斑点型锌指结构蛋白在体外负性调控HepG2细胞的转移
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摘要
目的探索斑点型锌指结构蛋白(SPOP)在体外对HepG2细胞迁移和侵袭的影响。方法利用质粒过表达和shRNA技术构建稳定过表达和敲低SPOP的稳定转染细胞,Western blotting检测SPOP过表达和敲低效果,然后利用体外Transwell实验检测SPOP对HepG2细胞的迁移和侵袭能力的影响,最后利用划痕实验再次检测SPOP对HepG2细胞迁移能力的影响。结果稳定转染HepG2中SPOP过表达和敲低效果明显;Transwell实验结果显示,过表达SPOP后HepG2细胞的迁移和侵袭能力明显下降,敲低后其迁移和侵袭能力明显上升。划痕实验结果显示,SPOP对HepG2细胞的迁移能力影响与Transwell实验一致。结论 SPOP在体外可以负性调控HepG2细胞的转移能力。
Objective To explore the effect of speckle-type POZ protein(SPOP) on migration and invasion of HepG2 cells in vitro. Methods Stable gain-and loss-of SPOP expression HepG2 cell lines were constructed using plasmid overexpression and shRNA technique. Western blotting was used to detect the overexpression and knockdown effect of SPOP, and the effects of SPOP on the migration and invasion of HepG2 cells was examined by Transwell assays and wound-healing assay was also used to detect HepG2 cells' migration in vitro. Results The effect of gain-and loss-of SPOP expression was obvious in constructed stable HepG2 cells. HepG2 cells' migration and invasion were significantly decreased by gain-SPOP expression, while the cells' migration and invasion were obviously enhanced in loss-SPOP expression HepG2 cells. Wound-healing assay exhibited that SPOP overexpression suppressed HepG2 cells' migration and that downregulation of SPOP promoted the migration of HepG2 cells. Conclusion SPOP could negatively regulate HepG2 cells' metastasis in vitro.
Objective To explore the effect of speckle-type POZ protein(SPOP) on migration and invasion of HepG2 cells in vitro. Methods Stable gain-and loss-of SPOP expression HepG2 cell lines were constructed using plasmid overexpression and shRNA technique. Western blotting was used to detect the overexpression and knockdown effect of SPOP, and the effects of SPOP on the migration and invasion of HepG2 cells was examined by Transwell assays and wound-healing assay was also used to detect HepG2 cells' migration in vitro. Results The effect of gain-and loss-of SPOP expression was obvious in constructed stable HepG2 cells. HepG2 cells' migration and invasion were significantly decreased by gain-SPOP expression, while the cells' migration and invasion were obviously enhanced in loss-SPOP expression HepG2 cells. Wound-healing assay exhibited that SPOP overexpression suppressed HepG2 cells' migration and that downregulation of SPOP promoted the migration of HepG2 cells. Conclusion SPOP could negatively regulate HepG2 cells' metastasis in vitro.
引文
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[14] Li G,Ci W,Karmakar S,et al.SPOP promotes tumorigenesis by acting as a key regulatory hub in kidney cancer[J].Cancer Cell,2014,25(4):455-468.
[15] Barbieri CE,Baca SC,Lawrence MS,et al.Exome sequencing identifies recurrent SPOP,FOXA1 and MED12 mutations in prostate cancer[J].Nat Genet,2012,44(6):685-689.
[16] Ding D,Song T,Jun W,et al.Decreased expression of the SPOP gene is associated with poor prognosis in glioma[J].Int J Oncol,2015,46(1):333-341.
[17] Luo J,Bao YC,Ji XX,et al.SPOP promotes SIRT2 degradation and suppresses non-small cell lung cancer cell growth[J].Biochem Biophys Res Commun,2017,483(2):880-884.
[18] Liu ChJ,Zhang XY,Shan ZhY,et al.Establishment of a tumor-bear mouse model carrying human HepG2 cells and exploration of immune tolerance[J].Acta Anatomica Sinica,2015,46(4):509-513.(in Chinese)刘春佳,张星宇,单智焱,等.人肝癌HepG2细胞小鼠荷瘤模型的建立及免疫耐受的探讨[J].解剖学报,2015,46(4):509-513.
