鸡TIGAR基因真核表达质粒的构建及其抗凋亡功能评价

doi:10.3864/j.issn.0578-1752.2019.06.013

Abstract

Abstract:

【Background】TP53-induced glycolysis and apoptosis regulator (TIGAR) is a target gene downstream of p53, which can regulate glycolysis level, remove reactive oxygen species (ROS) and reduce apoptosis induced by reactive oxygen species (ROS). 【Objective】The objective of this study was to construct the eukaryotic expression plasmid contain of chicken TIGAR gene, and to evaluate its resistance apoptosis effect in DF1 cells, in order to establish the cell line with stable expression of the chicken TIGAR gene. 【Method】According to design primers based on the predicted TIGAR gene published in Genbank (accession number: XM_417232.6), the TIGAR gene from SPF chicken spleen was amplified by RT-PCR, and then amplified products were cloned into expression vector (Flag-CMV14), finally the positive clone was verified by DNA sequencing. Then the phylogenetic tree of chicken TIGAR genes with other mammals and aquatic animals TIGAR gene was constructed to do homology analysis. The recombinant plasmid was transfected into DF1 cells, and infected with Newcastle disease virus to induce apoptosis. The expression of the TIGAR gene and the cleavage level of PARP protein were detected by Western Blot. At the same time the recombinant plasmid (Flag-TIGAR) was transfected into DF1 cells and incubated with apoptosis inducer staurosporine for 2 hours before sample collection, then cells were collected at 24 hours and 48 hours after transfection to evaluate apoptosis level by flow cytometry. 【Result】The TIGAR gene was amplified by RT-PCR, and a band appeared at 843 bp, which was consistent with the prediction. The constructed TIGAR eukaryotic expression plasmid (Flag-TIGAR) was sequenced, and the result showed that the sequence of amplified TIGAR gene were almost consistent with the predicted sequence published on GenBank. Western Blot results showed: cleavaged PARP bands were existed, and the expression level in transfected recombinant plasmid (Flag-TIGAR) group was significantly different (P<0.05) from that in non-transfected plasmid (MOCK) group and transfected empty vector (Flag-CMV14) group. Flow cytometry results showed that the total apoptotic rate was 11% (early apoptosis 7.8%, late apoptosis 3.2%) after transfection with Flag-CMV14 for 24 h, while the total apoptotic rate was decreased to 4% (early apoptosis 3.7%, late apoptosis 0.3%) in Flag-TIGAR transfected group. The early apoptotic rate of Flag-CMV14 transfected group was much higher than that of Flag-TIGAR transfection group, and the difference was very significant (P<0.01), while the late apoptotic rate was also higher than that of Flag-TIGAR transfected group and the difference was significant (P<0.05). The total apoptotic rate was 20.3% (early apoptosis 14.3%, late apoptosis 6.0%) after transfection with Flag-CMV14 for 48 h, while the total apoptotic rate was only 6.4% (early apoptosis 4.8%, late apoptosis 1.6%) after transfection with Flag-TIGAR for 48 h. Both the early apoptotic rate and late apoptotic rate of Flag-CMV14 transfected group were much higher than that of Flag-TIGAR transfected group and the difference was very significant (P<0.01). 【Conclusion】The chicken TIGAR gene was amplified and the eukaryotic expression plasmid was constructed successfully. It was also confirmed that overexpression of chicken TIGAR could obviously reduce the degree of apoptosis and increase cell survival rate.

Key words: TIGAR, Eukaryotic expression plasmid, Newcastle disease virus, Anti-apoptotic function

LI YongHua, CHE LuPing, QIU XuSheng, TAN Lei, SUN YingJie, LIU WeiWei, SONG CuiPing, LIAO Ying, DING Chan, WANG JinQuan, MENG ChunChun. Construction of Chicken TIGAR Gene Eukaryotic Expression Plasmid and Evaluation of Its Anti-Apoptotic Function[J].Scientia Agricultura Sinica, 2019, 52(6): 1102-1109.

