Western blot. The role of miR-7-5p in lung cancer growth was measured by MTS assays. To identify the delivery of miR-7-5p via PLGA, an release assay of PLGA-miR-7-5p was done. miR-7-5p was highly expressed whereas OGT showed low expression in H358, H827. However, miR-7-5p exhibited low expression while OGT had high expression in H522, H460, and H1299 cell lines. OGT were repressed by binding of miR-7a-5p to the 3'-UTR. Overexpression of miR-7-5p also diminished anaerobic glycolysis. miR-181a-5p transfection induced expression levels of OGT were diminished compared to those in the control group. O-GlcNAcylation was suppressed by miR-7-5p. Moreover, the overexpression of miR-7-5a suppressed lung cancer cell growth. miR-7-5p was released via PLGA for up to 10 days. In the present study, inhibition of OGT by miR-7-5p decreased the growth and cancer metabolism of lung cancer.

摘要

O-连接的N-乙酰葡糖胺转移酶（OGT）是一种调节O-连接的N-乙酰葡糖胺化（O-GlcNAc糖基化）的互补酶，在包括侵袭、迁移和代谢重编程在内的各种癌症表型中起关键作用。在我们之前的研究中，我们发现miR-7-5p在具有高转移能力的肺癌细胞中表达下调。在计算机模拟方法中，OGT是miR-7-5p的预测靶标。为了确定miR-7-5p在细胞生长和代谢中的作用，我们用miR-7-5p转染了各种肺癌细胞系。通过qRT-PCR在肺癌细胞系中证实了miR-7-5p的表达水平。进行蛋白质免疫印迹分析和qRT-PCR以证明miR-7-5p的作用。生物信息学分析表明OGT是miR-7-5p的直接靶标。通过荧光素酶报告基因测定验证了miR-7-5p在OGT 3'UTR中的结合位点。为了研究miR-7-5p在非小细胞肺癌（NSCLC）细胞癌症代谢中的作用，将miR-7-5p模拟物转染到NSCLC细胞中，并通过乳酸脱氢酶（LDH）测定、葡萄糖摄取和线粒体ATP合酶抑制剂测定分析miR-7-5p对癌症代谢的影响。通过蛋白质免疫印迹测定O-GlcNAc糖基化蛋白水平。通过MTS测定测量miR-7-5p在肺癌生长中的作用。为了鉴定通过聚乳酸-羟基乙酸共聚物（PLGA）递送miR-7-5p，进行了PLGA-miR-7-5p的释放测定。在H358、H827中，miR-7-5p高表达而OGT低表达。然而，在H522、H460和H1299细胞系中，miR-7-5p低表达而OGT高表达。OGT通过miR-7a-5p与3'-UTR的结合而被抑制。miR-7-5p的过表达也减少了无氧糖酵解。与对照组相比，miR-181a-5p转染诱导的OGT表达水平降低。O-GlcNAc糖基化被miR-7-5p抑制。此外，miR-7-5a的过表达抑制了肺癌细胞的生长。miR-7-5p通过PLGA释放长达10天之久。在本研究中，miR-7-5p对OGT的抑制降低了肺癌的生长和癌症代谢。