湖羊PSMB9基因克隆、序列分析及对成肌细胞增殖的影响

doi:10.16431/j.cnki.1671-7236.2025.01.001

Abstract

Abstract: 【Objective】 This study was aimed to investigate the sequence characteristics of coding region and promoter region of the proteasome 20S subunit beta 9 (PSMB9) gene in Hu sheep,and analyze its potential transcriptional regulation mechanism and its impact on myoblast cell proliferation. 【Method】 The CDS region of PSMB9 gene in Hu sheep was obtained by cloning and sequencing,the promoter region was determined by genome position in sheep,and the characteristics of CDS and promoter region were analyzed by bioinformatics method.The phylogenetic tree based on the amino acid sequence of PSMB9 was constructed using Neighbor-Joining (NJ) method of Mega 5.0 software.The tissue expression profile of PSMB9 gene in Hu sheep was detected by Real-time quantitative PCR.The overexpression vector of PSMB9 gene in Hu sheep was constructed by double enzyme digestion.CCK-8 kit were determined to detect the proliferative capacity,Real-time quantitative PCR was used to detect the overexpression effect of PSMB9 gene in C2C12 cells and the expression of proliferation gene PCNA. 【Result】 The results showed that the CDS region of PSMB9 gene in Hu sheep was 660 bp,encoding 219 amino acid residues,and the similarity of nucleotide and amino acid sequences with mammals including Homo sapiens,Bos taurus,Sus scrofa and Mus musculus were high.The active sites and beta subunit interaction sites of PSMB9 were highly conserved.Phylogenetic tree analysis found that Hu sheep first clustered with Bos taurus,and then clustered with Sus scrofa,Homo sapiens and Mus musculus,indicating that PSMB9 was relatively conserved in mammals.PSMB9 gene promoter contained one CpG island,two GC-boxes (CCGCCC) and two E-boxes (CANNTG),as well as potential binding sites for various transcription factors such as SP1,KLF4,STAT3,YY1 and CREB1.Tissue expression pattern showed PSMB9 gene was widely expressed in various tissues of Hu sheep,especially highly expressed in spleen and followed by muscle.Compared with control group,overexpression of PSMB9 gene in C2C12 cell lines could extremely significantly improve the proliferation ability of cells (P＜0.01),the expression of proliferation gene PCNA was extremely significantly upregulated (P＜0.01). 【Conclusion】 The sequence of PSMB9 gene in Hu sheep was successfully cloned in this study,PSMB9 gene was highly conserved in mammals,the promoter region of PSMB9 gene contained important regulatory elements,CpG island and potential binding sites of transcription factors.PSMB9 gene was highly expressed in spleen and muscle of Hu sheep,and overexpression of PSMB9 gene could promote the proliferation of myoblasts.The results provided a basis for further elucidating the molecular mechanism of PSMB9 gene regulation of muscle development in Hu sheep.

Key words: Hu sheep; PSMB9 gene; cloning; tissue expression pattern; C2C12 cell lines; cell proliferation

CLC Number:

S826
Q78

XIE Beiyiting, WANG Yue, MENG Chunhua, QIAN Yong, ZHANG Jun, ZHANG Jianli, WANG Huili, CAO Shaoxian, LI Yinxia. Cloning and Sequence Analysis of PSMB9 Gene in Hu Sheep and Its Effect on Myoblast Proliferation[J]. China Animal Husbandry and Veterinary Medicine, 2025, 52(1): 1-12.