[2] Andrade LE,Chan EK,Raska Ⅰ,et al.Human autoantibody to a novel protein of the nuclear coiled body:immunological characterization and cDNA cloning of p80-coilin[J].J Exp Med,1991,173(6):1407-1419.
[3] Li C,Ao J,Fu J,et al.Tumor-suppressor role for the SPOP ubiquitin ligase in signal-dependent proteolysis of the oncogenic co-activator SRC-3/AIB1[J].Oncogene,2011,30(42):4350-4364.
[4] An J,Wang C,Deng Y,et al.Destruction of full-length androgen receptor by wild-type SPOP,but not prostate-cancer-associated mutants[J].Cell Rep,2014,6(4):657-669.
[5] Zhang L,Peng S,Dai X,et al.Tumor suppressor SPOP ubiquitinates and degrades EglN2 to compromise growth of prostate cancer cells[J].Cancer Lett,2017,390:11-20.
[6] Hu X,Yang Z,Zeng M,et al.Speckle-type POZ (pox virus and zinc finger protein) protein gene deletion in ovarian cancer:fluorescence in situ hybridization analysis of a tissue microarray[J].Oncol Lett,2016,12(1):658-662.
[7] Zeng C,Wang Y,Lu Q,et al.SPOP suppresses tumorigenesis by regulating Hedgehog/Gli2 signaling pathway in gastric cancer[J].J Exp Clin Cancer Rese,2014,33:75.
[8] Gao K,Jin X,Tang Y,et al.Tumor suppressor SPOP mediates the proteasomal degradation of progesterone receptors (PRs) in breast cancer cells[J].Am J Cancer Res,2015,5(10):3210-3220.
[9] El-Serag HB,Rudolph KL.Hepatocellular carcinoma:epidemiology and molecular carcinogenesis[J].Gastroenterology,2007,132(7):2557-2576.
[10] Lau WY,Lai EC.Hepatocellular carcinoma:current management and recent advances[J].Hepatobiliary Pancreat Dis Int,2008,7(3):237-257.
[11] Dahmani R,Just PA,Perret C.The Wnt/beta-catenin pathway as a therapeutic target in human hepatocellular carcinoma[J].Clin Res Hepatol Gastroenterol,2011,35(11):709-713.
[12] Komposch K,Sibilia M.EGFR signaling in liver diseases[J].Int J Mol Sci,2015,17(1):pii E30.
[13] Sia D,Alsinet C,Newell P,et al.VEGF signaling in cancer treatment[J].Curr Pharm Des,2014,20(17):2834-2842.
[14] Li G,Ci W,Karmakar S,et al.SPOP promotes tumorigenesis by acting as a key regulatory hub in kidney cancer[J].Cancer Cell,2014,25(4):455-468.
[15] Barbieri CE,Baca SC,Lawrence MS,et al.Exome sequencing identifies recurrent SPOP,FOXA1 and MED12 mutations in prostate cancer[J].Nat Genet,2012,44(6):685-689.
[16] Ding D,Song T,Jun W,et al.Decreased expression of the SPOP gene is associated with poor prognosis in glioma[J].Int J Oncol,2015,46(1):333-341.
[17] Luo J,Bao YC,Ji XX,et al.SPOP promotes SIRT2 degradation and suppresses non-small cell lung cancer cell growth[J].Biochem Biophys Res Commun,2017,483(2):880-884.
[18] Liu ChJ,Zhang XY,Shan ZhY,et al.Establishment of a tumor-bear mouse model carrying human HepG2 cells and exploration of immune tolerance[J].Acta Anatomica Sinica,2015,46(4):509-513.(in Chinese)刘春佳,张星宇,单智焱,等.人肝癌HepG2细胞小鼠荷瘤模型的建立及免疫耐受的探讨[J].解剖学报,2015,46(4):509-513.