THE EFFECT OF SP600125 ON THE EXPRESSION OF SKELETAL GENES OF CRUCIAN CARP (CARASSIUS AURATUS VAR)
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摘要: 前期研究表明ATP竞争性小分子抑制剂SP600125在体外可以高效诱导鱼类细胞多倍化。研究首次尝试用SP600125直接孵育鲫受精卵以获得多倍体鱼的可能性。结果显示SP600125处理受精卵虽然能够引起部分胚胎倍性发生改变, 但也显著影响胚胎发育并导致胚胎高死亡率, 同时伴随着畸形鱼苗的发生, 其中主要表现为脊椎弯曲和尾缺失等骨骼发育异常。SP600125处理导致骨骼畸形在SP600125孵育鲫鱼苗试验中也同样获得验证。荧光定量PCR检测结果表明, 在体(受精卵和鱼苗)或离体(培养的尾鳍细胞)状态下, SP600125孵育会引起smad6、stat1、 lef1、bmp2和twist1等骨骼发育相关基因mRNA水平显著上调。相关结果为进一步调整SP600125诱导的鱼类倍性操作策略和方法提供了重要理论参考。Abstract: It has been shown that the ATP competitive small molecule inhibitor SP600125 can efficiently induce fish cell polyploidization in vitro. In this study, we used SP600125 to directly incubate the fertilized eggs of crucian carp to obtain the possibility of polyploid fish, and found that SP600125 caused partial embryo ploidy, significantly affected the embryonic development and led to high mortality of embryos. SP600125 also induced a large number of malformed fry after treating fertilized eggs, mainly skeletal dysplasia such as spinal curvature and tail loss. The skeletal malformation caused by SP600125 was further validated. Real-time PCR indicated that SP600125 significantly induced mRNA levels of skeletal development-related genes, such as smad6, stat1, lef1, bmp2 and twist1 in vivo (fertilized eggs and fry) or in vitro (cultured caudal fin cells). These results clearly help to develop a potential mode of fish polyploidy induced by SP600125.
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Keywords:
- SP600125 /
- Polyploidization /
- Skeletal development /
- Crucian carp
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图 1 SP600125孵育对鲫胚胎发育的影响
Figure 1. The effect of SP600125 on the embryonic development of crucian carp
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图 2 SP600125孵育对受精卵形态和倍性的影响
A. SP60015处理组正常鱼苗; B—D. SP600125处理组畸形鱼苗; E. 空白对照组孵化出膜鱼苗DNA含量检测结果; F—G. SP600125处理组孵化出膜鱼苗DNA含量检测结果; H. SP600125处理组3月龄鱼DNA含量检测结果。a. 心包水肿; b. 脊椎弯曲; c. 尾巴弯曲; d. 尾缺失
Figure 2. Effects of SP600125 on morphology and ploidy of fertilized egg
A. Normal fry in SP600125-treated fertilized egg group; B—D. Deformed fry in SP600125-treated fertilized egg group; E. DNA content of fry in blank control group; F—G. DNA content of fry in SP600125-treated fertilized egg group; H. DNA content of 3-month fish in SP600125-treated fertilized egg group. a. shown pericardium edema; b. shown spinal curvature; c. shown bent tail vertebrae; d. shown tail loss
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图 3 SP600125孵育鲫鱼苗导致骨骼发育畸形(A和B分别为鲫胚胎出膜后3d和7d的鱼苗)
Figure 3. Skeletal malformations caused by SP600125 in crucian carp (A and B were 3-day and 7-day fry after hatching, respectively)
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图 4 SP600126孵育对鲫骨骼发育相关基因表达的影响
A. SP600125孵育受精卵组的出膜第1天鱼苗; B. SP600125孵育鱼苗组的出膜第7天鱼苗
Figure 4. The effects of SP600125 on the expression of several skeletal development-related genes in crucian carp
A. 1-day fry in SP600125-treated fertilized egg group; B. 7-day fry in SP600125-treated fry group
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图 5 SP600125诱导的同源四倍体鲫细胞系(SP4N)(A)和SP600125孵育鲫尾鳍细胞(B)中几种骨骼发育相关基因表达
