Feng et al. Journal of Experimental & Clinical Cancer Research (2020) 39:157 https://doi.org/10.1186/s13046-020-01652-5 RESEARCH Open Access A SNP-mediated lncRNA (LOC146880) and microRNA (miR-539-5p) interaction and its potential impact on the NSCLC risk Tienan Feng1,2†, Nannan Feng1†, Tengteng Zhu1, Qiang Li1, Qi Zhang1, Yu Wang1, Ming Gao3, Baosen Zhou4, Herbert Yu5, Min Zheng6 and Biyun Qian1,7* Abstract Background: Many cancer-associated single nucleotide polymorphisms (SNPs) are located in the genomic regions of long non-coding RNAs (lncRNAs). Mechanisms of these SNPs in connection to cancer risk are not fully understood. Methods: Association of SNP (rs140618127) in lncRNA LOC146880 with non-small cell lung cancer (NSCLC) was evaluated in a case-control study of 2707 individuals. The mechanism of the SNP’s biologic influence was explored with in vitro and in vivo experiments, including plasmid transfection, siRNA knockdown, flow cytometry assessment, and assays of cell proliferation, migration, invasion, and colony formation. Results: Association analysis showed that A allele of SNP rs140618127 was associated with low risk of NSCLC in the Chinese population. Lab experiments indicated that SNP rs140618127 contained a binding site for miR-539-5p and the binding between miR-539-5p and LOC146880 resulted in declined phosphorylation of an oncogene, ENO1. The reduced phosphorylation of ENO1 led to decreased phosphorylation of PI3K and Akt, which is further linked to the decline in cell proliferation and tumor progression. Conclusion: The study demonstrates that SNP rs140618127 in lncRNA loc146880 provides an alternate binding site for microRNA miR-539-5p which affects the phosphorylation of ENO1 and activation of the PI3K and Akt pathway. Keywords: NSCLC, lncRNA, SNP, miRNA, ENO1 Background lung cancer cases. Genetic factors may play an important Lung cancer is the most commonly diagnosed cancer role in an individual’s susceptibility to NSCLC. Long (11.6% of the total cases) and the leading cause of cancer non-coding RNAs (lncRNAs) are a class of non-coding death (18.4% of the total cancer deaths) in the world [1]. transcripts with 200 nucleotides or more. Increasing The majority of lung cancer is non-small cell lung evidence suggests that lncRNAs are involved in the cancer (NSCLC), which accounts for around 85% of all occurrence of lung cancer due to their functions as oncogenes or tumor suppressors [2]. Our previous stud- ies indicated that a lncRNA on chromosome 17q24.3, * Correspondence: [email protected] †Tienan Feng and Nannan Feng contributed equally to this work. named LOC146880, was expressed higher in tumor 1Hongqiao International Institute of Medicine, Shanghai Tongren Hospital/ tissues than in adjacent normal tissues and high expres- Clinical Research Institute, Shanghai Jiao Tong University School of Medicine, sion was associated with poor prognosis of NSCLC [3]. Shanghai 200025, China 7Second Affiliated hospital of Chengdu Medical College, China National Single-nucleotide polymorphisms (SNPs) in the non- Nuclear Corporation 416 Hospital, Chengdu 610051, Sichuan, China coding regions of the genome have been shown to affect Full list of author information is available at the end of the article © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Feng et al. Journal of Experimental & Clinical Cancer Research (2020) 39:157 Page 2 of 12 cancer risk via regulating the transcription and/or chan- (Applied Biosystems). For quality control, we imple- ging the structure of lncRNA [4–7]. A previous study mented several measures in our genotyping assays, in- identified 495,729 SNPs in more than 30,000 human cluding 1) each plate contained both case and control lncRNAs, and a large number of SNPs were predicted to samples, 2) technicians were blinded to the case/control have a potential impact on the microRNA (miRNA)- status of the samples, 3) both positive- and negative- lncRNA interaction [8]. Here we report the identification control (no DNA template) samples were included in of SNP rs140618127 in LOC146880, as a new susceptible each 384-well plate, and 4) nearly 8% of the samples locus to NSCLC. Bioinformatics analysis predicts that were assayed in duplicate and the concordances were variant rs140618127 (the ‘A’ allele) in LOC146880 between 99.7 and 100%. provides an altered secondary structure which may create a binding site for microRNA miR-539-5p [8], Cell lines sequestering its action on other molecules. Shiraishi Human NSCLC cell lines (A549 and PC9) and human et al. conducted a