Author Manuscript Published OnlineFirst on November 17, 2020; DOI: 10.1158/0008-5472.CAN-20-0554 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 1 Title 2 circIGHG-induced epithelial-to-mesenchymal transition promotes oral squamous cell carcinoma 3 progression via miR-142-5p/IGF2BP3 signaling 4 5 Authors and affiliations 6 Jingpeng Liu, Xiao Jiang, Ailing Zou, Zhaoyi Mai, Zhijie Huang, Liying Sun, Jianjiang Zhao 7 Authors’ affiliation: Department of Oral Surgery, Stomatological Hospital, Southern Medical 8 University, Guangzhou, China. 9 10 Running title 11 circIGHG regulates OSCC via miR-142-5p/IGF2BP3 signaling 12 13 Correspondence: Jianjiang Zhao, Department of Oral Surgery, Stomatological Hospital, 14 Southern Medical University, China. 15 366 S Jiangnan Street, Haizhu District, Guangzhou City, Guangdong Province, China. 16 Tel: +86-020-84439500 17 Mobile: +86-13609645845 18 Email: [email protected] 19 20 Keywords 21 circular RNA, OSCC, EMT, metastasis, miRNA 22 23 Competing interest 24 The authors declare no potential conflicts of interest. 25 26 Abbreviations 27 OSCC, oral squamous cell carcinoma; circRNA, circular RNA; miRNA, microRNA; 28 EMT, epithelial-mesenchymal transition; IGF2BP3, Insulin like growth factor 2 mRNA binding 29 protein 3; IGHG, immunoglobulin heavy chain G; CCCP, carbonyl cyanide 3- 30 chlorophenylhydrazone; FISH, fluorescence in situ hybridization; RT-qPCR, real time 31 quantitative polymerase chain reaction; IHC, immunohistochemistry; IF, immunofluorescence; 32 GEO, Gene Expression Omnibus. 33 34 Word count: 4658 35 Number of figures: 6 36 Number of tables: 0 37 38 39 40 41 42 43 44 45 1 Downloaded from cancerres.aacrjournals.org on October 4, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on November 17, 2020; DOI: 10.1158/0008-5472.CAN-20-0554 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 1 Abstract 2 Circular RNAs are a new member of endogenously produced noncoding RNAs that have been 3 characterized as key regulators of gene expression in a variety of malignances. However, the role 4 of circRNA in oral squamous cell carcinoma (OSCC) remains largely unknown. In this study, we 5 identified unique circRNA that regulate OSCC progression and metastasis and pave roads for 6 future research in early diagnosis, prevention, and treatment of OSCC. Transcriptomic analyses 7 identified a circRNA derived from immunoglobulin heavy chain G locus (circIGHG) as 8 significantly upregulated in OSCC and positively associated with poor prognosis of OSCC. 9 circIGHG directly bound miR-142-5p and consequently elevated IGF2BP3 activity. Knockdown 10 of circIGHG led to impaired expression of IGF2BP3 and attenuated aggressiveness of OSCC 11 cells. EMT was the main mechanism through which circIGHG/IGF2BP3 promotes metastasis of 12 OSCC. Overall, these results demonstrate that circIGHG plays pivotal role in OSCC 13 development and metastasis and has potential to serve as a biomarker and therapeutic target for 14 early-stage diagnosis and treatment of OSCC. 15 16 Statement of significance 17 Findings broaden our insights regarding regulation of OSCC progression by circular RNA and 18 serve as a reference for future clinical research in OSCC diagnosis and treatment. 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 2 Downloaded from cancerres.aacrjournals.org on October 4, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on November 17, 2020; DOI: 10.1158/0008-5472.CAN-20-0554 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 1 Introduction 2 Oral cancer is overall the eighth most common malignancy and the most prevalent disease in 3 head and neck tumors, with 170,000 deaths and over 350,000 newly added cases in 2018 alone 4 worldwide(1,2). Squamous cell carcinoma accounts for over 95% of oral cancer, which is 5 characterized by robust aggressiveness and frequent metastasis and recurrence(3). Although 6 recent years have witnessed remarkable improvements in diagnosis and treatment, 5-year 7 survival rate of OSCC patients remains under 50% and lack of early detection is responsible for 8 the high mortality (4,5). Therefore, understanding the mechanisms of progression and metastasis 9 of OSCC is an important goal in OSCC intervention. 