收稿日期: 2019-07-09
网络出版日期: 2022-07-15
Functional study and detection of vimentin produced by miR-200c target gene in human gastric cancer BGC823 cells
Received date: 2019-07-09
Online published: 2022-07-15
目的： 采用RNA靶向干扰技术,研究人胃癌细胞BGC823中微小RNA（microRNA, miRNA, miR）-200c的靶点锌指E盒增强子结合蛋白2（zinc finger E-box enhancer binding protein 2, ZEB2）基因的表达情况,并检测相关的波形蛋白基因表达情况,观察miR-200c对胃癌BGC823细胞功能的影响。方法： 构建ZEB2基因表达的干扰质粒shZEB2,用Lipofectamine 2000转染胃癌BGC823细胞,用G418筛选稳定转染细胞株,进行实时定量聚合酶链反应（polymerase chain reaction,PCR）检测人BCG823细胞中ZEB2 mRNA、miR-200c及波形蛋白基因的表达水平,并用蛋白印迹法检测ZEB2蛋白、波形蛋白表达水平,噻唑蓝（methylthiazolyldiphenyl-tetrazolium bromide, MTT）法检测细胞增殖能力;Transwell小室检测细胞侵袭能力,划痕实验检测细胞迁移能力。结果： 干扰ZEB2分子表达后,miR-200c表达水平增高,而BGC823细胞的增殖、迁移及侵袭能力明显下降。结论： ZEB2是miR-200c的检测靶点,干扰ZEB2后,胃癌细胞BGC823的增殖、迁移及侵袭能力明显降低。
关键词： 胃癌; 锌指E盒增强子结合蛋白2; 波形蛋白
杨翠萍, 杨晓金, 杨燕萍, 张梦茵, 谢玲, 俞骁珺, 蔡波尔, 陈登宇, 陈平, 吴云林 . 人胃癌细胞BGC823中miR-200c靶基因产物波形蛋白的检测及功能研究[J]. 诊断学理论与实践, 2020 , 19(04) : 414 -419 . DOI: 10.16150/j.1671-2870.2020.04.017
Objective: To investigate the expression of zinc finger E-box enhancer binding protein 2(ZEB2) gene in miR-200c in human gastric cancer BGC823 cells by RNA targeted interference technique, and detect expression of vimentin, so as to observe the effect of miR-200c on the function of gastric cancer BGC823 cells. Methods: The interference plasmids shZEB2 was constructed and transfected to BGC-823 cells in mediation of Lipofectamine 2000. The stable transfectants were screened by G418, and ZEB2 mRNA, miR-200c and vimentin was detected by real-time PCR in BCG823 cells, while protein expression levels of ZEB2 and vimentin by Western blotting. Proliferation of BGC-823 cells was evaluated by MTT assay. Transwell assay and wound healing assay were used to observe the invasion and migration ability of BCG823 cells, respectively. Results: With interfering ZEB2 expression, miR-200c expression level increased while proliferation, migration and invasion ability of BGC823 cells decreased significantly. Conclusions: ZEB2 may be the target of miR-200c, and the ability of BGC823 cells including proliferation, migration and invasion is significantly decreased after interference with ZEB2.
| [1] | Kim SS, Ruiz VE, Carroll JD, Moss SF. Helicobacter pylori in the pathogenesis of gastric cancer and gastric lymphoma[J]. Cancer Lett, 2011, 305(2):228-238. |
| [2] | Yang L, Zheng R, Wang N, et al. Incidence and mortality of stomach cancer in China[J]. Chin J Cancer Res, 2018, 30(3):291-298. |
| [3] | Niinuma T, Suzuki H, Nojima M, et al. Upregulation of miR-196a and HOTAIR drive malignant character in gastrointestinal stromal tumors[J]. Cancer Res, 2012, 72(5):1126-1136. |
| [4] | Thiery JP, Acloque H, Huang RY, et al. Epithelial-mesenchymal transitions in development and disease[J]. Cell, 2009, 139(5):871-890. |
| [5] | Mani SA, Guo WJ, Liao MJ, et al. The epithelial-mesenchymal transition generates cells with properties of stem cells[J]. Cell, 2008, 133(4):704-715. |
| [6] | Ishibashi M, Kogo R, Shibata K, et al. Clinical significance of the expression of long non-coding RNA HOTAIR in primary hepatocellular carcinoma[J]. Oncol Rep, 2013, 29(3):946-950. |
| [7] | Wang J, Chen D, He X, et al. Downregulated lincRNA HOTAIR expression in ovarian cancer stem cells decreases its tumorgeniesis and metastasis by inhibiting epithelial-mesenchymal transition[J]. Cancer Cell Int, 2015, 15:24. |
| [8] | Christoffersen NR, Silahtaroglu A, Orom UA, et al. miR-200b mediates post-transcriptional repression of ZFHX1B[J]. RNA, 2007, 13(8):1172-1178. |
| [9] | Hurteau GJ, Carlson JA, Spivack SD, et al. Overexpression of the microRNA hsa-miR-200c leads to reduced expression of transcription factor 8 and increased expression of E-cadherin[J]. Cancer Res, 2007, 67(17):7972-7976. |
