ONCOLOGY LETTERS 22: 697, 2021 A micro‑peptide encoded by HOXB‑AS3 promotes the proliferation and viability of oral squamous cell carcinoma cell lines by directly binding with IGF2BP2 to stabilize c‑Myc FEI LENG, YAN‑YU MIU, YI ZHANG, HAO LUO, XIAO‑LI LU, HUI CHENG and ZHI‑GUO ZHENG The Affiliated Stomatological Hospital of Nanchang University, The Key Laboratory of Oral Biomedicine, Nanchang, Jiangxi 330006, P.R. China Received April 17, 2021; Accepted June 18, 2021 DOI: 10.3892/ol.2021.12958 Abstract. HOXB‑AS3 is a long non‑coding RNA and that increased HOXB‑AS3 expression was associated with recent studies have shown that the HOXB‑AS3‑encoded poor prognosis in OSCC. This indicated that HOXB‑AS3 and micro‑peptide was associated with the progression of colon its encoded protein promoted OSCC cell proliferation and cancer tumorigenesis; however, the biofunction of HOXB‑AS3 viability by maintaining c‑Myc mRNA stability by directly varies in different types of cancer and the potential function in binding to IGF2BP2. oral squamous cell carcinoma (OSCC) is still unknown. The Cancer Genome Atlas (TCGA) database was searched and the Introduction expression patterns of HOXB‑AS3 in head and neck carci‑ noma were analyzed. Reverse transcription‑quantitative PCR Oral cancer is the 6th most common malignancy worldwide and and western blot analysis was used to measure the mRNA oral squamous cell carcinoma (OSCC) represents 90% of all and protein expression level of HOXB‑AS3 in patients with cancer cases (1). The current therapy is only effective for early OSCC, respectively. Next, HOXB‑AS3 was knocked down stage OSCC, and the 5‑year overall survival rate was <50% in in 2 OSCC cell lines to investigate the biological function 2020 (2). Studies have indicated that tobacco addiction, exces‑ of the HOXB‑AS3‑encoded protein using a Cell Counting sive alcohol consumption and human papillomavirus (HPV) Kit‑8 and colony formation assays. To further identify the infection are the main risk factors for tumorigenesis and the potential mechanism of the HOXB‑AS3‑encoded protein, development of OSCC (3). Current targeted therapies, such as co‑immunoprecipitation was also used to detect the interaction targeting PD1/PDL1, are far from satisfactory (4); therefore; between HOXB‑AS3 and IGF2BP2, while HOXB‑AS3 was new predictive biomarkers and therapeutic targets are urgently re‑expressed to determine whether the HOXB‑AS3‑encoded required. protein and not HOXB‑AS3 exerted its function in OSCC. Long non‑coding (lnc)RNAs are 200 nucleotides HOXB‑AS3 was upregulated in OSCC tissues, in both TCGA in length (5). With the application of high‑throughput database and in patients with OSCC recruited into the present sequencing, lncRNAs have been reported to be associated study. HOXB‑AS3 was associated with poor prognosis in with the progression of numerous types of cancer. Rs6695584 OSCC. The proliferation and viability decreased in the 2 OSCC single nucleotide polymorphism‑induced lncRNA‑SLCC1 cell lines following knock down of HOXB‑AS3. HOXB‑AS3 drove colorectal cancer by activating glycolysis signaling (6). was also found to encode a protein that directly interacted lncRNA lnc030 maintained breast cancer stemness by stabi‑ with IGF2BP2 and thereby promoted the stability of c‑myc. lizing SQLE mRNA and therefore promoted cholesterol Taken together, the results from the present study indicated synthesis (7). lncRNA CRNDE attenuated chemoresistance in gastric cancer by SRSF6‑regulated alternative splicing of PICALM (8). HOXB‑AS3 was previously reported to encode a micro‑peptide and to suppress tumor progression in colon cancer (9). However, biofunction varies in different types of Correspondence to: Professor Zhi‑Guo Zheng, The Affiliated cancer (10,11). Its potential function and underlying mecha‑ Stomatological Hospital of Nanchang University, The Key nism in OSCC are still unknown. Laboratory of Oral Biomedicine, 49 Fuzhou Road, Nanchang, Jiangxi 330006, P.R. China In the present study, the expression level of HOXB‑AS3 E‑mail: [email protected] in patients with OSCC was analyzed and its biological func‑ tions and underlying mechanism in OSCC cell lines was Key words: lncRNA, HOXB‑AS3, OSCC, proliferation, c‑Myc, also investigated. The results demonstrated, to the best of IGF2BP2 knowledge, for the first time that HOXB‑AS3 and its encoded protein were upregulated in OSCC tumors. Knocking down HOXB‑AS3 repressed OSCC cell proliferation and viability, 2 LENG et al: HOXB‑AS3 PROMOTES THE PROGRESSION OF OSCC and a Co‑immunoprecipitation (IP) assay indicated that the TCC CTC CAA AAT ‑3' and reverse 5'‑GGC TGT TGT CAT ACT HOXB‑AS3‑encoded protein interacted with IGF2BP2; there‑ TCT CAT GG‑3' and the expression level was normalized to the fore, stabilized c‑Myc. control (13). Materials and methods Cell culture and transfection. The Cal‑27 and UM2 cell lines were gifts from Professor Wang from the Department of Oral