TPX2通过p38 MAPK信号通路诱导直肠癌HR-8348细胞凋亡
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摘要
目的:研究下调非洲爪蟾驱动蛋白样蛋白2靶蛋白(TPX2)对直肠癌细胞凋亡的影响及机制。方法:用TPX2小干扰RNA(si RNA)转染直肠癌HR-8348细胞,记为TPX2 si RNA组;以不做转染的细胞作为正常对照(control)组;以转染si RNA阴性对照(si RNA-NC)的细胞作为si RNA-NC组;用p38 MAPK抑制剂处理敲减TPX2表达后的直肠癌HR-8348细胞记为TPX2 si RNA+SB203580组。RT-qPCR和Western blot测定TPX2的表达水平,MTT法测定细胞存活率,流式细胞术测定细胞凋亡,Western blot测定细胞中p38 MAPK、p-p38 MAPK、cleaved caspase-3和Bcl-2的蛋白水平。结果:TPX2 si RNA转染后HR-8348细胞中TPX2的m RNA和蛋白表达水平显著下降(P <0. 05),而转染si RNA-NC对HR-8348细胞中TPX2的m RNA和蛋白水平没有影响。敲减TPX2表达后的直肠癌HR-8348细胞存活率降低,凋亡率升高,细胞中的cleaved caspase-3、p-p38 MAPK/p38 MAPK蛋白水平明显升高,Bcl-2水平水平降低,与control组比较,差异有统计学意义(P <0. 05)。与TPX2 si RNA组相比,TPX2 si RNA+SB203580组的HR-8348细胞凋亡率、cleaved caspase-3水平和p-p38 MAPK/p38 MAPK蛋白水平明显降低,存活率明显升高(P <0. 05)。结论:TPX2表达下调可以通过激活p38 MAPK促进直肠癌HR-8348细胞凋亡。
AIM: To study the effect of targeting protein for Xenopus kinesin-like protein 2( TPX2) expression knockdown on the apoptosis of rectal cancer HR-8348 cells. METHODS: The HR-8348 cells transfected with TPX2 small interfering RNA( si RNA) served as TPX2 si RNA group. The non-transfected cells were used as control group. The cells transfected with si RNA negative control( si RNA-NC) were used as si RNA-NC group. The TPX2 si RNA-transfected cells exposed to p38 MAPK inhibitor SB203580 served as TPX2 si RNA + SB203580 group. The expression of TPX2 at m RNA and protein levels was determined by RT-qPCR and Western blot. The cell viability was measured by MTT assay,the apoptosis was analyzed by flow cytometry. The protein levels of p38 MAPK,p-p38 MAPK,cleaved caspase-3 and Bcl-2 in the HR-8348 cells were determined by Western blot. RESULTS: After transfection,the expression of TPX2 at m RNA and protein levels was decreased in TPX2 si RNA-transfected cells( P < 0. 05). Transfection with si RNA-NC had no effect on TPX2 m RNA and protein levels in the cells. After knockdown of TPX2 expression,the viability of rectal cancer HR-8348 cells and the expression of Bcl-2 were decreased,while the apoptotic rate and the protein levels of cleaved caspase-3 and pp38 MAPK/p38 MAPK were increased significantly reduced( P < 0. 05). Compared with TPX2 si RNA group,the apoptotic rate and the protein levels of cleaved caspase-3 and p-p38 MAPK/p38 MAPK in TPX2 si RNA + SB203580 group were significantly decreased,while the viability was significantly increased( P < 0. 05). CONCLUSION: Knockdown of TPX2 expression promotes apoptosis of rectal cancer HR-8348 cells by activating p38 MAPK signaling pathway.