References

[1] ABBAS R,LARISCH S.Killing by degradation:Regulation of apoptosis by the ubiquitin-proteasome system[J].Cells,2021,10(12):3465.
[2] BASHORE C,PRAKASH S,JOHNSON M C,et al.Targeted degradation via derect 26S proteasome recruitment[J].Nature Chemical Biology,2023,19(1):55-63.
[3] YERLIKAYA A,KANBUR E,STANLEY B A,et al.The ubiquitin-proteasome pathway and epigenetic modifications in cancer[J].Anticancer Agents in Medicinal Chemistry,2021,21(1):20-32.
[4] MAO Y.Structure,dynamics and function of the 26S proteasome[J].Sub-Cellular Biochemistry,2021,196:1-151.
[5] LUNDH M,BUGLIANI M,DAHLBY T,et al.The immunoproteasome is induced by cytokines and regulates apoptosis in human islets[J].Journal of Endocrinology, 2017,233(3):369-379.
[6] ELEFTHERIADIS T,PISSAS G,ANTONIADI G,et al.Proteasome or immunoproteasome inhibitors cause apoptosis in human renal tubular epithelial cells under normoxic and hypoxic conditions[J].International Urology Nephrology,2016,48(6):907-915.
[7] ROUETTE A,TROFIMOV A,HABERL D,et al.Expression of immunoproteasome genes is regulated by cell-intrinsic and -extrinsic factors in human cancers[J].Science Reports, 2016,6:34019.
[8] SHEN G,MAO Y,SU Z,et al.PSMB8-AS1 activated by ELK1 promotes cell proliferation in glioma via regulating miR-574-5p/RAB10[J].Biomedicine & Pharmacotherapy, 2020,122:109658.
[9] YANG B Y,SONG J W,SUN H Z,et al.PSMB8 regulates glioma cell migration,proliferation,and apoptosis through modulating ERK1/2 and PI3K/Akt signaling pathways[J].Biomedicine & Pharmacotherapy, 2018,100:205-212.
[10] DENG S,YANG C,NIE K,et al.Host cell protein PSMB10 interacts with viral NS3 protein and inhibits the growth of Classical swine fever virus[J].Virology, 2019,537:74-83.
[11] 牛佳兰.免疫蛋白酶体亚基PSMB10诱导急性髓系白血病细胞衰老抵抗[D].武汉:华中科技大学,2022. NIU J L.Immunoproteasome subunit PSMB10 induces cellular senescence resistance in acute myeloid leukemia[D].Wuhan:Huazhong University of Science and Technology,2022.(in Chinese)
[12] KIM S,SRRWA R A,SAMLUK L,et al.Immunoproteasome-specific subunit PSMB9 induction is required to regulate cellular proteostasis upon mitochondrial dysfunction[J].Nature Communications,2023,4(1):4092.
[13] HU X,HU Z,ZHANG H,et al.Deciphering the tumor-suppressive role of PSMB9 in melanoma through multi-omics and single-cell transcriptome analyses[J]. Cancer Letters, 2024,581:21646.
[14] CUI Z,HWANG S M,GOMES A V.Identification of the immunoproteasome as a novel regulator of skeletal muscle differentiation[J].Molecular and Cellular Biology,2014,34(1):96-109.
[15] GU Z C,ENENKEL C.Proteasome assembly[J]. Cellular and Molecular Life Sciences,2014,71(24):4729-4745.
[16] 李媛,梁俊玉,武国凡,等.真核细胞蛋白酶体研究进展[J].中国细胞生物学学报,2017,39(4):515-522. LI Y,LIANG J Y,WU G F,et al.Research progress on eukaryotic proteasome[J].Chinese Journal of Cell Biology,2017,39(4):515-522.(in Chinese)
[17] KASTHURI S R,UMASUTHAN N,WHANG I,et al.Molecular characterization and expressional affirmation of the beta proteasome subunit cluster in rock bream immune defense[J].Molocular Biology Reports,2014,41(8):5413-5427.
[18] TUNDO G R,CASCIO P,MILARDI D,et al.Targeting immunoproteasome in neurodegeneration:A glance to the future[J].Pharmacology & Therapeutics, 2023,241:108329.
[19] WANG X,ZHANG H,WANG Y,et al.DNA sensing via the cGAS/STING pathway activates the immunoproteasome and adaptive T-cell immunity[J].EMBO Journal,2023,42(8):e110597.