Figure 5. The effects of SP600125 on the expression of several skeletal development-related genes in autotetraploid crucian carp cell line (SP4N) (A) and caudal fin cells in crucian carp (B)
表 1 PCR引物序列
Table 1 Sequences of PCR primer
基因Gene 上游引物Upstream primer (5′—3′) 下游引物Downstream primer (5′—3′) stat1 CTGCGCTCTGATGGTTCTG GTTGCTGTTGTTCTGCTCCG Smad6 TGACAGTGACAGAGCGGTT AGTTGTGGAGTCTCAAGGCG lef1 CAATGTGGTGGCTGAATG CTGGCCTGTACCTGAAGC bmp2 ATCACCCGCTCGTATTCGTC GCTACCATGCCTTTTACTGCC twist1 ACGCACAAAAGGAGCA TCCTCTTGCATCGGCTCT ptk7 CAGTACGATTCGCTCAGCCT CTTCCAGTGTGTAGCGTC opg TGAGCGTCACACATCCC GCACTGAAACACACA stat3 CTCGACCTTGTGGCATG CTGGATGAGCTGGTAGGA β-actin CATCTACGAGGGTTACGCCC AATTTCCCTCTCGGCTGTGG 
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[1] Bennett B L, Sasaki D T, Murray B W, et al. SP600125, an anthrapyrazolone inhibitor of Jun N-terminal kinase [J]. Proceedings of the National Academy of Sciences of the United States of American, 2001, 98(24): 13681-13686. doi: 10.1073/pnas.251194298
[2] Zhou Y, Wang M, Jiang M, et al. Autotetraploid cell Line induced by SP600125 from crucian carp and its developmental potentiality [J]. Scientific Reports, 2016(6): 21814.
[3] Zhou L, Gui J F. Natural and artificial polyploids in aquaculture [J]. Aquaculture and Fisheries, 2017, 2(3): 103-111. doi: 10.1016/j.aaf.2017.04.003
[4] Xu K, Duan W, Xiao J, et al. Development and application of biological technologies in fish genetic breeding [J]. Science China-Life Sciences, 2015, 58(2): 187-201. doi: 10.1007/s11427-015-4798-3
[5] 王延晖. 鱼类人工多倍体育种及其在水产养殖中的应用 [J]. 河南水产, 2017(6): 3-5. Wang Y H. Artificial polyploidy breeding of fishes and its application in aquaculture [J]. Henan Fisheries, 2017(6): 3-5. [
[6] Valesio E, Zhang H H, Zhang C B. Exposure to the JNK inhibitor SP600125 (anthrapyrazolone) during early zebrafish development results in morphological defects [J]. Journal of Applied Toxicology, 2013, 33(1): 32-40. doi: 10.1002/jat.1708
[7] Tajima K, Takaishi H, Takito J, et al. Inhibition of STAT1 accelerates bone fracture healing [J]. Journal of Orthopaedic Research, 2010, 28(7): 937-941. doi: 10.1002/jor.21086
[8] 杨建, 佟广香, 郑先虎, 等. 肌间刺缺失对斑马鱼骨骼发育的影响 [J]. 水生生物学报, 2020, 44(3): 546-553. doi: 10.1074/jbc.M702100200x Yang J, Tong G X, Zheng X H, et al. Comparative analysis of skeletal development between wildtype zebrafish and intermuscular bone-deficient mutants [J]. Acta Hydrobiologica Sinica, 2020, 44(3): 546-553. [ doi: 10.1074/jbc.M702100200x
[9] Quarto N, Senarath-Yapa K, Renda A, et al. Twist1mm silencing enhances in vitro and in vivo osteogenic differentiation of human adipose-derived stem cells by triggering activation of BMP-ERK/FGF signaling and TAZ upregulation [J]. Stem Cells, 2015, 33(3): 833-847. doi: 10.1002/stem.1907
[10] Galindo M, Kahler R A, Teplyuk N M, et al. Cell cycle related modulations in Runx2 protein levels are independent of lymphocyte enhancer-binding factor 1 (Lef1) in proliferating osteoblasts [J]. Journal of Molecular Histology, 2007, 38(5): 501-506. doi: 10.1007/s10735-007-9143-0
[11] Reddi A H. BMPs: From bone morphogenetic proteins to body morphogenetic proteins [J]. Cytokine and Growth Factor Reviews, 2005, 16(3): 249-250.
