|Table of Contents|

Construction of lentiviral plasmid with over-expressing MINDY2 and investigation its effect on cell proliferation，migration and apoptosis in lung cancer A549 cells

Journal Of Modern Oncology[ISSN:1672-4992/CN:61-1415/R]

Issue:
2023 07
Page:
1187-1194
Research Field:
Publishing date:

Info

Title:
Construction of lentiviral plasmid with over-expressing MINDY2 and investigation its effect on cell proliferation，migration and apoptosis in lung cancer A549 cells
Author(s):
ZHANG Luyao; LI Zeyan; WANG Xiaoxuan; SHI Xingyun; LUO Wanzheng; SONG Liangpeng; SUN Yuling; LI Deyi; MA Xueyan
School of Basic Medical,Dali University,Yunnan Dali 671000,China.
Keywords:
MINDY2; A549 cells; proliferation; apoptosis; cell cycle; migration
PACS:
R73-3
DOI:
10.3969/j.issn.1672-4992.2023.07.002
Abstract:
Objective:To construct MINDY2 lentiviral plasmid and transfected into HEK293T cells to detect the expression of MINDY2.A549 cell line with stable overexpression of MINDY2 was obtained,and the effects of overexpression of MINDY2 on the proliferation，migration and apoptosis of lung cancer A549 cells were observed.Methods:pLVX-Flag-MINDY2 overexpressed plasmid was constructed by homologous recombination method,and the recombinant plasmid was identified by colony PCR and sequencing.HEK293T cells were instantaneously transfected with pLVX-Flag-MINDY2 plasmid,and the expression of Flag-MINDY2 fusion protein was detected by Western blot assay.HEK293T cells were packed with recombinant lentiviral vector,and the virus supernatant was obtained to infect A549 cells.After purinomycin screening,the expression of Flag-MINDY2 fusion protein was detected by Western blot assay.CCK8 assay,Transwell assay and scratch assay were used to detect the effects of MINDY2 overexpression on proliferation and migration of A549 cells.Cell apoptosis and cycle were detected by flow cytometry.Results:Sequencing results proved that the overexpressed lentivirus vector MINDY2 was successfully constructed.The expression of Flag-MINDY2 fusion protein could be detected by transient transfection of MINDY2 recombinant plasmid into HEK293T cells.Western blot results showed that MINDY2 was overexpressed stably in A549 cells.CCK8 assay showed that overexpression of MINDY2 could inhibit the proliferation of A549 cells.Flow cytometry showed that overexpression of MINDY2 significantly increased the apoptosis rate of A549 cells,and the cell cycle was arrested in G0/G1 phase.Transwell and scratch assay showed that overexpression of MINDY2 could inhibit the migration of A549 cells.Conclusion:The lentiviral plasmid expressing MINDY2 and the A549 cell line stably expressing MINDY2 were successfully constructed.MINDY2 can inhibit the proliferation and migration of lung cancer cell line A549 and promote cell apoptosis.

References:

［1］CHANDRASEKARAN AP,SURESH B,KIM HH,et al.Concise review:fate determination of stem cells by deubiquitinating enzymes［J］.Stem Cells(Dayton,Ohio),2017,35(1):9-16.
［2］SNYDER NA,SILVA GM.Deubiquitinating enzymes(DUBs):Regulation,homeostasis,and oxidative stress response［J］.J Biol Chem，2021，297(3):101077.
［3］ZHU H,YAN F,YUAN T,et al.USP10 promotes proliferation of hepatocellular carcinoma by deubiquitinating and stabilizing YAP/TAZ［J］.Cancer Res，2020，80(11):2204-2216.
［4］LI Y,YUAN J.Role of deubiquitinating enzymes in DNA double-strand break repair［J］.J Zhejiang Univ Sci B，2021，22(1):63-72.
［5］FRAILE JM,QUESADA V,RODRIGUEZ D,et al.Deubiquitinases in cancer:new functions and therapeutic options［J］.Oncogene,2012,31(19):2373-2388.
［6］DAS T,SHIN SC,SONG EJ,et al.Regulation of deubiquitinating enzymes by post-translational modifications［J］.Int J Mol Sci,2020,21(11):4028.
［7］PROULX J,BORGMANN K,PARK IW.Role of virally-encoded deubiquitinating enzymes in-regulation of the virus life cycle［J］.Int J Mol Sci,2021,22(9):4438.
［8］NIJMAN SM,LUNA-VARGAS MP,VELDS A,et al.A genomic and functional inventory of deubiquitinating enzymes［J］.Cell,2005,123(5):773-786.
［9］LI Y,REVERTER D.Molecular mechanisms of DUBs regulation in signaling and disease［J］.Int J Mol Sci,2021,22(3):986.
［10］DEY A,SESHASAYEE D,NOUBADE R,et al.Loss of the tumor suppressor BAP causes myeloid transformation［J］.Science(New York),2012,337(6101):1541-1546.
［11］BIGNELL GR,WARREN W,SEAL S,et al.Identification of the familial cylindrmatosis tumour-suppressor gene［J］.Nature Genetics,2000,25(2):160-165.
［12］MASUDA-ROBENS JM,KUTNEY SN,QI H,et al. The TRE17 oncogene encodesa component of a novel effector pathway for Rho GTPases Cdc42 and Rac1 and stimulates actin remodeling［J］.Molecular and Cellular Biology,2003,23(6):2151-2161.
［13］ABDUL REHMAN SA,ARMSTRONG LA,LANGE SM,et al.Mechanism of activation and regulation of deubiquitinase activity in MINDY1 and MINDY2［J］.Molecular Cell,2021,81(20):4176-4190.
［14］KOMANDAR D,CLAGUEl MJ,URBE S.Breaking the chains:structure and function of the deubiquitinases［J］.Nat Revmol Cell Biol,2009,10(8):550-563.
［15］AMERIK AY,HOCHSTRASSER M.Mechanism and function of deubiquitinating enzymes［J］.Biochim Biophys Acta,2004,1695(1-3):189-207.
［16］戴陈新,袁健,郑亚新.去泛素化酶与肿瘤发生及发展关系的研究进展［J］.中国癌症杂志,2013,23(7):547-550. DAI CX,YUAN J,ZHENG YX.Advances in the study of deubiquitinating enzymes in cancer research［J］．China Oncology,2013,23(7):547-550.
［17］WOO B,BAEK KH.Regulatory interplay between deubiquitinating enzymes and cytokines［J］.Cytokine Growth Factor Rev,2019,48:40-51.
［18］CSIZMADIA T,LOW P.The role of deubiquitinating enzymes in the various forms of autophagy［J］.Int J Mol Sci,2020,21(12):4196.
［19］CLAGUE MJ，URBE S，KOMANDER D.Breaking the chains:deubiquitylating enzyme spec-ificity begets function［J］．Nat Rev Mol Cell Biol，2019,20(6):338-352．
［20］逯越,王万里,宋巧炎.去泛素化酶USP33对胃癌上皮细胞-间质转化的影响及对癌细胞迁移、侵袭的作用［J］.肿瘤学杂志,2022,28(4):272-279. LU Y,WANG WL,SONG QY.Effect of deubiquitinating enzyme USP33 on epithelial-mesenchymal transition,migration and invasion of gastric cancer cells［J］.Journal of Chinese Oncology,2022,28(4):272-279.
［21］QIAN M,YAN F,WANG W,et al.Deubiquitinase JOSD2 stabilizes YAP/TAZ to promote c-holangiocarcinoprogression［J］.Acta Pharm Sin B,2021,11(12):4008-4019.
［22］韩祯祯,刘禹辰,王建.去泛素化酶JOSD2促进肝癌细胞的存活和转移［J］.中国生物化学与分子生物学报,2022,38(6):762-771. HAN ZZ,LIU YC,WANG J.Deubiquitinase JOSD2 promotes the survival and metastasis of hepatoma cells［J］.Chinese Journal of Biochemistry and Molecular Biology,2022,38(6):762-771.
［23］LIU S,GONZALEZ-PRIETO R，ZHANG M，et al.Deubiquiti nase activity profiling identifies UCHL1 as a candidate oncoprotein that promotes TGFβ-induced breast cancer metastasis［J］.Clin Cancer Res,2020,26(6):1460-1473.
［24］陈茂鹤,陈肖霞,黄文洋,等.去泛素化酶USP29调控CBX6蛋白质稳定性［J］.中国生物化学与分子生物学报,2021,37(11):1474-1481. CHEN MH,CHEN XX,HUANG WY,et al.Regulation of CBX6 protein stability by the deubiquitination enzyme USP29［J］.Chinese Journal of Biochemistry and Molecular Biology,2021,37(11):1474-1481.
［25］陈兆波,方敏,薛浩然,等.去泛素化酶USP35促进非小细胞肺癌细胞迁移和侵袭［J］.山东大学学报(医学版),2022,60(4):30-37. CHEN ZB,FANG M,XUE HR,et al.Deubiquitinase USP35 promotes the cells migration and invasion of non-small cell lung cancer［J］.Journal of Shandong university（Health sciences）,2022,60(4):30-37.
［26］BURROWS JF,KELVIN AA,MCFARLANE C,et al.USP17 regulates ras activation and cell proliferation by blocking RCE1 activity［J］.The Journal of Biologicalchemistry,2009,284(14):9587-9595.
［27］张超，姜斌，高丰厚.去泛素化酶-USP7通过影响相关癌蛋白参与肿瘤的发生与发展［J］.现代肿瘤医学，2016,24（22）：3684-3687. ZHANG C,JIANG B,GAO FH.Deubiquitinating enzyme-USP7 involves in tumor occurrence and development through the interaction with the related oncoproteins［J］.Modern Oncology，2016,24（22）：3684-3687.
［28］王德成,高洪.细胞凋亡信号转导途径及调控的研究进展［J］.动物医学进展,2003,24(6):4-7. WANG DC，GAO H.Research progress of apoptosis signal transduction pathway and regulation［J］.Progress in Veterinary Medicine,2003,24(6):4-7.
［29］谭秀君,叶玲,汪成林.c-Jun N-末端激酶信号转导通路调节细胞迁移的机制［J］.国际口腔医学杂志,2015,42(5):586-591. TAN XJ,YE L,WANG CL.Mechanism of c-Jun N-terminal kinase signal transduction pathway regulating cell migration［J］.International Journal of Stomatology,2015,42(5):586-591.

Memo

Memo:
National Natural Science Foundation of China（No.82160504）;国家自然科学基金（编号：82160504）；云南省科技厅科技计划项目（编号：202101AU070136）；大学生创新创业训练计划项目（编号：S201110679014）
Last Update: 2023-02-28