[20] CRUZ M,ELENICH L A,SMOLAREK T A,et al.DNA sequence,chromosomal localization,and tissue expression of the mouse proteasome subunit LMP10 (Psmb10) gene[J].Genomics,1997,45(3):618-622.
[21] ZANKER D,PANG K,OVEISSI S,et al.LMP2 immunoproteasome promotes lymphocyte survival by degrading apoptotic BH3-only proteins[J]. Immunology Cell Biology,2018,96(9):981-993.
[22] LIU Q,HU W,ZHANG Y L,et al.Anti-viral immune response in the lung and thymus:Molecular characterization and expression analysis of immunoproteasome subunits LMP2,LMP7 and MECL-1 in pigs[J].Biochemical Biophysical Research Communications,2018,502(4):472-478.
[23] HANSEN J D,STRASSBURGER P,THORGAARD G H,et al.Expression,linkage,and polymorphism of MHC-related genes in rainbow trout,Oncorhynchus mykiss[J].Journal of Immunology,1999,163(2):774-786.
[24] KALAORA S,LEE J S,BARNEA E,et al.Immunoproteasome expression is associated with better prognosis and response to checkpoint therapies in melanoma[J].Nature Communications,2020,11(1):896.
[25] YANG Y,LI Y,ZHANG S,et al.miR-199a-5p from bone marrow mesenchymal stem cell exosomes promotes the proliferation of neural stem cells by targeting GSK-3beta[J]. Acta Biochimica et Biophysica Sinica,2023,55(5):783-794.
[26] GONZALEZ-MAGANA A,BLANCO F J.Human PCNA structure,function and interactions[J].Biomolecules,2020,10(4):570.
[27] 韦瑶,张瑞门,安强,等.circCEP85L对牛肌肉干细胞增殖、成肌细胞分化的调控[J].中国农业科学,2023,56(18):3670-3681. WEI Y,ZHANG R M,AN Q,et al.circCEP85L regulates the proliferation and myogenic differentiation of bovine MUSCs[J].Scientia Agricultura Sinica,2023,56(18):3670-3681.(in Chinese)
[28] CARDANO M,TRIBIOLI C,PROSPERI E.Targeting proliferating cell nuclear antigen (PCNA) as an effective strategy to inhibit tumor cell proliferation[J].Current Cancer Drug Targets,2020,20(4):240-252.
[29] TENGOWSKI M W,FENG D,SUTOVSKY M,et al.Differential expression of genes encoding constitutive and inducible 20S proteasomal core subunits in the testis and epididymis of theophylline- or 1,3-dinitrobenzene-exposed rats[J].Biology of Reproduction,2007,76(1):149-163.
[30] MAIA FALCÃO R,KOKARAKI G,DE WISPELAERE W,et al.The expression of the immunoproteasome subunit PSMB9 is related to distinct molecular subtypes of uterine leiomyosarcoma[J].Cancers (Basel),2022,14(20):5007.
[31] CHOI J H,JO H S,LIM S,et al.Mtorc1 accelerates retinal development via the immunoproteasome[J].Nature Communications,2018,9(1):2502.
[32] 苏红,侯晓慧,李世杰.牛Gtl2基因cDNA序列分析及启动子预测[J].河北农业大学学报,2011,24(4):75-80. SU H,HOU X H,LI S J.Sequencing analysis of bovine Gtl2 cDNA and promoter prediction[J].Journal of Hebei Agricultural University,2011,24(4):75-80.(in Chinese)
[33] 李清春,孙晓梅,孙敬礼,等.猪miR-23A-27A-24簇启动子预测及活性分析[J].石河子大学学报(自然科学版),2023,41(3):279-285. LI Q C,SUN X M,SUN J L,et al.Prediction and activity analysis of the promoter of the porcine miR-23A-27A-24 cluster[J].Journal of Shihezi University (Natural Science),2023,41(3):279-285.(in Chinese)
[34] YUAN J,TANF Z,YANF S,et al.CRABP2 promotes myoblast differentiation and is modulated by the transcription factors MyoD and Sp1 in C2C12 cells[J].PLoS One,2013,8(1):e55479.
[35] CAI S,WANG X,XU R,et al.KLF4 regulates skeletal muscle development and regeneration by directly targeting P57 and Myomixer[J].Cell Death & Disease,2023,14(9):612.

