MOLECULAR MEDICINE REPORTS 14: 5637-5643, 2016 GRIN3A and MAPT stimulate nerve overgrowth in macrodactyly XU SHI1*, LU LU2*, XIU JIN3, BIN LIU4, XIGUANG SUN4, LAIJIN LU4 and YANFANG JIANG1,5 1Department of Genetic Diagnosis Center, Central Laboratory; Departments of 2Breast Surgery, 3Burn Surgery and 4Hand and Foot Surgery, The First Hospital of Jilin University, Changchun, Jilin 130000; 5Key Laboratory of Zoonosis Research, Ministry of Education, Institute of Zoonosis, Jilin University, Changchun, Jilin 130000, P.R. China Received September 30, 2015; Accepted October 12, 2016 DOI: 10.3892/mmr.2016.5923 Abstract. As an uncommon and congenital condition, to abnormal nerve proliferation and underpin the pathogenesis macrodactyly is characterized by an increase in the size of all of macrodactyly, and provide potential application targets in the elements or structures of the digits or toes; however, the nerve tissue regeneration engineering. underlying pathogenesis remains to be fully elucidated. In the present study, the gene expression profiles of abnormal nerves Introduction were examined in three patients with macrodactyly using microarray analysis to identify potential genes contributing Macrodactyly is an uncommon congenital condition character- to nerve overgrowth. Gene expression profiling in the nerve ized by an increase in the size of all the elements or structures tissue samples were scanned using the microarray and the of the digits or toes, including phalanges, tendons, vessels, differentially expressed genes were verified at the transcrip- subcutaneous fat and finger nails. The malformation often tion level using reverse transcription-quantitative polymerase occurs unilaterally or asymmetrically and affects more than chain reaction analysis. Western blot analysis was used to one digit or toe. The clinical conditions associated with this determine the expression of target genes at the translational deformity are carpal tunnel syndrome, syndactylism, neuro- level. To confirm the upregulated genes during the process of fibromatosis type 1 cafe au lait spots, lipoma and nevi (1-3). nerve proliferation, SH-SY5Y cells were induced to differ- The malformations in the fingers or toes are considered entiate into a neuronal-like phenotype using retinoic acid. A to be associated with other syndromes of macrodactyly, total of 165 genes showed significant changes ≥( 5-fold) in gene including vascular malformations, multiple enchondromatosis, expression, which may be associated with the development of maffuci syndrome, tuberous sclerosis and neurofibromatosis limbs in macrodactyly. Glutamate ionotropic receptor NMDA type 1 (4,5). The exact pathogenesis underlying this condi- 3A (GRIN3A) and microtubule‑associated protein tau (MAPT) tion and, in particular, the role of nerve growth stimulators were identified as important contributors in promoting nerve in macrodactyly, remains to be fully elucidated. The present overgrowth. Furthermore, it was identified that GRIN3A and study aimed to determine nerve overgrowth stimuli using gene MAPT were regulated by the cAMP-protein kinase A and expression profiling was performed in malformed enlarged extracellular signal-regulated kinase 1/2 pathways, respec- nerve tissues from the digits or toes of patients with macrodac- tively. The identification of genes expressed at high levels in tyly. A total of six overexpressed (>10‑fold) genes, including macrodactyly may reveal potential factors, which contribute creatine kinase, mitochondrial 2 (CKMT2), vasoactive intes- tinal peptide (VIP), FXYD domain-containing ion transport regulator 3 (FXYD3), Glutamate ionotropic receptor NMDA 3A (GRIN3A), GSTT1 and microtubule-associated protein tau (MAPT) were identified for their potential contribution to Correspondence to: Professor Yanfang Jiang, Department of abnormal nerve overgrowth. In addition, MAPT and GRIN3A Genetic Diagnosis Center, Central Laboratory, The First Hospital were identified as key regulators of nerve outgrowth. These of Jilin University, 71 Xinmin Street, Changchun, Jilin 130000, P. R. Ch i na nerve growth stimulators may contribute to nerve regeneration E-mail: [email protected] and reconstruction following nerve injury. Professor Laijin Lu, Department of Hand and Foot Surgery, The Materials and methods First Hospital of Jilin University, 71 Xinmin Street, Changchun, Jilin 130000, China. Patients. E-mail: [email protected] The present study was reviewed and approved by the Ethics Committee of the First Hospital of Jilin Univer- *Contributed equally sity, (Changchun, China). Three male patients between the ages of 17 and 25 were recruited from the First Hospital of Key words: macrodactyly, nerve overgrowth, gene expression, Jilin University between May 2011 and September 