[12] Hayes M, Naito M, Daulat A, et al. Ptk7 promotes non-canonical Wnt/PCP-mediated morphogenesis and inhibits Wnt/β-catenin-dependent cell fate decisions during vertebrate development [J]. Development, 2013, 140(8): 1807-1818. doi: 10.1242/dev.090183
[13] Qin S, Zhang Q, Zhang L. Effect of OPG gene mutation on protein expression and biological activity in osteoporosis [J]. Experimental and Therapeutic Medicine, 2017, 14(2): 1475-1480.
[14] Bubici C, Papa S. JNK signalling in cancer: in need of new, smarter therapeutic targets [J]. British Journal of Pharmacology, 2014, 171(1): 24-37. doi: 10.1111/bph.12432
[15] Chambers J W, Pachori A, Howard S, et al. Small molecule c-jun-N-terminal Kinase inhibitors protect dopaminergic neurons in a model of parkinson’s disease [J]. ACS Chemical Neuroscience, 2011, 2(4): 198-206. doi: 10.1021/cn100109k
[16] Mingo-Sion A M, Marietta P M, Koller E, et al. Inhibition of JNK reduces G2/M transit independent of p53, leading to endoreduplication, decreased proliferation, and apoptosis in breast cancer cells [J]. Oncogene, 2004, 23(2): 596-604. doi: 10.1038/sj.onc.1207147
[17] Jacobs-Helber S M, Sawyer S T. Jun N-terminal kinase promotes proliferation of immature erythroid cells and erythropoietin-dependent cell lines [J]. Blood, 2004, 104(3): 696-703. doi: 10.1182/blood-2003-05-1754
[18] 隋华, 周利红, 殷佩浩, 等. JNK信号通路介导MDR1/P-gp调控人结肠癌多药耐药 [J]. 世界华人消化杂志, 2011, 19(9): 892-898. Sui H, Zhou L H, Yin P H, et al. JNK signal transduction pathway regulates MDR1/P-glycoprotein-mediated multidrug resistance in colon carcinoma cells [J]. World Chinese Journal of Digestology, 2011, 19(9): 892-898. [
[19] 李佳殷, 林丽珠. JNK信号通路在恶性肿瘤中的研究进展 [J]. 实用中医内科杂志, 2012(18): 10-11. Li J Y, Lin L Z. Advances of JNK pathway in malignant tumors [J]. Journal of Practical Traditional Chinese Internal Medicine, 2012(18): 10-11. [
[20] Tiwari V K, Stadler M B, Wirbelauer C, et al. A chromatin-modifying function of JNK during stem cell differentiation [J]. Nature Genetics, 2012, 44(1): 94-100. doi: 10.1038/ng.1036
[21] Zhou Y, Jiang M, Wang M, et al. Effect of SP600125 on proliferation of embryonic stem cell [J]. American Journal of Molecular Biology, 2013, 3(2): 67-71. doi: 10.4236/ajmb.2013.32009
[22] Miyamoto-Yamasaki Y, Yamasaki M, Yamada K. Induction of endoreduplication by a JNK inhibitor SP600125 in human lung carcinom A 549 cells [J]. Cell Biology International, 2007, 31(12): 1501-1506. doi: 10.1016/j.cellbi.2007.07.002
[23] Vlacic-Zischke J, Hamlet S M, Friis T, et al. The influence of surface microroughness and hydrophilicity of titanium on the up-regulation of TGFβ/BMP signalling in osteoblasts [J]. Biomaterials, 2011, 32(3): 665-671. doi: 10.1016/j.biomaterials.2010.09.025
[24] Guimond J C, Mathieu Lévesque, Michaud P L, et al. BMP-2 functions independently of SHH signaling and triggers cell condensation and apoptosis in regenerating axolotl limbs [J]. BMC Developmental Biology, 2010, 10(1): 1-16. doi: 10.1186/1471-213X-10-1
[25] Paik S, Jung H S, Lee S, et al. Mir-449a regulates the chondrogenesis of human mesenchymal stem cells through direct targeting of lymphoid enhancer-binding factor-1 [J]. Stem Cells and Development, 2012, 21(18): 3298-3308. doi: 10.1089/scd.2011.0732
[26] Tommy N, Yankel G, Jon C, et al. Lef1 haploinsufficient mice display a low turnover and low bone mass phenotype in a gender- and age-specific manner [J]. PLoS One, 2009, 4(5): 5434-5438. doi: 10.1371/journal.pone.0005434
[27] Kahler R A, Galindo M, Lian J, et al. Lymphocyte enhancer-binding factor 1 (Lef1) inhibits terminal differentiation of osteoblasts [J]. Journal of Cellular Biochemistry, 2006, 97(5): 969-983. doi: 10.1002/jcb.20702
[28] Connerney J, Andreeva V, Leshem Y, et al. Twist1 homodimers enhance FGF responsiveness of the cranial sutures and promote suture closure [J]. Developmental Biology, 2008, 318(2): 323-334. doi: 10.1016/j.ydbio.2008.03.037
[29] Hayashi M, Nimura K, Kashiwagi K, et al. Comparative roles of Twist-1 and Id1 in transcriptional regulation by BMP signaling [J]. Journal of Cell Science, 2007, 120(8): 1350-1357. doi: 10.1242/jcs.000067
[30] Peradziryi H, Kaplan N A, Podleschny M, et al. Ptk7/Otk interacts with Wnts and inhibits canonical Wnt signalling [J]. Embo Journal, 2011, 30(18): 3729-3740. doi: 10.1038/emboj.2011.236
[31] Hayes M, Gao X, Yu L X, et al. Ptk7 mutant zebrafish models of congenital and idiopathic scoliosis implicate dysregulated Wnt signalling in disease [J]. Nature Communications, 2014, 5(6): 4777.
