Biological Characteristics of Chicken (Gallus gallus) ITM2A Gene and Its Role in Myoblast Proliferation and Differentiation
JIA Qi-Hui1,*, CAO Yu-Zhu1,*, XING Yu-Xin1, MA Cheng-Lin1, GUAN Hong-Bo1, WANG Qian1, KANG Xiang-Tao1,2,3, TIAN Ya-Dong1,2,3, LI Zhuan-Jian1,2,3, LIU Xiao-Jun1,2,3,**, LI Hong1,2,3,**
1 College of Animal Science and Technology, Henan Agricultural University, Zhengzhou 450046, China; 2 International Joint Research Laboratory for Poultry Breeding of Henan, Zhengzhou 450046, China; 3 Key Laboratory of Innovation and Utilization of Chicken Germplasm Resources in Henan Province, Zhengzhou 450046, China
Abstract The integral membrane protein 2A (ITM2A) plays an important role in various biological processes such as muscle development. Previous research results showed that the ITM2A gene is significantly related to chicken (Gallus gallus) body weight. However, the biological function of ITM2A in the proliferation and differentiation process of chicken skeletal muscle is not clear. In this study, bioinformatics analysis of chicken ITM2A protein was performed using online software. Six 7-day-old Arbor Acres Plus (AA) broilers were selected, and samples of heart, liver, spleen, lung, kidney, breast muscle, leg muscle were collected for tissue expression profile analysis. Breast muscle and leg muscle tissues at different developmental stages were selected for temporal expression analysis. The relative expression of ITM2A were detected using qPCR, and the ITM2A gene coding sequence was amplified using PCR technology to construct a recombinant plasmid for overexpression. Chicken primary myoblasts (CPMs) were isolated, and the relative expression of ITM2A at different stages of CPM induction differentiation were measured. The biological function of ITM2A was evaluated by assessing the expression of cell proliferation and differentiation marker genes in CPM cells overexpressing ITM2A. The bioinformatics analysis results showed that the ITM2A protein consisted of 251 amino acid residues, predominantly composed of α-helices (38.25%) and β-sheets (37.75%). It was an unstable, hydrophilic, non-secretory protein with a transmembrane structure, mainly located on the cell membrane, and might interact with proteins such as fin bud initiation factor homolog (FIBIN), ribosomal protein lateral stalk subunit P0 (RPLP0), and catenin alpha like 1 (CTNNAL1). It exhibited the highest homology with turkeys (Meleagris gallopavo) and the closest evolutionary relationship. Tissue expression analysis revealed that ITM2A was widely expressed in the examined tissues, with the highest expression in the lung and spleen and the lowest expression in pectoral and leg muscles. Temporal expression analysis showed that ITM2A expression gradually decreased with increasing days post-hatch, reaching its lowest point at 1 week post-hatch. Compared to the control group, the expression of ITM2A in the experimental group was extremely significantly increased (P<0.01). There were no significant changes in the relative expression levels of cell proliferation-related marker genes, while the expression levels of cell differentiation-related marker genes, such as myosin heavy chain (MYHC), myoblast determination (MYOD), and myomarker (MYMK), were significantly upregulated (P<0.05) in the overexpression group. The expression of the ITM2A gene showed a decreasing trend during CPM proliferation and an increasing trend with induction days. This study demonstrated that ITM2A could promote the differentiation of chicken myoblasts, providing fundamental information for further elucidating the molecular mechanisms through which ITM2A influences muscle development in chickens.
JIA Qi-Hui,CAO Yu-Zhu,XING Yu-Xin, et al. Biological Characteristics of Chicken (Gallus gallus) ITM2A Gene and Its Role in Myoblast Proliferation and Differentiation[J]. 农业生物技术学报, 2024, 32(11): 2593-2602.