10 11 Circular RNAs (circRNAs) are a recently re-discovered type of endogenous non-coding RNA 12 that are generated from back-splicing of precursor mRNAs and features distinguishable single- 13 stranded, covalently closed loops, lacking 5’-3’ polarity(6,7). Extensively distributed in 14 cytoplasm, this unique structure makes circRNA more resistant to exonucleases and more stable 15 than their linear RNA counterparts(8). For decades, circRNAs were nearly unexplored due to 16 facts that circRNA-specific back-splicing junction reads do not map to a linear reference genome 17 and circRNAs do not have poly(A) tails and are therefore not detectable in most early RNA-seq 18 datasets where poly(A)-enrichment is part of the library preparation protocol. Over the past few 19 years, circRNAs have gained enormous investigations. Despite controversy and skepticism still 20 hovering over circRNA research realm(9), there are increasing amount of studies showing that 21 circRNAs have great potential as miRNA sponges in a complementary base-pairing manner, 22 suppressing miRNA activity and thereby promoting translation and stability of downstream 23 target genes(10). Such a circRNA/miRNA/mRNA signaling axis has been demonstrated to exert 24 multiple functions in a broad variety of physiological and pathological conditions, particularly in 25 cancers(11-13). Accumulating research has identified numerous non-coding RNAs that 26 participate in OSCC progression regulation, including miR-23a-3p which suppresses tumor 27 growth in OSCC via targeting fibroblast growth factor 2 (FGF-2)(14) and lncRNA-p23154 28 whose inhibitory action over miR-378a-3 facilitates GLUT-1 expression, therefore promoting 29 invasion and metastasis of OSCC(15). With a growing body of reports that indicate emerging 30 roles and functions of circular RNAs in OSCC, how circRNAs pathways delicately regulate 31 OSCC progression still remains largely enigmatic and needs to be further explored. 32 33 Human immunoglobulin heavy chain G (IGHG) gene, located in chromosome region 14q32, is 34 known for polymorphism across populations and critically involved in immune response and 35 human survival(16,17). Although not quite often associated to cancers, dysregulation of IGHG 36 was reported in multiple malignancies, including lung cancer, breast cancer and gastric 37 cancer(18-20). In this study, we identified a unique IGHG-derived circular RNA, 38 hsa_circ_0000579 (termed circIGHG), whose upregulated expression was frequently observed in 39 OSCC and associated with poor prognosis. Further investigations demonstrated that circIGHG 40 facilitates epithelial-mesenchymal transition (EMT) by regulating IGF2BP3 via directly 41 sponging miR-142-5p, thereby leading to increased aggressiveness of OSCC. Those findings 42 endow circIGHG promising potentials in serving as a novel diagnostic and therapeutic marker in 43 OSCC. 44 Materials and Methods 45 RNA isolation, RNase R treatment, and PCR 3 Downloaded from cancerres.aacrjournals.org on October 4, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on November 17, 2020; DOI: 10.1158/0008-5472.CAN-20-0554 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 1 Total RNA was isolated using RNAprep Pure kits (DP430, DP431, Tiangen, China) according to 2 the manufacturer’s guidelines from either liquid nitrogen-frozen tissues or cultured cells. 3 Concentration and purity for each RNA extract was checked by 260/280 ratio on a NanoDrop 4 2000 instrument (Thermofisher, US) using RNA-specific protocol. First strand cDNA synthesis 5 was achieved by using FastKing RT Kit (KR116, Tiangen, China). Quantitative PCR was 6 performed with the Talent qPCR premix Kit (FP209, Tiangen, China) following the product’s 7 instructions. Primers used in this study were designed using Primer Bank 8 (https://pga.mgh.harvard.edu/primerbank/) and Primer3Plus (V2.5.0). Primer sequences were 9 listed in Supplementary file 1: Table S1. Three technical replicates were set for each single 10 reaction to ensure reliability and validity. Reactions were programmed for 1 cycle of 11 denaturation for 1.5 minutes at 95°C, followed by 40 cycles of extension including 30 seconds at 12 95°C, 50 seconds at 57°C and 1.5 minutes at 72°C. The ΔΔCT value of the target genes’ 13 expression was measured and assessed against the value of the reference gene GAPDH. 14 15 RNase R treatment was performed by incubating 2μg of total RNA mixture at 37°C for 10 16 minutes, as suggested by the product’s manual (RNR07250, Lucigen, US). Following removal of 17 linear forms, RT-qPCR qualification was performed to confirm the circular structure of 18 circIGHG. Sanger sequencing covering the back-splicing junction sequence was performed using 19 the RT-qPCR product of circIGHG (Geneseed, China). 1% agarose gel was used for DNA 20 electrophoresis. 21 22 RNA FISH 23 RNA FISH was performed using the Fluorescent in situ Hybridization Kit (C10910, RiboBio, 24 China) according to the manufacturer’s instructions. Cy3-labeled probes targeting circIGHG and 25 Dig-labeled miR-142-5p probes (see Supplementary file 1: Table S2) were synthesized by 26 RiboBio Technology Co. Ltd (China). Fluorescence was excited and recorded with a Zeiss 27 confocal laser scanning microscope (LSM 880 with Airyscan, Carl Zeiss, Germany). 28 29 RNA pull-down assay 30 RNA pull-down assay was performed referring to Lal A’s introduction(21). Biotinylated 31 circIGHG and miR-142-5p probes were synthesized and transfected into CAL-27 cells, 32 according to the manufacturer’s protocol (RiboBio, Guangzhou, China).