Patient information and clinical sample collection. Paired and Maxillofacial Surgery, Affiliated Hospital of Jiangxi OSCC samples and adjacent normal tissues were randomly University of Traditional Chinese Medicine (Jiangxi, China). collected from patients who underwent resection surgery at The cells were cultured with complete DMEM (cat. no. D0697; the Affiliated Stomatological Hospital of Nanchang University Sigma‑Aldrich; Merck KGaA) containing 10% fetal bovine (Jiangxi, China) between January 2012 and December 2013. The serum (cat. no. F8687; Invitrogen; Thermo Fisher Scientific, following inclusion criteria was used: Diagnosed with OSCC Inc.) and 1% penicillin/streptomycin (cat. no. V900929; by pathologists between January 2012 and December 2013 at Sigma‑Aldrich; Merck KGaA). the Affiliated Stomatological Hospital of Nanchang University. To establish a stable cell line, lentivirus was obtained from Patients with metastasis were excluded. A total of 20 cases GenScript, and the following short hairpin (sh)RNAs were (age range, 42‑68 years; 15 male and 5 female) were divided used in pCDH‑CMV‑MCS‑EF1 + Puro (SBI pCD513B‑1): into 4 groups, according to their clinical features and the TNM Scrambled negative control (NC) 5'‑GTA CCT ACG TA‑3', stage system (12). There were 5 patients with stage I, 3 patients shRNA1, 5'‑CCA TCC AAG TCT A‑3' and shRNA2, 5'‑TAA with stage II, 8 patients with stage III and 4 cases with stage 4. TGC AGC GTA AG‑3'. ORF, the open read frame of HOXB‑AS3 Adjacent normal tissues were collected >5 cm from the tumor encoding the protein but not the whole lncRNA. Plasmids site and confirmed by a pathologist as normal tissue. All the (4 µg) were co‑transfected with packaging vectors psPAX2 OSCC tissue samples were confirmed by pathology. The (Addgene, Inc.) (1 µg) and pMD2G (Addgene, Inc.) (3 µg) into present study was approved by the Ethics Committee of The 293T (cat. no. CRL‑11268; ATCC) cells for lentivirus produc‑ Affiliated Stomatological Hospital of Nanchang University tion using Lipofectamine 3000® (Thermo Fisher Scientific (Jiangxi, China). The clinical samples were collected from Inc.) in accordance to the manufacturer's instructions. After patients following written informed consent. 72 h, the suspension was collected. The lentivirus was stored at 1x108/ml. The cells were seeded into the 6‑well plate with 40% Survival analysis. Using data from The Cancer Genome Atlas density and incubated overnight at 37˚C, following which 10 µl (TCGA) database, the time of the end point of each patient (12 MOI) each lentivirus was added into the corresponding was collected, this was defined as the survival end point of well and incubated at 37˚C for 3 days to successfully establish each patient, all the survival end points in of the whole cohort the stable cell line. No selection method was used due to the was defined as overall survival. The overall survival analysis high transfection rate. was thus automatically determined using the Gene Expression Profiling Interactive Analysis online tool by analyzing Western blot analysis. The cells were harvested and washed these data (gepia.cancer‑pku.cn) by inputing the gene name 3 times with ice cold PBS and lysed with RIPA buffer (HOXB‑AS3) and cancer type (OSCC), 519 patients were (cat. no. 20‑188; Sigma‑Aldrich; Merck KGaA). The protein enrolled and 44 normal tissues were collected. For the patients was quantified using the BCA kit (Beyotime Institute of recruited into the present study, the survival time was collected Biotechnology), according to the manufacturer's protocol. between 2012 and 2018. From the patients recruited into the Equal amounts of protein (40 µg/lane) were separated using present study, they were divided into 2 groups according to 12% SDS‑PAGE, then transferred onto a PVF membrane and the mean mRNA expression (3.56) and Kaplan‑Meier survival blocked with 5% skimmed milk for 1 h at room temperature. analysis was performed and the GraphPad software v.7.0 The membranes were subsequently incubated with the primary (GraphPad Software, Inc.). antibodies overnight at 4˚C, washed with 0.1% TBS‑Tween‑20, then incubated with the donkey anti mouse/rabbit secondary Reverse transcription‑quantitative analysis (RT‑qPCR) antibody (1:10,000; cat. no. 5724; KPL, Inc.) for 1 h at room analysis. Tissues and cells were harvested and lysed using temperature. The target proteins were detected using an ECL TRIzol® (cat. no. 93289; Sigma‑Aldrich; Merck KGaA) (EMD Millipore) kit. The following primary antibodies were following the manufacturer's protocol. After purification and used: HOXB‑AS3 (1:1,000; cat. no. SBSN250; Genscript), reverse transcription (60˚C for 5 min, 37˚C for 15 min and β‑actin (cat. no. 3700; 1:1,000; Cell Signaling Technology, 85˚C for 5 sec) cDNA was collected. Specific primers were Inc.), IGF2BP2 (cat. no. ab109284; 1:1,000; Abcam), and used for qPCR using the SYBR‑Green RT‑PCR system with c‑Myc (cat.