[1] Abuderman A, Harb O, Gertallah L.2020. Prognostic and clinicopathological values of tissue expression of MFAP5 and ITM2A in triple-negative breast cancer: An immunohistochemical study[J]. Contemporary Oncology/Współczesna Onkologia, 24(2): 87-95. [2] Andrews S J, Rothnagel J A.2014. Emerging evidence for functional peptides encoded by short open reading frames[J]. Nature Reviews Genetics, 15(3): 193-204. [3] Barber T D, Barber M C, Tomescu O, et al.2002. Identification of target genes regulated by PAX3 and PAX3-FKHR in embryogenesis and alveolar rhabdomyosarcoma[J]. Genomics, 79(3): 278-284. [4] Barker S A, Fish F P, Tomana M, et al.1983. Gas chromatographic/mass spectrometric evidence for the identification of a heptitol and an octitol in human and Octodon degu eye lenses[J]. Biochemical and Biophysical Research Communications, 116(3): 988-993. [5] Cai B, Ma W, Ding F, et al.2018. The long noncoding RNA CAREL controls cardiac regeneration[J]. Journal of the American College of Cardiology, 72(5): 534-550. [6] Dave V D P, Joseph M.2004. Constitutive overexpression of the integral membrane protein Itm2A enhances myogenic differentiation of C2C12 cells[J]. Cell Biology International, 28(3): 199-207. [7] Dooley B J, Verma A, Ding R, et al.2020. Urinary cell transcriptome profiling and identification of ITM2A, SLAMF6, and IKZF3 as biomarkers of acute rejection in human kidney allografts[J]. Transplantation Direct, 6(8): e588. [8] Guerra San Juan I, Nash L A, Smith K S, et al.2022. Loss of mouse Stmn2 function causes motor neuropathy[J]. Neuron, 110(10): 1671-1688. [9] Guo Y, Zhang K, Geng W, et al.2023. Evolutionary analysis and functional characterization reveal the role of the insulin-like growth factor system in a diversified selection of chickens (Gallus gallus)[J]. Poultry Science, 102(3): 102411. [10] Lee N, Cho W, Shin H, et al.2020. SUN-724 Associations of GPR174 and ITM2A Genes on X Chromosome with Early Onset Autoimmune Thyroid Disease in Korean[J]. Journal of the Endocrine Society, 4(Supplement_1): SUN-724. [11] Liu D, Black B L and Derynck R.2001. TGF-β inhibits muscle differentiation through functional repression of myogenic transcription factors by Smad3[J]. Genes & Development, 15(22): 2950-2966. [12] Liu G, Ge C, Zhang X, et al.2008. Isolation, sequence identification and tissue expression distribution of three novel porcine genes-RAB14, S35A3 and ITM2A[J]. Molecular Biology Reports, 35: 201-206. [13] Mancinelli R, Pietrangelo T, Burnstock G, et al.2012. Transcriptional profile of GTP-mediated differentiation of C2C12 skeletal muscle cells[J]. Purinergic Signalling, 8: 207-221. [14] Mueller A C, Cichewicz M A, Dey B K, et al.2014. MUNC, a long noncoding RNA that facilitates the function of MyoD in skeletal myogenesis[J]. Molecular and Cellular Biology, 35(3): 498-513. [15] Namkoong S, Lee K I, Lee J I, et al.2015. The integral membrane protein ITM2A, a transcriptional target of PKA-CREB, regulates autophagic flux via interaction with the vacuolar ATPase[J]. Autophagy, 11(5): 756-768. [16] Nguyen T M H, Shin I W, Lee T J, et al.2016. Loss of ITM2A, a novel tumor suppressor of ovarian cancer through G2/M cell cycle arrest, is a poor prognostic factor of epithelial ovarian cancer[J]. Gynecologic Oncology, 140(3): 545-553. [17] Pittois K, Wauters J, Bossuyt P, et al.1999. Genomic organization and chromosomal localization of the Itm2a gene[J]. Mammalian Genome, 10: 54-56. [18] Tai T S, Pai S Y and Ho I C.2014. Itm2a, a target gene of GATA-3, plays a minimal role in regulating the development and function of T cells[J]. PLOS ONE, 9(5): e96535. [19] Zehentner B K, Leser U and Burtscher H.2000. BMP-2 and sonic hedgehog have contrary effects on adipocyte-like differentiation of C3H10T1/2 cells[J]. DNA and Cell Biology, 19(5): 275-281. [20] Zhu W, Lin Y, Liao H, et al.2015. Selection of reference genes for gene expression studies related to intramuscular fat deposition in Capra hircus skeletal muscle[J]. PLOS ONE, 10(3): e0121280.