Cloning and Sequence Analysis of PSMB9 Gene in Hu Sheep and Its Effect on Myoblast Proliferation

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 15

Recommended Articles

Metrics

Comments

[1]	LI Jinnan, WANG Yahui, DENG Tianyu, LIANG Mang, DU Lili, LI Keanning, XUE Qingqing, QIAN Li, GAO Xue, ZHANG Lupei, ZHU Bo, CHEN Yan, WANG Zezhao, LI Junya, GAO Huijiang. Cloning,Bioinformatics Analysis and Tissue Expression Profile of Coding Region of Bovine MED28 Gene [J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(8): 3225-3236.
[2]	RAN Yang, QIU Jie, SHEN Xiaoyun. Response of Fattening Hu Sheep to Cecropin Antimicrobial Peptides [J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(8): 3385-3393.
[3]	TANG Tian, WANG Zhen, YU Menghuan, XU Kun, HE Ruili, QIN Tianzhe, CHEN Chuangfu, MA Zhongchen, WANG Yong. Effect of Mycoplasma bovis Infection on Mitochondrial Pathway Mediated Apoptosis in Bovine Macrophages [J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(8): 3625-3634.
[4]	ZHANG Yan, MA Sen, LI Shouren, HUANGFU Weikang, LA Shaokai, MA Jixiang, WANG Zhichang, SHI Yinghua. Effects of Alfalfa Saponin Extract on Immunity,Antioxidant Capacity and Rumen Microflora of Weaned Hu Sheep [J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(7): 2799-2809.
[5]	ZHANG Xuedi, XU Kai, YIN Shuo, LIU Jing, WANG Zhonghao, QIN Jianhua, WU Pengyuan, WANG Chuanwen. Cloning,Sequence Analysis and Expression Characteristics of Carboxylesterase 2 Gene in Dermanyssus gallinae [J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(7): 2973-2983.
[6]	JI Guoshang, SHENG Hui, ZHANG Junxing, FENG Xue, HU Chunli, WANG Yachun, MA Yanfen, LI Li, YANG Wenfei, MA Yun. Study on the Function of PCYOX1L Gene Regulating LPS-induced Bovine Endometrial Epithelial Cells [J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(7): 2984-2997.
[7]	SUN Shiyu, LI Jinlan, XING Jiani, DONG Yaohui, LI Yanyan, WANG Youli, LIN Yaqiu. Cloning,Bioinformatics Analysis and Spatiotemporal Expression of CKMT2 Gene in Jianzhou Big-eared Goats [J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(6): 2342-2353.
[8]	WU Bowen, YAN Peiyu, WU Mishan. Mechanism of the Active Ingredients of Ephedra sinica Stapf Dried Grass Stems in the Treatment of Vasospasm Based on Network Pharmacology [J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(6): 2680-2696.
[9]	HUANG Qiaoyan, LI Wei, WANG Xinkun, XING Feng. Cloning,Bioinformatics and Tissue Differential Expression Analysis of PROKR2 Gene in Dolang Sheep [J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(4): 1339-1348.
[10]	WANG Yan, ZHANG Ying, ZHAO Yani, YI Lin, SHI Zhen, ZHOU Changming, ZHOU Wanrong, JIANG Lili, FAN Zhaobin. Cloning,Bioinformatics and Tissue Expression Characteristics of STING Gene in White Leghorn Chickens [J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(4): 1372-1381.
[11]	YAN Zunqiang, LIANG Yuhao, SONG Kelin, GUN Shuangbao, WANG Pengfei. Cloning,Bioinformatics Analysis and Tissue Expression of SIRT3 Gene in Hezuo Pigs [J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(4): 1390-1399.
[12]	MA Rong, LA Yongfu, BAO Pengjia, GUO Xian, LU Jianwei, ZHA Lao, ZHAO Xue, LIANG Chunnian, CHENG Shuru. Cloning,Bioinformatics Analysis and Tissue Expression of MYL9 Gene in Meiren Yak [J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(4): 1410-1419.
[13]	QING Yubo, CHENG Wen, JIAO Deling, LIU Cheng, HAO Zhiqiang, GUO Jianxiong, LI Yuying, ZHANG Xiaolin, LI Zhen, LI Xiaoyu, WANG Wen, WEI Hongjiang. Study on the Application of Propagation of Superfine-type Erlangshan Cashmere Goat by Somatic Cell Cloning Technology [J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(4): 1420-1427.
[14]	WANG Yu, SHI Song, JIN Panhao, LI Jinxin, ZHANG Mingxi, LI Tuo, LI Longping, QU Lei, DONG Shuwei. Isolation,Identification and Detection of Resistance and Virulence Genes of Pathogenic Bacteria Causing Mastitis in Some Areas of Yulin [J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(4): 1746-1756.
[15]	SONG Xingchao, MENG Jinzhu, ZHAO Yuanyuan, WU Zhenyang, AN Qingming. Cloning and Bioinformatics Analysis of myogenin Gene in Guizhou White Goat [J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(3): 926-935.