[1]董云朋,程远,陈晋.沉默APN对胶质瘤细胞U251增殖、侵袭和凋亡的影响[J].第三军医大学学报,2020,42(09):908-914.
 ONG Yunpeng,CHENG Yuan,CHEN Jin.Effects of silencing APN gene on proliferation, invasion and apoptosis of human glioma U251 cells[J].J Third Mil Med Univ,2020,42(09):908-914.
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沉默APN对胶质瘤细胞U251增殖、侵袭和凋亡的影响(/HTML )
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《第三军医大学学报》[ISSN:1000-5404/CN:51-1095/R]

卷:
42卷
期数:
2020年第09期
页码:
908-914
栏目:
基础医学
出版日期:
2020-05-15

文章信息/Info

Title:
Effects of silencing APN gene on proliferation, invasion and apoptosis of human glioma U251 cells
作者:
董云朋程远陈晋
重庆医科大学附属第二医院神经外科
Author(s):
ONG Yunpeng CHENG Yuan CHEN Jin

Department of Neurosurgery, the Second Affiliated Hospital of Chongqing Medical University, Chongqing, 400010, China

关键词:
胶质瘤aminopeptidase NRNA干扰STAT3细胞凋亡侵袭
Keywords:
glioma aminopeptidase N RNAi STAT3 apoptosis invasion
分类号:
R394.3; R730.23; R730.264
文献标志码:
A
摘要:

目的探究aminopeptidase N(APN)对胶质瘤细胞U251增殖能力、迁移侵袭能力和凋亡等生物学行为的影响及可能的作用机制。方法通过RNA干扰技术沉默胶质瘤细胞U251中APN的表达后,运用RT-qPCR的方法检测APN mRNA表达,采用Western blot检测APN蛋白表达水平,CCK-8检测各组U251细胞的增殖活性;采用流式细胞仪检测细胞凋亡和细胞周期;采用细胞划痕实验检测U251细胞的迁移能力,Transwell实验检测细胞侵袭能力;采用Western blot检测STAT3通路蛋白t-STAT3、p-STAT3、Bcl-2和MMP-2的蛋白表达水平。结果与空白对照组和阴性对照组相比,沉默U251中APN的表达后APN在基因和蛋白表达水平下降(P<0.01),沉默APN后U251的增殖活性明显下降(P<0.05),细胞周期停滞在G0/G1期,并且凋亡率明显增加(P<0.01);另外沉默APN的表达后U251细胞的迁移能力和侵袭能力明显下降(P<0.01);沉默APN后STAT3磷酸化水平下降,引起下游蛋白Bcl-2和MMP-2等蛋白表达下降(P<0.05)。结论沉默APN可通过STAT3信号途径抑制胶质瘤细胞U251增殖、迁移和侵袭,诱导凋亡。

Abstract:

ObjectiveTo determine the effect of aminopeptidase N (APN) on the proliferation, invasion, and apoptosis in human glioma U251 cells and to investigate the possible mechanism. MethodsAfter APN gene expression was silenced in glioma cells U251 by RNAi, its expression at mRNA and protein levels was detected by RT-qPCR and Western blotting, respectively. Then, CCK-8 assay, flow cytometry, wound healing test and transwell assay were used to detect the proliferation ability, cell cycle and apoptosis, migration and invasion activity in the U251 cells. The protein expression of STAT3 signal pathway proteins t-STAT3, p-STAT3, MMP-2 and Bcl-2 was measured by Western blotting. ResultsCompared with blank control and NC-siRNA cells, the expression of APN was decreased at mRNA and protein levels in APN-siRNA cells (P<0.01). Silencing of APN gene also resulted in inhibited proliferation ability (P<0.05), cell cycle arrested at G0/G1 phase, enhanced apoptotic rate (P<0.01), and decreased migration and invasion abilities (P<0.01) in U251 cells. What’s more, the inhibited expression of p-STAT3 caused the decreased expression of Bcl-2 and MMP-2 (all P<0.05). ConclusionSilencing of APN could inhibit the proliferation, migration and invasion, and induce apoptosis in glioma U251 cells via STAT3 signaling pathway.

参考文献/References:

[1]DIXON J, KAKLAMANIS L, TURLEY H, et al. Expression of aminopeptidase-n (CD 13) in normal tissues and malignant neoplasms of epithelial and lymphoid origin[J]. J Clin Pathol, 1994, 47(1): 43-47. DOI:10.1136/jcp.47.1.43.
[2]RESHEQ Y J, MENZNER A K, BOSCH J, et al. Impaired transmigration of myeloid-derived suppressor cells across human sinusoidal endothelium is associated with decreased expression of CD13[J]. J Immunol, 2017, 199(5): 1672-1681. DOI:10.4049/jimmunol.1600466.
[3]YAMANAKA C, WADA H, EGUCHI H, et al. Clinical significance of CD13 and epithelial mesenchymal transition (EMT) markers in hepatocellular carcinoma[J]. Jpn J Clin Oncol, 2018, 48(1): 52-60. DOI:10.1093/jjco/hyx157.
[4]MARTNEZ J M, PRIETO I, RAMREZ M J, et al. Aminopeptidase activities in breast cancer tissue[J]. Clin Chem, 1999, 45(10): 1797-1802. DOI:10.1093/clinchem/45.10.1797.
[5]ISHII K, USUI S, SUGIMURA Y, et al. Aminopeptidase N regulated by zinc in human prostate participates in tumor cell invasion[J]. Int J Cancer, 2001, 92(1): 49-54.
[6]ZHANG J, FANG C Y, QU M H, et al. CD13 inhibition enhances cytotoxic effect of chemotherapy agents[J]. Front Pharmacol, 2018, 9: 1042. DOI:10.3389/fphar.2018.01042.
[7]AFFRONTI M L, HEERY C R, HERNDON J E 2nd, et al. Overall survival of newly diagnosed glioblastoma patients receiving carmustine wafers followed by radiation and concurrent temozolomide plus rotational multiagent chemotherapy[J]. Cancer, 2009, 115(15): 3501-3511. DOI:10.1002/cncr.24398.
[8]LIAO A H, CHOU H Y, HSIEH Y L, et al. Enhanced therapeutic epidermal growth factor receptor (EGFR) antibody delivery via pulsed ultrasound with targeting microbubbles for glioma treatment[J]. J Med Biol Eng, 2015, 35(2): 156-164. DOI:10.1007/s40846-015-0032-9.
[9]WELLER M, WICK W, ALDAPE K, et al. Glioma[J]. Nat Rev Dis Primers, 2015, 1: 15017. DOI:10.1038/nrdp.2015.17.
[10]HAMID O, ROBERT C, DAUD A, et al. Safety and tumor responses with lambrolizumab (anti-PD-1) in melanoma [J]. N Engl J Med, 2013, 369(2):134-144. DOI: 10.1056/NEJMoa1305133.
[11]BALAR A V, WEBER J S. PD-1 and PD-L1 antibodies in cancer: current status and future directions[J]. Cancer Immunol Immunother, 2017, 66(5): 551-564. DOI:10.1007/s00262-017-1954-6.
[12]FILLEY A C, HENRIQUEZ M, DEY M. Recurrent glioma clinical trial, CheckMate-143: the game is not over yet[J]. Oncotarget, 2017, 8(53): 91779-91794. DOI:10.18632/oncotarget.21586.
[13]GRUPP S A, KALOS M, BARRETT D, et al. Chimeric antigen receptor-modified T cells for acute lymphoid leukemia[J]. N Engl J Med, 2013, 368(16): 1509-1518. DOI:10.1056/NEJMoa1215134.
[14]SCHUSTER S J, SVOBODA J, CHONG E A, et al. Chimeric antigen receptor T cells in refractory B-cell lymphomas[J]. N Engl J Med, 2017, 377(26): 2545-2554. DOI:10.1056/nejmoa1708566.
[15]WEI X R, LAI Y X, LI J, et al. PSCA and MUC1 in non-small-cell lung cancer as targets of chimeric antigen receptor T cells[J]. Oncoimmunology, 2017, 6(3): e1284722. DOI:10.1080/2162402x.2017.1284722.
[16]HECKLER M, DOUGAN S K. Unmasking pancreatic cancer: epitope spreading after single antigen chimeric antigen receptor T-cell therapy in a human phase I trial[J]. Gastroenterology, 2018, 155(1): 11-14. DOI:10.1053/j.gastro.2018.06.023.
[17]陈峰, 郑晓红, 李文斌. 脑胶质瘤免疫治疗进展[J]. 首都医科大学学报, 2019, 40(6): 966-971.
 CHEN F, ZHENG X H, LI W B. Immunotherapy for the treatment of glioma[J]. J Cap Med Univ, 2019, 40(6): 966-971.
[18]PIEDFER M, DAUZONNE D, TANG R P, et al. Aminopeptidase-N/CD13 is a potential proapoptotic target in human myeloid tumor cells[J]. FASEB J, 2011, 25(8): 2831-2842. DOI:10.1096/fj.11-181396.
[19]WANG X Q, LIU Y, LIU W, et al. Ubenimex, an APN inhibitor, could serve as an antitumor drug in RT112 and 5637 cells by operating in an Aktassociated manner[J]. Mol Med Rep, 2018,17: 4531-4539. DOI:10.3892/mmr.2018.8402.
[20]TERAUCHI M, KAJIYAMA H, SHIBATA K, et al. Inhibition of APN/CD13 leads to suppressed progressive potential in ovarian carcinoma cells[J]. BMC Cancer, 2007, 7: 140. DOI:10.1186/1471-2407-7-140.
[21]WANG R, SUN Q, WANG P, et al. Notch and Wnt/β-catenin signaling pathway play important roles in activating liver cancer stem cells [J]. Oncotarget, 2016, 7(5): 5754-5768. DOI:10.18632/oncotarget.6805.
[22]SAIKI I, FUJII H, YONEDA J, et al. Role of aminopeptidase N (CD13) in tumor-cell invasion and extracellular matrix degradation[J]. Int J Cancer, 1993, 54(1): 137-143. DOI:10.1002/ijc.2910540122.
[23]BRANTLEY E C, BENVENISTE E N. Signal transducer and activator of transcription-3: a molecular hub for signaling pathways in gliomas[J]. Mol Cancer Res, 2008, 6(5): 675-684. DOI:10.1158/1541-7786.mcr-07-2180.

更新日期/Last Update: 2020-05-06