0
/ / Recommend

Add to citation manager EndNote|Reference Manager|ProCite|BibTeX|RefWorks

URL: https://www.chinaagrisci.com/EN/10.3864/j.issn.0578-1752.2019.06.013

https://www.chinaagrisci.com/EN/Y2019/V52/I6/1102

Figures/Tables 5

Fig. 1

Fig. 2

Fig. 3

Fig. 4

Fig. 5

References 33

[1]	RAVINDRA P V, TIWARI A K, RATTA B, BAIS M V, CHATURVEDI U, PALIA S K, SHARMA B, CHAUHAN R S . Time course of Newcastle disease virus-induced apoptotic pathways. Virus Research, 2009,144(1-2):350-354. doi: 10.1016/j.virusres.2009.05.012 pmid: 19501124
[2]	RAVINDRA P V, TIWARI A K, RATTA B, CHATURVEDI U, PALIA S K, CHAUHAN R S . Newcastle disease virus-induced cytopathic effect in infected cells is caused by apoptosis. Virus Research, 2009,141(1):13-20. doi: 10.1016/j.virusres.2008.12.008 pmid: 19152817
[3]	KERR J F, WYLLIE A H, CURRIE A R . Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. British Journal of Cancer, 1972, 26(4):239-257. doi: 10.1038/bjc.1972.33 pmid: 4561027
[4]	赵明, 冯婧, 刘勇, 杨玲麟 . TIGAR调节肺癌细胞的增殖和侵袭能力研究. 国际检验医学杂志, 2016(06):754-755. doi: 10.3969/j.issn.1673-4130.2016.06.014
	ZHAO M, FENG J, LIU Y, YANG L L . TIGAR promotes proliferation and invasiveness of lung cancer cells.International Journal of Laboratory Medicine, 2016(06):754-755. (in Chinese) doi: 10.3969/j.issn.1673-4130.2016.06.014
[5]	GREEN D R , CHIPUK J E . p53 and metabolism: Inside the TIGAR.Cell, 2006, 126(1):30-32. doi: 10.1016/j.cell.2006.06.032 pmid: 16839873
[6]	VOGELSTEIN B, LANE D, LEVINE A J . Surfing the p53 network. Nature, 2000,408(6810):307-310. doi: 10.1038/35042675
[7]	SHEN M, ZHAO X, ZHAO L, SHI L, AN S, HUANG G, LIU J . Met is involved in TIGAR-regulated metastasis of non-small-cell lung cancer. Molecular Cancer, 2018,17(1):88. doi: 10.1186/s12943-018-0839-4 pmid: 29753331
[8]	ROMEO M, HUTCHISON T, MALU A, WHITE A, KIM J, GARDNER R, SMITH K, NELSON K, BERGESON R, MCKEE R, HARROD C, RATNER L, LUSCHER B, MARTINEZ E, HARROD R . The human T-cell leukemia virus type-1 p30(II) protein activates p53 and induces the TIGAR and suppresses oncogene-induced oxidative stress during viral carcinogenesis. Virology, 2018,518(10):3-15.
[9]	BENSAAD K, TSURUTA A, SELAK M A, VIDAL M N, NAKANO K, BARTRONS R, GOTTLIEB E, VOUSDEN K H . TIGAR, a p53-inducible regulator of glycolysis and apoptosis. Cell, 2006, 126(1):107-120. doi: 10.1016/j.cell.2006.05.036 pmid: 16839880
[10]	YU H P, XIE J M, LI B, SUN Y H, GAO Q G, DING Z H, WU H R, QIN Z H . TIGAR regulates DNA damage and repair through pentosephosphate pathway and Cdk5-ATM pathway. Scientific Reports, 2015. DOI: 10.1038/srep09853
[11]	ZHOU J H, ZHANG T T, SONG D D, XIA Y F, QIN Z H, SHENG R . TIGAR contributes to ischemic tolerance induced by cerebral preconditioning through scavenging of reactive oxygen species and inhibition of apoptosis. Scientific Reports, 2016. DOI: 10.1038/srep27096. doi: 10.1038/srep27096 pmid: 27256465
[12]	JIANG L B, CAO L, MA Y Q, CHEN Q, LIANG Y, YUAN F L, LI X L, DONG J, CHEN N . TIGAR mediates the inhibitory role of hypoxia on ROS production and apoptosis in rat nucleus pulposus cells. Osteoarthritis and Cartilage, 2018,26(1):138-148. doi: 10.1016/j.joca.2017.10.007 pmid: 29061494
[13]	KIM J, DEVALARAJA-NARASHIMHA K, PADANILAM B J . TIGAR regulates glycolysis in ischemic kidney proximal tubules. American Journal of Physiology Renal Physiology, 2015, 308(4).DOI: 10.1152/ajprenal.00459.2014F298-308. doi: 10.1152/ajprenal.00459.2014 pmid: 25503731
