Microarray Identifies a Key Carcinogenic Circular RNA 0008594 That Is Related to Non-Small-Cell Lung Cancer Development and Lymph Node Metastasis and Promotes NSCLC Progression by Regulating the miR-760-Mediated PI3K/AKT and MEK/ERK Pathways.

Qiushi Wang,Chunhua Yan,Guanghua Li,Guangquan Xu,Pengfei Zhang,Libo Wu,Ruidong Zhu,Hanbing Wang

doi:10.3389/fonc.2021.757541

Abstract

PurposeThis study aimed to explore the circular RNA (circRNA/circ) profile engaged in non-small cell lung cancer (NSCLC) development and metastasis and to investigate potentially key carcinogenic circRNAs related to NSCLC.MethodsCircRNA profiles between 10 NSCLC tissues and 10 adjacent tissues and between five NSCLC tissues with lymph node metastasis (LNM) and five NSCLC tissues without LNM were detected by Arraystar Human circRNA Array followed by bioinformatics. Circ_0008594 knockdown, circ_0004293 overexpression, and circ_0003832 overexpression plasmids were transfected into H23 and H460 cells to sort potential oncogenic circRNA. Then circ_0008594 overexpression and knockdown plasmids were transfected, followed by that circ_0008594 knockdown plus miR-760 knockdown plasmids were transfected into these cells. Cell proliferation, apoptosis, invasion, stemness, and pathways were detected. In addition, xenograft mice models were constructed via injecting H23 cells with circ_0008594 overexpression or knockdown to validate the findings.ResultsA total of 455 dysregulated circRNAs in NSCLC tissues versus adjacent tissues and 353 dysregulated circRNAs in NSCLC tissues with LNM versus those without LNM were discovered. Via cross-analysis, 19 accordant circRNAs were uncovered, among which three candidate circRNAs (circ_0008594, circ_0004293, circ_0003832) were chosen for functional experiments, during which it was observed that circ_0008549 affected H23 and H460 cell proliferation and apoptosis more obviously than circ_0004293 and circ_0003832. Subsequent experiments showed that circ_0008594 promoted H23 and H460 cell proliferation and invasion but affected stemness less and negatively regulated miR-760 via direct binding. Furthermore, miR-760 attenuated the effect of circ_0008549 on regulating H23 and H460 cell functions and the PI3K/AKT and MEK/ERK pathways. In vivo experiments further confirmed that circ_0008549 increased tumor volume, epithelial-mesenchymal transition, and the PI3K/AKT and MEK/ERK pathways while reducing tumor apoptosis and miR-760 NSCLC xenograft models.ConclusionOur study identifies several valuable circRNAs related to NSCLC development and LNM. Furthermore, as a key functional circRNA, circ_0008594 was observed to promote NSCLC progression by regulating the miR-760-mediated PI3K/AKT and MEK/ERK pathways.

Highlights

Lung cancer remains the deadliest and second most frequent cancer worldwide, accounting for 11.4% of newly diagnosed cancer cases and 18.0% of cancer-related deaths in 2020 [1]
The circRNA profile could differentiate non-small-cell lung cancer (NSCLC) tissues from adjacent non-cancerous tissues by principal component analysis (PCA) and heatmap analyses, with 173 upregulated and 282 downregulated circRNAs identified in NSCLC tissues by volcano plot, and their enriched bioprocesses and pathways are presented in Supplementary Figures 1A–E
The circRNA profile could distinguish NSCLC tissues with LNM from those without LNM, with 183 upregulated and 170 downregulated circRNAs discovered in NSCLC tissues with LNM by volcano plot, and their enriched bioprocesses and pathways are presented at the end of this paper (Supplementary Figures 1F–J)

Summary

Introduction

Lung cancer remains the deadliest and second most frequent cancer worldwide, accounting for 11.4% of newly diagnosed cancer cases and 18.0% of cancer-related deaths in 2020 [1]. NSCLC treatment has been greatly promoted in recent years, benefiting from measures such as early screening programs, treatment strategy improvement, individualized and precise medicine, and novel targeted drug development [4,5,6,7]. Circular RNA (circRNA/circ), a recently identified noncoding RNA with a loop construction, regulates numerous cell functions and biological processes and participates in the pathogenesis of almost all cancers [11,12,13,14,15]. In terms of NSCLC, several specific functional circRNAs have been discovered to regulate NSCLC growth, metastasis, and drug sensitivity via various oncogenes and carcinogenic pathways [16,17,18,19]. There remain a large number of circRNAs whose functions or involvement in NSCLC need to be evaluated, and only a very limited number of studies have explored the comprehensive circRNA profile engaged in NSCLC etiology [20, 21]

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