2013. All microarray enrolled patients underwent surgical management for isolated nonsyndromic macrodactyly. Normal nerve tissue samples, which served as healthy controls, were obtained from five 5638 SHI et al: GRIN3A AND MAPT STIMULATE NERVE OVERGROWTH Table I. Primer sequences used in reverse transcription-polymerase chain reaction analysis. Gene Accession no. Forward primer Reverse primer CKMT2 NM_001825.2 5'-GCCGCAATGCTTCTCTG-3' 5'-GGCCATCCCAGCACAT-3' VIP NM_003381.3 5'-TCCTTGTGCTCCTGACTC-3' 5'-CTGCTCCTCTTTCCATTC -3' FXYD3 NM_005971.3 5'-GTGACCCTGGGCCTGCT-3' 5'-CAGCTTTGGGCTGAGCCT-3' GRIN3A NM_133445.2 5'-GGAGGTAGATGATGAAGGC-3' 5'-AAACAAGAGGGCATAACAG-3' GSTT1 NM_000853.2 5'-ATCTTTGCCAAGAAGAACG-3' 5'-TGTGAGGACCAGTAAGGAAG-3' MAPT NM_016835.4 5'-GCCAAAGGGCAGGATG-3' 5'-TTCGGGAAGTGACAGAAGAG-3' GAPDH NM_002046 5'-GGAGTCAACGGATTTGGTC-3' 5'-CCCCAGCCTTCTCCAT-3' CKMT2, creatine kinase, mitochondrial 2; VIP, vasoactive intestinal peptide; FXYD3, FXYD domain-containing ion transport regulator 3; GRIN3A, glutamate ionotropic receptor NMDA 3A; GSTT1, glutathione S-transferase θ 1; MAPT, microtubule-associated protein tau. patients undergoing elective surgery for unrelated reasons, the differentiated cells were treated with protein kinase A including road traffic accidents or fires. Written informed (PKA) inhibitor H89 or extracellular signal‑regulated kinase consent was obtained from guardians on the behalf of all (ERK)1/2 inhibitor PD98059 (Beyotime Institute of Biotech- participants. nology, Haimen, China) for up to 4 h at 37˚C. Tissue treatment and reverse transcription‑quantitative Western blot analysis. The cells were collected and treated polymerase chain reaction (RT‑qPCR) analysis. A sample w it h lysi s bu f fe r ( Beyot i m e I n st it ut e of Biot e ch nolog y) sup ple - (~10 mg) of enlarged nerve tissue from a digit or toe was mented with 1% protease inhibitor mixture (Sigma‑Aldrich; obtained from each of the patients with macrodactyly, which Merck Millipore). Subsequently, cell lysates were centrifuged were sectioned into smaller sections (~3 mm3), snap frozen at 10,000 x g for 15 min at 4˚C. Protein quantification was in liquid nitrogen and stored at ‑80˚C until further use. The performed using BCA Protein Quantification kit (Thermo frozen tissues were homogenized in cold normal saline (0.9% Fisher Scientific, Inc.) and 30 µg protein per lane were sepa- NaCl), using a ULTRA-TURRAX Tube Drive Workstation rated on 10% SDS PAGE gel and then transferred onto PVDF (IKA Werke GmbH & Co. KG, Staufen, Germany). Next, membranes (Invitrogen; Thermo Fisher Scientific, Inc.). The RNA was extracted from the nerve tissue samples using membranes were incubated with 5% skimmed dry milk for TRIzol (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, 30 min and washed three times with TBST (10 mM Tris, MA USA) according to the manufacturer's protocol. A total of 150 mM NaCl and 0.1% Tween‑20). Next, the membranes 500 ng RNA was used for reverse transcription was performed were incubated with rabbit anti-human GRIN3A polyclonal using the GoScript Reverse Transcription system (Promega antibody (cat. no. sc-98986; 1:5,000) and rabbit anti-human Corporation, Madison, WI, USA). Next, the products were MAPT polyclonal antibody (cat. no. sc‑32828; 1:1,000) at amplified using Power SYBR Green Master Mix (containing 4˚C overnight. Then the goat anti‑rabbit IgG‑HRP secondary SYBR Green I Dye, AmpliTaq Gold® DNA Polymerase, antibody (cat. no. sc‑2302; 1:1,000) was used to incubate the dNTPs, passive reference and optimized buffer) on an ABI membranes for 2 h at 37˚C. All antibodies were purchased 7300 (Applied Biosystems; Thermo Fisher Scientific, Inc.). from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). RT‑qPCR was performed with an initial 10 min at 95˚C Densitometry scores were determined using Quantity One followed by 40 cycles of 95˚C for 15 sec, and 60˚C for 1 min. software, version 4.6.9 (Bio‑Rad Laboratories, Inc., CA, All experiments were repeated three times. Relative gene USA). expression was calculated with the 2-ΔΔCq method (6) following normalization to the expression of GAPDH. The primers used Microarray. The RNA from the nerve tissue samples was for PCR are listed in Table I. hybridized and scanned using the Agilent Microarray Scanner (Agilent Technologies, Inc., Santa Clara, CA, USA) at the Cell culture. The SH-SY5Y cell line was obtained from the National Engineering Center for Biochip (Shanghai, China), Cell Bank of the Chinese Academy of Sciences (Shanghai, according to the manufacturer's protocol. The raw data were China), and cultured in Dulbecco's modified Eagle's medium obtained using Feature Extraction 10.7 software (Agilent supplemented with 10% fetal bovine serum (all from Gibco; Technologies,