| [10] | Park SM, Gaur AB, Lengyel E, et al. The miR200 family determines the epithelial phenotype of cancer cells by targeting the E-cadherin repressors ZEB1 and ZEB2[J]. Genes Dev, 2008, 22(7):894-907. |
| [11] | Gregory PA, Bert AG, Paterson EL, et al. The miR-200 family and miR-205 regulate epithelial to mesenchymal transition by targeting ZEB1 and SIP1[J]. Nat Cell Biol, 2008, 10(5):593-601. |
| [12] | Korpal M, Lee ES, Hu G, et al. The miR-200 family inhibits epithelial-mesenchymal transition and cancer cell migration by direct targeting of E-cadherin transcriptional repressors ZEB1 and ZEB2[J]. J Biol Chem, 2008, 283(22):14910-14914. |
| [13] | Brabletz S, Brabletz T. The ZEB/miR-200 feedback loop—A motor of cellular plasticity in development and cancer?[J] EMBO Rep, 2010, 11(9):670-677. |
| [14] | Burk U, Schubert J, Wellner U, et al. A reciprocal repression between ZEB1 and members of the miR-200 family promotes EMT and invasion in cancer cells[J]. EMBO Rep, 2008, 9(6):582-589. |
| [15] | Bracken CP, Gregory PA, Kolesnikoff N, et al. A double-negative feedback loop between ZEB1- SIP1 and the microRNA-200 family regulates epithelial-mesenchymal transition[J]. Cancer Res, 2008, 68(19):7846-7854. |
| [16] | Olson P, Lu J, Zhang H, et al. MicroRNA dynamics in the stages of tumorigenesis correlate with hallmark capabilities of cancer[J]. Genes Dev, 2009, 23(18):2152-2165. |
| [17] | Korpal M, Ell BJ, Buffa FM, et al. Direct targeting of Sec23a by miR-200s influences cancer cell secretome and promotes metastatic colonization[J]. Nat Med, 2011, 17(9):1101-1108. |
| [18] | Fanelli MF, Chinen LT Sr, Begnami MD, et al. The influence of CD44v6, TGF-α, COX-2, MMP-7, and MMP-9 on clinical evolution of patients with gastric cancer[J]. J Clin Oncol, 2011, 29(4 Suppl):21. |
| [19] | Hill L, Browne G, Tulchinsky E. ZEB/miR-200 feedback loop: at the crossroads of signal transduction in cancer[J]. Int J Cancer, 2013, 132(4):745-754. |
| [20] | Liu S, Tetzlaff MT, Cui R, et al. miR-200c inhibits melanoma progression and drug resistance through down-regulation of BMI-1[J]. Am J Pathol, 2012, 181(5):1823-1835. |
| [21] | Okugawa Y, Toiyama Y, Hur K, et al. Metastasis-associated long non-coding RNA drives gastric cancer development and promotes peritoneal metastasis[J]. Carcinogenesis, 2014, 35(12):2731-2739. |
| [22] | Kurashige J, Mima K, Sawada G, et al. Epigenetic modulation and repression of miR-200b by cancer-associated fibroblasts contribute to cancer invasion and peritoneal dissemination in gastric cancer[J]. Carcinogenesis, 2015, 36(1):133-141. |
| [23] | Sundararajan V, Gengenbacher N, Stemmler MP, et al. The ZEB1/miR-200c feedback loop regulates invasion via actin interacting proteins MYLK and TKS5[J]. Oncotarget, 2015, 6(29):27083-27096. |
| [24] | Fanelli MF, Chinen LT Sr, Begnami MD, et al. The influence of CD44v6, TGF-α, COX-2, MMP-7, and MMP-9 on clinical evolution of patients with gastric cancer[J]. J Clin Oncol, 2011, 29(4_suppl):21. |
| [25] | Hur H, Kim JY, Kim YB, et al. Effect of Helicobacter pylori on prognosis of curatively resected gastric cancers in a population with high prevalence: short-term results of a prospective study[J]. J Clin Oncol, 2011, 29(4 Suppl):152b. |
| [26] | Corso G, Marrelli D, Pascale V, et al. Oncogenic mutations in MAPK cascade as novel molecular biomarkers for treatment of gastric cancer patients with EGFR inhibitors[J]. J Clin Oncol, 2011, 29(4_suppl):39. |
| [27] | Noormohammad M, Sadeghi S, Tabatabaeian H, et al. Upregulation of miR-222 in both Helicobacter pylori-infected and noninfected gastric cancer patients[J]. J Genet, 2016, 95(4):991-995. |
| [28] | Shamsdin SA, Alborzi A, Rasouli M, et al. The importance of TH22 and TC22 cells in the pathogenesis of Helicobacter pylori-associated gastric diseases[J/OL]. [2016-12-19]. https://pubmed.ncbi.nlm.nih.gov/27990709/. |
/
| 〈 |
|
〉 |