AIM: To study the effect of targeting protein for Xenopus kinesin-like protein 2( TPX2) expression knockdown on the apoptosis of rectal cancer HR-8348 cells. METHODS: The HR-8348 cells transfected with TPX2 small interfering RNA( si RNA) served as TPX2 si RNA group. The non-transfected cells were used as control group. The cells transfected with si RNA negative control( si RNA-NC) were used as si RNA-NC group. The TPX2 si RNA-transfected cells exposed to p38 MAPK inhibitor SB203580 served as TPX2 si RNA + SB203580 group. The expression of TPX2 at m RNA and protein levels was determined by RT-qPCR and Western blot. The cell viability was measured by MTT assay,the apoptosis was analyzed by flow cytometry. The protein levels of p38 MAPK,p-p38 MAPK,cleaved caspase-3 and Bcl-2 in the HR-8348 cells were determined by Western blot. RESULTS: After transfection,the expression of TPX2 at m RNA and protein levels was decreased in TPX2 si RNA-transfected cells( P < 0. 05). Transfection with si RNA-NC had no effect on TPX2 m RNA and protein levels in the cells. After knockdown of TPX2 expression,the viability of rectal cancer HR-8348 cells and the expression of Bcl-2 were decreased,while the apoptotic rate and the protein levels of cleaved caspase-3 and pp38 MAPK/p38 MAPK were increased significantly reduced( P < 0. 05). Compared with TPX2 si RNA group,the apoptotic rate and the protein levels of cleaved caspase-3 and p-p38 MAPK/p38 MAPK in TPX2 si RNA + SB203580 group were significantly decreased,while the viability was significantly increased( P < 0. 05). CONCLUSION: Knockdown of TPX2 expression promotes apoptosis of rectal cancer HR-8348 cells by activating p38 MAPK signaling pathway.
引文
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[22]万强,杨玉萍,刘中勇.丹参酮ⅡA通过抑制p38MAPK通路减轻PM2.5对血管内皮细胞的损伤[J].中国病理生理杂志,2016,32(4):597-601.
[23]Lu T,Dang S,Rui Z,et al.Adamts18 deficiency promotes colon carcinogenesis by enhancingβ-catenin and p38MAPK/ERK1/2 signaling in the mouse model of AOM/DSS-induced colitis-associated colorectal cancer[J].Oncotarget,2017,8(12):18979-18990.
[24]He H,Chang R,Zhang T,et al.ATM mediates DAB2IP-deficient bladder cancer cell resistance to ionizing radiation through the p38MAPK and NF-κB signaling pathway[J].Mol Med Rep,2017,16(2):1216-1222.
[25]吴池华,李一,杨麟翰,等.TPX2基因调控p38MAPK信号通路抑制乳腺癌细胞增殖及促进凋亡的机制研究[J].实用预防医学,2017,24(8):941-945.
[2]Glaser ZA,Love HD,Guo S,et al.TPX2 as a prognostic indicator and potential therapeutic target in clear cell renal cell carcinoma[J].Urol Oncol,2017,35(5):286-293.
[3]Harb OA,Hegazy AA,Gertallah LM,et al.Prognostic value of immunohistochemical expressions of the stem cell biomarker“LGR5”and the proliferation biomarker“TPX2”in colorectal carcinoma[J].J Tumor,2016,4(3):426-434.
[4]Hsu PK,Chen HY,Yeh YC,et al.TPX2 expression is associated with cell proliferation and patient outcome in esophageal squamous cell carcinoma[J].J Gastroenterol,2014,49(8):1231-1240.
[5]刘万伟,李恩亮,邬林泉.TPX2在细胞间期参与DNA损伤反应及其与肿瘤发生关系的研究进展[J].广东医学,2016,37(8):1245-1248.
[6]Liang B,Zheng W,Fang L,et al.Overexpressed targeting protein for Xklp2(TPX2)serves as a promising prognostic marker and therapeutic target for gastric cancer[J].Cancer Biol Ther,2016,17(8):824-832.
[7]Xie D,Wu X,Lan L,et al.Downregulation of TFAM inhibits the tumorigenesis of non-small cell lung cancer by activating ROS-mediated JNK/p38MAPK signaling and reducing cellular bioenergetics[J].Oncotarget,2016,7(10):11609-11624.