[32] 任洁, 卢建华. RANKL/RANK/OPG系统与绝经后骨质疏松模型的研究 [J]. 浙江中西医结合杂志, 2015, 25(9): 892-895. doi: 10.3969/j.issn.1005-4561.2015.09.035 Ren J, Lu J H. Study on RANKL / RANK / OPG System and postmenopausal osteoporosis model [J]. Zhejiang Journal of Integrated Traditional Chinese and Western Medicine, 2015, 25(9): 892-895. [ doi: 10.3969/j.issn.1005-4561.2015.09.035
[33] 吴佳莹, 李岳泽, 刘红, 等. “肾主骨”机理研究——左归丸对Hepcidin、Fpn1及OPG/RANKL mRNA表达的影响 [J]. 中国中医基础医学杂志, 2017, 23(11): 1548-1551. Wu J Y, Li Y Z, Liu H, et al. Study on the mechanism of “kidney governing bone”——The effect of Zuogui Wan on Hepcidin, Fpn1 and OPG/RANKL mRNA expression [J]. Chinese Journal of Basic Medicine in Traditional Chinese Medicine, 2017, 23(11): 1548-1551. [
[34] Ishida W, Hamamoto T, Kusanagi K, et al. Smad6 is a Smad1/5-induced smad inhibitor-characterization of bone morphogenetic protein-responsive element in the mouse Smad6 promoter [J]. Journal of Biological Chemistry, 2000, 275(9): 6075-6079. doi: 10.1074/jbc.M702100200
[35] 刘猛, 董伟, 冯晓洁, 等. Smad6信号干扰对MSCs骨向分化中Smad5及Smurf1基因表达的影响 [J]. 河北医药, 2012, 34(10): 1445-1447. Liu M, Dong W, Feng X J, et al. The influence of Smad6 RNA interference on gene expression of Smad5 and Smurf1 during osteogenic differentiation of MSCs [J]. Hebei Medical Journal, 2012, 34(10): 1445-1447. [
[36] 邓洋洋, 郑洪新, 林庶茹, 等. 益肾填精中药对骨质疏松症模型大鼠的骨组织中Smad1/5的表达影响 [J]. 中国骨质疏松杂志, 2009, 15(8): 563-567. doi: 10.3969/j.issn.1006-7108.2009.08.003 Deng Y Y, Zheng H X, Lin S R, et al. Regulative effect of some Chinese herbal drugs on expression of signal transduction molecules Smad1/5 in the femur tissues of osteoporosis in rats after ovariectomy [J]. Chinese Journal of Osteoporosis, 2009, 15(8): 563-567. [ doi: 10.3969/j.issn.1006-7108.2009.08.003
[37] Fujie A, Funayama A, Miyauchi Y, et al. Bcl6 promotes osteoblastogenesis through Stat1 inhibition [J]. Biochemical and Biophysical Research Communications, 2015, 457(3): 451-456. doi: 10.1016/j.bbrc.2015.01.012
[38] Li J, He X, Wei W, et al. MicroRNA-194 promotes osteoblast differentiation via downregulating STAT1 [J]. Biochemical and Biophysical Research Communications, 2015, 460(2): 482-488. doi: 10.1016/j.bbrc.2015.03.059
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