INTERNATIONAL JOURNAL OF ONCOLOGY 57: 1157-1168, 2020 lncRNA LUNAR1 accelerates colorectal cancer progression by targeting the miR‑495‑3p/MYCBP axis JIAJIE QIAN1, ALOK GARG2, FUQIANG LI3, QIANYUN SHEN1 and KE XIAO4 1Department of Gastrointestinal Surgery, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, Zhejiang 310003, P.R. China; 2Department of Surgery, City Hospital Braunschweig, D-38118 Braunschweig, Germany; 3Department of Thyroid Surgery, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, Zhejiang 310003, P.R. China; 4Institute of Molecular and Translational Therapeutic Strategies (IMTTS), Hannover Medical School, D-30625 Hannover, Lower Saxony, Germany Received April 16, 2020; Accepted September 14, 2020 DOI: 10.3892/ijo.2020.5128 Abstract. Colorectal cancer (CRC) is a tumor type and functional research showed that LUNAR1 accelerated characterized by high patient morbidity and mortality. It has CRC progression via the miR-495-3p/MYCBP axis. In been reported that long non-coding (lncRNA) LUNAR1 conclusion, LUNAR1 accelerates CRC progression via the (LUNAR1) participates in the regulation of tumor progression, miR-495-3p/MYCBP axis, indicating that LUNAR1 may such as diffuse large B-cell lymphoma. However, its role and serve as a prognostic biomarker for CRC patients. underlying mechanisms in CRC progression have not been elucidated. The present study was designed to investigate Introduction the underlying mechanisms by which LUNAR1 regulates CRC progression. RT-qPCR and Pearson's correlation Colorectal cancer (CRC) is a tumor type characterized by analysis revealed that LUNAR1 was highly expressed and high patient morbidity and mortality (1). At present, surgery, was negatively associated with the overall survival of CRC radiotherapy and chemotherapy are the primary strategies for patients. Moreover, CCK-8, clone formation, wound-healing CRC treatment (2). Although some progress has been made migration, Transwell chamber and FACs assay analyses in regards to CRC treatment on account of improvement in showed that LUNAR1 knockdown inhibited CRC cell the medical level, the strong metastatic tendency of CRC still proliferation, migration and invasion, while accelerating cell increases the risk of patient death (3). Notably, drug resistance apoptosis. Additionally, LUNAR1 was found to function as and the generation of side effects are the primary obstacles for a sponge of miR-495-3p, which was predicted by TargetScan CRC treatment (4). Therefore, investigating the pathological and confirmed by luciferase reporter assay. Furthermore, progression of CRC is essential and beneficial to investigate functional studies indicated that miR-495-3p overexpression new diagnostic and treatment strategies for CRC. inhibited CRC cell proliferation, migration and invasion, Long non-coding RNAs (lncRNAs), working as pivotal while accelerating cell apoptosis. In addition, bioinformatics intracellular regulatory molecules, have functional bioactivity and luciferase reporter assays showed that miR-495-3p was in various physiological processes (5). Crucially, lncRNAs have found to negatively target Myc binding protein (MYCBP), been proven to be pivotal regulators in tumor progression. In gastric cancer, lncRNA MAGI2-AS3 was found to be primarily distributed in the cytoplasm and was found to accelerate tumor progression by interacting with miR-141/200a (6). In cervical Correspondence to: Dr Ke Xiao, Institute of Molecular and cancer, lncRNA SOX21-AS1 overexpression was found Translational Therapeutic Strategies (IMTTS), Hannover Medical to inhibit cervical cancer progression, and its methylation School, D-30625 Hannover, Lower Saxony, Germany was found to have clinical prognostic value (7). In prostate E-mail: [email protected] cancer, lncRNA SNHG7 was demonstrated to regulate tumor progression via the miR-324-3p/WNT2B axis (8). Consistently, Abbreviations: CRC, colorectal cancer; lncRNA, long non‑coding reports indicate that multiple lncRNAs participate in RNA; LUNAR1, lncRNA LUNAR1; ceRNA, competitive endogenous RNA; DMEM, Dulbecco's modified Eagle's the regulation of CRC processes. For example, lncRNA medium; FBS, fetal bovine serum; miRNA, microRNA; Cox‑2, LEF1-AS1 was found to accelerate CRC progression by cyclooxygenase 2; MMP‑2, matrix metalloprotein 2; MMP‑9, matrix targeting the miR-489/DIAPH1 axis (9). lncRNA MIR503HG metalloprotein 9; MYCBP, Myc binding protein alleviated CRC progression by downregulating TGF-β2 expression (10). In addition, lncRNA TTN-AS1 accelerated Key words: LncRNA LUNAR1, miR-495-3p, MYCBP, colorectal CRC progression by sponging miR-376a-3p (11). LUNAR1 is cancer a 491-nucleotide transcript located at 15q26.3, with 4 exons and a poly(A) tail (12). Increasing