[8]Fu J,Bian M,Xin G,et al.TPX2 phosphorylation maintains metaphase spindle length by regulating microtubule flux[J].J Cell Biol,2015,210(3):373-383.
[9]Sanchez-Pulido L,Perez L,Kuhn S,et al.The C-terminal domain of TPX2 is made of alpha-helical tandem repeats[J].BMC Struct Biol,2016,16:17.
[10]Petry S,Groen AC,Ishihara K,et al.Branching microtubule nucleation in Xenopus egg extracts mediated by augmin and TPX2[J].Cell,2013,152(4):768-777.
[11]Wei JH,Zhang ZC,Wynn RM,et al.GM130 regulates Golgi-derived spindle assembly by activating TPX2 and capturing microtubules[J].Cell,2015,162(2):287-299.
[12]Cáceres-Gorriti KY,Carmona E,Barrès V,et al.RANnucleo-cytoplasmic transport and mitotic spindle assembly partners XPO7 and TPX2 are new prognostic biomarkers in serous epithelial ovarian cancer[J].PLo S One,2014,9(3):e91000.
[13]Rennie YK,McIntyre PJ,Akindele T,et al.A TPX2proteomimetic has enhanced affinity for Aurora-A due to hydrocarbon stapling of a helix[J].ACS Chem Biol,2016,11(12):3383-3390.
[14]Aguirre-Portolés C,Bird AW,Hyman A,et al.Tpx2 controls spindle integrity,genome stability,and tumor development[J].Cancer Res,2012,72(6):1518-1528.
[15]Liu HC,Li YY,Liu YH.Expression of TPX2 m RNA and its correlation with clinical pathology in esophageal cancer[J].Chin J Clin Exp Pathol,2010,26(2):151-153.
[16]Schneider MA,Christopoulos P,Muley T,et al.AURKA,DLGAP5,TPX2,KIF11 and CKAP5:five specific mitosis-associated genes correlate with poor prognosis for nonsmall cell lung cancer patients[J].Int J Oncol,2017,50(2):365-372.
[17]刘红春.TPX2在食管鳞癌组织中的表达及其与浸润转移关系的研究[D].郑州:郑州大学,2010.
[18]刘洁,李湘洲,张居民,等.TPX2和TP在结直肠癌中的表达及临床意义[J].潍坊医学院学报,2013,35(1):38-41.
[19]Vafaiyan Z,Gharaei R,Asadi J.The correlation between telomerase activity and Bax/Bcl-2 ratio in valproic acidtreated MCF-7 breast cancer cell line[J].Iran J Basic Med Sci,2015,18(7):700-704.
[20]Yang X,Gao F,Ma F,et al.Association of the functional BCL-2 rs2279115 genetic variant and small cell lung cancer[J].Tumor Biol,2016,37(2):1693-1698.
[21]Han B,Wang TD,Shen SM,et al.Annonaceous acetogenin mimic AA005 induces cancer cell death via apoptosis inducing factor through a caspase-3-independent mechanism[J].BMC Cancer,2015,15:139.
[22]万强,杨玉萍,刘中勇.丹参酮ⅡA通过抑制p38MAPK通路减轻PM2.5对血管内皮细胞的损伤[J].中国病理生理杂志,2016,32(4):597-601.
[23]Lu T,Dang S,Rui Z,et al.Adamts18 deficiency promotes colon carcinogenesis by enhancingβ-catenin and p38MAPK/ERK1/2 signaling in the mouse model of AOM/DSS-induced colitis-associated colorectal cancer[J].Oncotarget,2017,8(12):18979-18990.
[24]He H,Chang R,Zhang T,et al.ATM mediates DAB2IP-deficient bladder cancer cell resistance to ionizing radiation through the p38MAPK and NF-κB signaling pathway[J].Mol Med Rep,2017,16(2):1216-1222.
[25]吴池华,李一,杨麟翰,等.TPX2基因调控p38MAPK信号通路抑制乳腺癌细胞增殖及促进凋亡的机制研究[J].实用预防医学,2017,24(8):941-945.