Effects of silencing UBE2O on proliferation and invasion of human non-small cell lung cancer cells
-
摘要:
目的 研究泛素结合酶E2O（ubiquitin-conjugating enzyme E2O，UBE2O）蛋白在非小细胞肺癌（non-small cell lung cancer，NSCLC）细胞增殖、细胞周期、迁移、侵袭中的作用。 方法 通过慢病毒介导的shRNA靶向抑制人NSCLC细胞株A549中UBE2O基因的表达，应用CCK-8法、流式细胞术分别检测UBE2O对A549细胞增殖、细胞周期和凋亡的影响，应用Transwell实验检测下调UBE2O表达后对A549细胞迁移、侵袭能力的影响，应用Western blot检测细胞上皮间质转化（epithelial-mesenchymal transition，EMT）两种标志分子E-cadherin蛋白、N-cadherin蛋白和PI3K-Akt信号通路相关蛋白表达量的变化。 结果 下调UBE2O表达后，A549细胞UBE2O mRNA和蛋白表达均下调（均P < 0.01），CCK-8实验结果显示沉默UBE2O可以抑制A549的增殖（P < 0.001），Transwell实验显示下调UBE2O的表达能够抑制A549细胞的迁移、侵袭（均P < 0.001）。Western blot结果表明下调UBE2O的表达可使A549细胞中的E-cadherin蛋白表达水平升高、p-Akt蛋白表达水平下降。 结论 UBE2O蛋白能够促进NSCLC细胞侵袭和迁移，作用机制可能是通过诱导肺癌细胞发生EMT和促进pI3K-Akt信号通路的激活。 Abstract:Objective To study the role of ubiquitin-conjugating enzyme E2O (UBE2O) in the proliferation, cell cycle, migration, and invasion of non-small cell lung cancer (NSCLC) cells. Methods CCK-8 assay and flow cytometry were used to detect the effects of UBE2O on A549 cell proliferation, cell cycle, and apoptosis by inhibiting UBE2O gene expression through lentivirus-mediated shRNA targeting in the human NSCLC cell line A549. Transwell assay was used to examine the effect of UBE2O downregulation on the migration and invasion of A549 cells. Western blot was used to detect the expression of E-cadherin and N-cadherin, which are epithelial-mesenchymal transition (EMT) markers and the main proteins of the PI3K-Akt signaling pathway. Results The mRNA and protein expression levels of UBE2O in A549 cells were downregulated (P < 0.01). CCK-8 assay showed that UBE2O silencing inhibited the proliferation of A549 cells (P < 0.001). Transwell assay showed that UBE2O knockdown inhibited the migration and invasion of A549 cells (P < 0.001). Western blot showed that UBE2O downregulation increased E-cadherin expression and decreased p-PI3K and p-Akt expression in A549 cells. Conclusions UBE2O overexpression promotes the invasion and migration of NSCLC cells by inducing EMT and activating the PI3K-Akt signaling pathway. -
图 5 靶向沉默UBE2O对A549细胞周期的影响
A：流式检测细胞周期分布；B：细胞周期统计；C：Western blot检测细胞周期相关分子Cyclin D1、Cyclin A1表达情况
-
[1] Zakowski MF. Analytic inquiry: Molecular testing in lung cancer[J]. Cancer Cytopathol, 2017, 125(S6): 470-476. doi: 10.1002/cncy.21866 [2] Vergnenègre A, Chouaïd C. Review of economic analyses of treatment for non-small cell lung cancer (NSCLC)[J]. Expert Rev Pharmacoecon Outcomes Res, 2018, 18(5): 519-528. doi: 10.1080/14737167.2018.1485099 [3] Berleth ES, Pickart CM. Mechanism of ubiquitin conjugating enzyme E2-230K: catalysis involving a thiol relay[J]? Biochemistry, 1996, 35(5): 1664-1671. doi: 10.1021/bi952105y [4] Yokota T, Nagai H, Harada H, et al. Identification, tissue expression, and chromosomal position of a novel gene encoding human ubiquitinconjugating enzyme E2-230k[J]. Gene, 2001, 267(1): 95-100. doi: 10.1016/S0378-1119(01)00407-3 [5] Zhang X, Zhang J, Bauer A, et al. Fine-tuning BMP7 signalling in adipogenesis by UBE2O/E2-230K-mediated monoubiquitination of SMAD6 [J]. EMBO J, 2013, 32(7): 996-1007. doi: 10.1038/emboj.2013.38 [6] Zhang X, Zhang J, Zhang L, et al. UBE2O negatively regulates TRAF6- mediated NF-κB activation by inhibiting TRAF6 polyubiquitination[J]. Cell Res, 2013, 23(3): 366-377. doi: 10.1038/cr.2013.21 [7] Chen S, Yang J, Zhang Y, et al. Ubiquitin-conjugating enzyme UBE2O regulates cellular clock function by promoting the degradation of the transcription factor BMAL1[J]. J Biol Chem, 2018, 293(29): 11296- 11309. doi: 10.1074/jbc.RA117.001432 [8] Lin M, Smith LT, Smiraglia DJ, et al. DNA copy number gains in head and neck squamous cell carcinoma[J]. Oncogene, 2006, 25(9): 1424-1433. doi: 10.1038/sj.onc.1209166 [9] Rice K L, Lin X, Wolniak K, et al. Analysis of genomic aberrations and gene expression profiling identifies novel lesions and pathways in myeloproliferative neoplasms[J]. Blood Cancer J, 2011, 1(11): e40. doi: 10.1038/bcj.2011.39 [10] Toffoli S, Bar I, Abdel-Sater F, et al. Identification by array comparative genomic hybridization of a new amplicon on chromosome 17q highly recurrent in BRCA1 mutated triple negative breast cancer[J]. Breast Cancer Res, 2014, 16(6): 466. doi: 10.1186/s13058-014-0466-y [11] Xu L, Fei M, Chunxiao L, et al. UBE2O promotes the proliferation, EMT and stemness properties of breast cancer cells through the UBE2O/AMPKα2/mTORC1-MYC positive feedback loop[J]. Cell Death Dis, 2020, 11(1): 1-17. doi: 10.1038/s41419-019-2182-0 [12] Gao J, Aksoy BA, Dogrusoz U, et al. Integrative analysis of complex cancer genomics and clinical profiles using the cBioPortal[J]. Sci Signal, 2013, 6(269): pl1. http://www.tandfonline.com/servlet/linkout?suffix=CIT0052&dbid=8&doi=10.1080%2F10409238.2018.1442408&key=23550210 [13] Jiang WG, E-cadherin and its associated protein catenins, cancer invasion and metastasis[J]. Br J Surg, 1996, 83(4): 437-446. http://www.ncbi.nlm.nih.gov/pubmed/8665230 [14] Brazil DP, Yang Z, Hemmings BA. Advances in protein kinase B signalling: AKTion on multiple fronts[J]. Trends Biochem Sci, 2004, 29(5): 233-242. doi: 10.1016/j.tibs.2004.03.006 [15] Manning BD, Cantley LC. AKT/PKB signaling: navigating downstream[J]. Cell, 2007, 129(7): 1261-1274 doi: 10.1016/j.cell.2007.06.009 -
下载:
点击查看大图
图(8)
计量
- 文章访问数: 233
- HTML全文浏览量: 20
- PDF下载量: 36
- 被引次数: 0