evidence indicates that LUNAR1 participates in tumor progression. In diffuse large 1158 QIAN et al: ROLE OF lncRNA LUNAR1 IN CRC PROGRESSION B-cell lymphoma, LUNAR1 was found to serve as a candidate consent of the patients was obtained. Multivariate analysis was prognostic biomarker via growth regulation (13). Moreover, performed to identify factors associated with overall survival the novel Notch-induced LUNAR1 promoted the proliferation using the Cox proportional hazards model. of cancer cells, and may serve as a prognostic marker for CRC (14). These findings showed that LUNAR1 may act as an Tissue specimens. Tumor tissues or corresponding paracan- oncogene and plays an important role in cancer progression. cerous tissues were obtained by surgical extraction from However, to date, the roles of LUNAR1 in CRC progression 15 CRC patients (age range, 25‑60 years, average age, 42; have not been fully elucidated. 7 males and 8 females) at The First Affiliated Hospital, College A microRNA (miRNA/mRNA) is defined as a series of of Medicine, Zhejiang University (Hangzhou, Zhejiang, China) non-coding RNA sequences of approximately 18-22 bp (15). between March 2018 and March 2019. All experimental Reports indicate that miRNAs play essential roles in gene protocols were approved by the Ethics Committee of The First expression via regulating post-transcriptional translation, Affiliated Hospital, College of Medicine, Zhejiang University which may be involved in the regulation of the progression (Zhejiang, China; ethical approval no. PRO20180916‑R1) and of various diseases (16). Research has demonstrated that experimental procedures were conducted according to the miR-495-3p participates in the regulation of the progression Declaration of Helsinki Principles. of multiple diseases. For example, miR-495-3p was found to inhibit rifampicin-induced SULT2A1 expression via accel- Cell culture. CRC cells lines, including HT29, LoVo, SW480, erating the degradation of mRNAs, which in turn reduces SW620 cells and normal HIEC cells which served as the hepatocellular toxicity (17). Increasing evidence indicates control were obtained from Kunming Medical University that miRNAs might be sponged by lncRNAs to regulate (Kunming, Yunnan, China). Dulbecco's modified Eagle's disease-related gene expression, resulting in the regulation of medium (DMEM; Roche) supplemented with 10% fetal disease progression. Hence, lncRNAs may serve as competitive bovine serum (FBS) (Roche) and 1% penicillin‑streptomycin endogenous RNAs (ceRNAs) (18). For example, miR-495-3p is solution (Solarbio) was applied to the cultured cells in a humid sponged by lncRNA NEAT1 to regulate STAT3 expression, incubator containing 5% CO2 at 37˚C. resulting in alleviation of septicemia‑related inflammatory response (19). Moreover, it is worth noting that miR-495-3p Cell transfection. The transfection doses for pLKO.1 plasmid serves as an inhibitory regulator in CRC (20). However, the shRNAs targeting lncRNA LUNAR1, MYCBP and its negative relationship between LUNAR1 and miR-495-3p, and the control sh-NC (synthesized by Sangon Biotech) were 500 ng specific mechanisms involved remain unclear. for cells in each well of 6-well plates. The transfection doses C-myc binding protein (MYCBP) plays a vital role of miR-495-3p mimics or inhibitors (synthesized by Sangon in disease progression. MYCBP binds to the N-terminal Biotech), as well as their corresponding controls were 100 nM for region of MYC corresponding to the transactivation domain cells in each well of 6-well plates. The transfection was performed via its C-terminal region and stimulates the activation of E using Lipofectamine™ 3,000 Transfection Reagent (Takara). box-dependent transcription by c-MYC (21). In esophageal Following a 48-h transfection, the SW480 and LoVo cells were cancer, miR-26a and miR-26b inhibit tumor cell proliferation applied to subsequent experiments. Detailed sequences for these by inhibition of MYCBP expression (22). Overexpression of shRNAs, mimics and inhibitors are presented in Table I. MYCBP binding protein was found to promote the invasion and migration of gastric cancer (23). These findings indicate RT‑qPCR. Trizol reagent (Takara) was applied to extracted that MYCBP plays a carcinogenic role in most cancers. In the total RNA from CRC cell lines or tissues. M-MLV Reverse present study, we further investigated the specific mechanism Transcriptase (RNase H) kit (Takara) was performed to of MYCBP in CRC. synthesize cDNA. RT-qPCR was performed as previously In this research, we aimed to explore the role of LUNAR1 described (24). Primers applied to this research are shown in in CRC progression and the underlying mechanisms by evalu- Table II. ating the proliferation, migration,