GWAS on lung adenocarcinoma and lung epithelial BEAS-2B cells were purchased from the identified SNP rs7216064 in BPTF (17q24.3) in associ- Cell Bank of Type Culture Collection at the Chinese ation with the cancer risk (OR = 1.20, p = 7e-11) [9]. Academy of Sciences Shanghai Institute of Biochemistry Seow et al. confirmed that SNP rs7216064 was associ- and Cell Biology. These cell lines were passaged for ated with the risk of lung cancer based on a GWAS fewer than 6 months. All the cells were tested for myco- study of Asian female non-smokers [10]. We found that plasma and were found to be free from infection. The SNP rs140618127 was in strong linkage disequilibrium cells were maintained in DMEM supplemented with with SNP rs7216064 (LD; r2 > 0.80), and this lncRNA 10% FBS and grown without antibiotics in an atmos- SNP was associated with lung cancer risk (OR = 0.38, phere of 5% CO2 and 99% relative humidity at 37 °C. p = 0.007) in our case-control study of 2707 individuals. To explore the molecular mechanism of SNP 5′ and 3′ RACE and coding prediction of LOC146880 rs140618127 in NSCLC development and progression, We used 5′ and 3′ RACE to determine the transcrip- we evaluated that the biological consequence of tional initiation and termination sites of LOC146880 LOC146880 and miR-539-5p interaction, and found that with a SMARTe RACE cDNA Amplification kit (Clon- the microRNA behaved like a tumor suppressor [11], tech). The Alignment File of a full-length sequence of which prevented LOC146880 from interacting with LOC146880 obtained from 5′ and 3′ RACE is available protein ENO1, an oncogene product [12], reducing its upon request. phosphorylation. As a result, the phosphorylation of PI3K/AKT was also reduced after the suppression of Construction of reporter plasmids, transient transfections ENO1 phosphorylation [13], which further inhibited and luciferase assays tumor growth and metastasis, leading to a better A reporter plasmid in the psiCHECK-2 vector (Promega) prognosis of NSCLC. was created which contains a 1000-bp LOC146880 exon region flanking rs140618127 [G] or rs140618127 [A] Materials and methods with the restriction enzymes XhoI and NotI (Fermentas). Study populations A549 and PC9 cells were seeded at 1 × 105 cells per well Suspected NSCLC individuals had histopathological or in 24-well plates, and 800 ng of the reporter plasmid and cytologically confirmed diagnosis according to the 40 pmol of miR-539-5p mimic (Ambion) were co- World Health Organization classification. These study transfected into the cells 16 h later using Lipofectamine subjects including suspected individuals diagnosed with 2000 (Invitrogen). These cells were collected 24 h after lung cancer or normal were recruited from the China transfection. Renilla luciferase activity was measured and Medical University (CMU). Distributions of the basic used to normalize the efficiency of transfection. characteristics of the study subjects are provided in Table 1. At recruitment, an informed consent was RNA extraction and qRT-PCR analysis Illuminated. Only if the subject agreed, he/she was Total RNA from the NSCLC tissue specimens and cell included. This study was approved by the Institutional lines used in this study was extracted using the TRIzol Review Board at CMU. reagent. First-strand cDNA was synthesized using the SuperScript II reverse transcriptase kit (Invitrogen). SNP selection and genotyping Relative RNA levels determined by qPCR were measured SNPs with r2 > 0.8 were considered to be in the same LD on an ABI 7500 sequence detection system (Applied block. With this criterion, one SNP was selected in each Biosystems) using the SYBR Green method. Βeta-actin LD block and genotyped using the TaqMan genotyping was employed as an internal control for the quantifica- method in the ABI 7500 Real-Time PCR system tion of LOC146880 and the mRNA levels of other genes. Feng et al. Journal of Experimental & Clinical Cancer Research (2020) 39:157 Page 3 of 12 For miRNA quantification, small nuclear RNA U6 was Plasmid construction and transfection used as an endogenous control. The relative expression To construct a lentiviral vector expressing human of RNA was calculated using the comparative Ct LOC146880 (NR_026899), a full-length of LOC146880 method. cDNA containing rs140618127 [G] or rs140618127 [A] was commercially synthesized (GeneChem) and sub- Subcellular fractionation cloned into the AgeI and NheI sites of the GV367-IRES- Cytosolic and nuclear fractions of A549 and BEAS-2B Puromycin lentiviral expression vector (GeneChem).