[14]	付托 . 过表达人TIGAR的CHO细胞株构建及其抗凋亡机制研究[D]. 苏州: 苏州大学, 2013.
	FU T . Establishment of CHO cell line overexpressing hTIGAR and mechanism of anti-apoptosis effect[D]. Suzhou: Suzhou University, 2013. (in Chinese)
[15]	HWANG S O, LEE G M . Nutrient deprivation induces autophagy as well as apoptosis in Chinese hamster ovary cell culture. Biotechnology and Bioengineering, 2008,99(3):678-685. doi: 10.1002/bit.21589 pmid: 17680685
[16]	WANG H, CHENG Q, LI X, HU F, HAN L, ZHANG H, LI L, GE J, YING X, GUO X, WANG Q . Loss of TIGAR induces oxidative stress and meiotic defects in oocytes from obese mice. Molecular & Cellular Proteomics, 2018. DOI: 10.1074/mcp.RA118.000620. doi: 10.1074/mcp.RA118.000620 pmid: 29776966
[17]	LI B, WANG Z, XIE J M, WANG G, QIAN L Q, GUAN X M, SHEN X P, QIN Z H, SHEN G H, LI X Q, GAO Q G . TIGAR knockdown enhanced the anticancer effect of aescin via regulating autophagy and apoptosis in colorectal cancer cells. Acta Pharmacologica Sinica, 2018. DOI: 10.1038/s41401-018-0001-2. doi: 10.1038/s41401-018-0001-2 pmid: 29769743
[18]	KUMAR B, IQBAL M A, SINGH R K, BAMEZAI R N . Resveratrol inhibits TIGAR to promote ROS induced apoptosis and autophagy. Biochimie, 2015, 118:26-35. doi: 10.1016/j.biochi.2015.07.016 pmid: 26212201
[19]	YE L, ZHAO X, LU J, QIAN G, ZHENG J C, GE S . Knockdown of TIGAR by RNA interference induces apoptosis and autophagy in HepG2 hepatocellular carcinoma cells. Biochemical and Biophysical Research Communications, 2013, 437(2):300-306. doi: 10.1016/j.bbrc.2013.06.072 pmid: 23817040
[20]	CHEUNG E C, LEE P, CETECI F, NIXON C, BLYTH K, SANSOM O J, VOUSDEN K H . Opposing effects of TIGAR- and RAC1- derived ROS on Wnt-driven proliferation in the mouse intestine. Genes & Development, 2016, 30(1):52-63. doi: 10.1101/gad.271130.115 pmid: 26679840
[21]	XIE J M, LI B, YU H P, GAO Q G, LI W, WU H R, QIN Z H . TIGAR has a dual role in cancer cell survival through regulating apoptosis and autophagy. Cancer Research, 2014, 74(18):5127-5138. doi: 10.1158/0008-5472.CAN-13-3517 pmid: 25085248
[22]	CHEN J, ZHANG D M, FENG X, WANG J, QIN Y Y, ZHANG T, HUANG Q, SHENG R, CHEN Z, LI M, QIN Z H . TIGAR inhibits ischemia/reperfusion-induced inflammatory response of astrocytes. Neuropharmacology, 2018,131:377-388. doi: 10.1016/j.neuropharm.2018.01.012 pmid: 29331305
[23]	CHEUNG E C, LUDWIG R L, VOUSDEN K H . Mitochondrial localization of TIGAR under hypoxia stimulates HK2 and lowers ROS and cell death. Proceedings of the National Academy of Sciences of the United States of America, 2012,109(50):20491-20496. doi: 10.1073/pnas.1206530109 pmid: 23185017
[24]	FENG J, LUO L, LIU Y, FU S, CHEN J, DUAN X, XIANG L, ZHANG Y, WU J, FAN J, WEN Q, ZHANG Y, YANG J, PENG J, ZHAO M, YANG L . TP53-induced glycolysis and apoptosis regulator is indispensable for mitochondria quality control and degradation following damage. Oncology Letters, 2018, 15(1):155-160. doi: 10.3892/ol.2017.7303 pmid: 5766069
[25]	KO Y H, DOMINGO-VIDAL M, ROCHE M, LIN Z, WHITAKER- MENEZES D, SEIFERT E, CAPPARELLI C, TULUC M, BIRBE R C, TASSONE P, CURRY J M, NAVARRO-SABATE A, MANZANO A, BARTRONS R, CARO J, MARTINEZ-OUTSCHOORN U . TP53- inducible glycolysis and apoptosis regulator (TIGAR) metabolically reprograms carcinoma and stromal cells in breast cancer. The Journal of Biological Chemistry, 2016, 291(51):26291-26303. doi: 10.1074/jbc.M116.740209 pmid: 27803158
[26]	张腾, 袁梅, 俞同福 . 肿瘤中TP53诱导的糖酵解和凋亡调节因子及其靶向治疗研究进展. 肿瘤, 2016(12):1383-1388.
	ZHANG T, YUAN M, YU T F . Advances in TP53-induced glycolysis and apoptosis regulator in tumor and its targeted therapy.TUMOR, 2016(12):1383-1388. (in Chinese)
[27]	顾志冬, 倪培华, 吴蓓颖, 吴华成, 樊绮诗, 肖家诚 . TIGAR基因真核表达载体的构建及在HepG_2细胞中的作用初探. 诊断学理论与实践, 2009(6):617-622.
	GU Z D, NI P H, WU B Y, WU H C, FAN Y S, XIAO J C . Construction of TIGAR gene eukaryotic expression vector and effect of TIGAR expression in HepG2 cells.Journal of Diagnostics Concepts & Practice, 2009(6):617-622. (in Chinese)
[28]	MA T, ZHANG Y, ZHANG C, LUO J G, KONG L Y . Downregulation of TIGAR sensitizes the antitumor effect of physapubenolide through increasing intracellular ROS levels to trigger apoptosis and autophagosome formation in human breast carcinoma cells. Biochemical Pharmacology, 2017, 143:90-106. doi: 10.1016/j.bcp.2017.07.018 pmid: 28774732
[29]	周骏浩 . TIGAR 通过清除活性氧和抑制凋亡参与脑预适应介导缺血耐受[D]. 苏州: 苏州大学, 2016.
	ZHOU J H . TIGAR contributes to ischemic tolerance induced by cerebral preconditioning through scavenging of reactive oxygen species and inhibition of apoptosis[D]. Suzhou: Suzhou University, 2016. (in Chinese)
[30]	冯婧 . TIGAR对鼻咽癌细胞自噬、凋亡及线粒体降解作用研究[D]. 泸州: 西南医科大学, 2018.
	FENG J . Study on autophagy, apoptosis and mitochondrial degradation in nasopharyngeal carcinoma cells induced by TIGAR[D]. Luzhou: Southwest Medical University, 2018. ( in Chinese)
[31]	梁思佳, 黄金丽, 魏东轩, 刘洪波, 石春薇 . 细胞自噬与人肠道病毒感染的研究进展. 华中科技大学学报(医学版), 2018,47(01):126-130. doi: 10.3870/j.issn.1672-0741.2018.01.025
	LIANG S J, HUANG J L, WEI D X, LIU H B, SHI C W . Advances in the study of autophagy and human enterovirus infection. Acta Medicinae Universitatis Scientiae et Technologiae Huazhong, 2018,47(01):126-130. (in Chinese) doi: 10.3870/j.issn.1672-0741.2018.01.025
[32]	孟春春 . ClassⅠ新城疫弱毒的毒力演化及自噬在新城疫病毒感染肿瘤细胞中的作用[D]. 北京: 中国农业科学院, 2012.
	MENG C C . ClassⅠvirulence evolution of attenuated Newcastle Disease virus and the role of autophagy in Newcastle Disease virus infection with tumor cells[D]. Beijing: Chinese Academy of Agricultural Sciences, 2012. ( in Chinese)
[33]	陶冶, 任晓峰 . 细胞自噬与病毒感染. 病毒学报, 2013,29(01):76-84.
	TAO Y, REN X F . Autophagy and virus infection. Chinese Journal of Virology, 2013, 29(01):76-84. (in Chinese)

Metrics

Viewed

Full text

Abstract

Cited

Shared

Discussed

Comments

Recommended 0

No Suggested Reading articles found!

Construction of Chicken TIGAR Gene Eukaryotic Expression Plasmid and Evaluation of Its Anti-Apoptotic Function

RichHTML

PDF

Abstract

Cite this article

share this article

Figures/Tables 5

References 33

Related Articles 3

Metrics

Comments

Recommended 0

[1]	. Generation of recombinant Newcastle disease virus LaSota vaccine strain expressing VP2 gene of very virulent infectious bursal disease virus isolate from cDNA clone by reverse genetic [J]. Scientia Agricultura Sinica, 2008, 41(1): 243-251 .
[2]	,,,,. Pathotyping of Newcastle Disease Virus by SYBR Green I Real-Time RT-PCR [J]. Scientia Agricultura Sinica, 2006, 39(01): 215- .
[3]	,,. Construction and Immunogenicity of Recombinant Phage Expressing the F Gene of Newcastle Disease Virus [J]. Scientia Agricultura Sinica, 2005, 38(06): 1270-1274 .