TIMP-3基因真核表达载体的构建及其对乳腺癌MDA-MB-453细胞生长及侵袭的抑制作用

doi:10.13481/j.1671-587x.20160118

摘要/Abstract

摘要：

目的: 构建组织基质金属蛋白酶抑制剂3(TIMP-3)真核表达载体并转染乳腺癌MDA-MB-453细胞,探讨TIMP-3对MDA-MB-453细胞生长及侵袭的抑制作用。方法: 采用RT-PCR法从人新鲜胎盘组织中获得TIMP-3 cDNA,连接至pMD18-T载体,EcoRⅠ、XbaⅠ双酶切pMD18-T-TIMP3重组质粒及pcDNA3.1载体并连接,构建pcDNA-TIMP-3真核载体,酶切鉴定、测序。将乳腺癌MDA-MB-453细胞分为正常对照组、pcDNA空载体组(只转染pcDNA空载体)和pcDNA-TIMP-3组(转染pcDNA-TIMP-3)。Western blotting法测定蛋白表达,采用MTT法和Boyden小室侵袭实验测定各组细胞的增殖活性及侵袭能力。结果: 重组载体经过EcoRⅠ 和XbaⅠ酶切后,产生633bp的TIMP-3目的片段和pcDNA3.1线性化载体,经自动测序仪测定TIMP-3序列完全正确。转染重组载体后MDA-MB-453细胞稳定表达了TIMP-3,与正常对照组和pcDNA空载体组比较,pcDNA-TIMP-3组细胞增殖活性明显降低(P<0.05),穿透以Ⅰ型胶原(Col Ⅰ)、层黏连蛋白(LN)、纤维连接蛋白(FN)和玻璃连接蛋白(VN)包被的Matrigel膜的侵袭细胞数明显减少(P<0.05)。结论: 成功构建pcDNA3.1-TIMP-3真核表达载体,可在MDA-MB-453细胞中高表达TIMP-3蛋白,并对MDA-MB-453细胞生长及侵袭性有抑制作用。

关键词: 组织基质金属蛋白酶抑制剂3, 真核表达载体, MDA-MB-453细胞系

Abstract:

Objective: To construct a mammalian tissue inhibitor of metalloproteinase-3 (TIMP-3)recombinant eukaryotic expression vecor and transfect the breast cancer MDA-MB-453 cells,and to explore the inhibitory effect of TIMP-3 gene on the growth and invasion of MDA-MB-453.Methods: The TIMP-3 cDNA was obtained from the human fresh placenta by RT-PCR. TIMP-3 gene was connected to pMD18-T vector.The recombinant pMD18-T vector and pcDNA3.1 were digested by EcoR Ⅰ and XbaⅠ. Then TIMP-3 gene was subcloned into pcDNA3.1 vetor to construct pcDNA-TIMP-3 recombinant vector and enzyme digestion identification and sequencing.The MDA-MB-453 cells were divided into normal control group,pcDNA group (transferted with pcDNA)and pcDNA-TIMP-3 group (transfected with pcDNA3.1-TIMP-3). The proliferation activities and invasion abilities of the MDA-453 cells in various groups were determined by MTT method and Boyden invasion experiment. Results: The correct construction of pcDNA-TIMP-3 was identified by means of restriction enzyme EcoRⅠ and XbaⅠ. The gene fragment of 633 bp and linearized vector were obtained.The result of TIMP-3 gene sequencing was correct.The Western blotting results showed that the transfected MDA-MB-453 cells expressed TIMP-3. The proliferation activity and invasion ability of MDA-MB-453 cells in pcDNA-TIMP-3 group were reduced significantly compared with normal control group and pcDNA group(P<0.05). Conclusion: The pcDNA-TIMP-3 eukaryotic expression vector is constructed successfully and TIMP-3 can be expressed in the MDA-MB-453 cells.The pcDNA-TIMP-3 has inhibitory effect on the growth and invasion of MDA-MB-453 cell lines.

Key words: tissue inhibitor of metalloproteinase-3, eukaryotic expression vector, MDA-MB-453 cell line

中图分类号:

R737.9

王丹, 王振宇, 石光, 尹光浩. TIMP-3基因真核表达载体的构建及其对乳腺癌MDA-MB-453细胞生长及侵袭的抑制作用[J]. 吉林大学学报(医学版), 2016, 42(01): 89-93.

WANG Dan, WANG Zhenyu, SHI Guang, YIN Guanghao. Construction of eukaryotic expression vector of TIMP-3 gene and its inhibitory effect on growth and invasion of MDA-MB-453 cells[J]. Journal of Jilin University Medicine Edition, 2016, 42(01): 89-93.

参考文献

[1] D'addazio G, Artese L,Piccirilli M,et al. Role of matrix metalloproteinases in radicular cysts and periapical granulomas[J].Minerva Stomatol,2014,63(11/12):411-420.

[2] Kim GE,Lee JS,Choi YD,et al.Expression of matrix metalloproteinases and their inhibitors in different immunohistochemical-based molecular subtypes of breast cancer[J].BMC Cancer,2014,14:959.

[3] Suarez-Roa ML,Asbun-Bojalil J,Ruiz-Godoy LM,et al.Immunoexpression of matrix metalloproteinases and their inhibitors in different areas of oral squamous cell carcinoma[J].Aust Dent J,2012,57(3):300-307.

[4] Kornfeld JW,Meder S,Wohlberg M,et al.Overexpression of TACE and TIMP3 mRNA in head and neck cancer:association with tumour development and progression[J].Br J Cancer, 2011,104(1):138-145.

[5] Liu Ww,Liu Y,Liang S,et al.Hypoxia and radiation-induced overexpression of Smac by an adenoviral vector and its effects on cell cycle and apoptosis in MDA-MB-231 human breast cancer cells[J].Exp Ther Med,2013,6(6):1560-1564.

[6] Li Y,Guo C,Wang Z,et al.Enhanced effects of TRAIL-endostatin-based double-gene-radiotherapy on suppressing growth,promoting apoptosis and inducing cell cycle arrest in vascular endothelial cells[J].J Huazhong Med Sci,2012,32(2):167-172.

[7] 尉承泽,江泽飞.乳腺癌治疗基本原则和临床选择[J].中国实用外科杂志,2009,29(9):774-776.

[8] 刘政红.乳腺癌早期筛查临床意义探讨[J].当代医学,2011,17(33):154-155.

[9] 范松青,魏启幼,李美容,等.基质金属蛋白酶及其抑制剂在乳腺癌中的表达及其临床意义[J].癌症,2003,22(9):968-973.

[10] Murphy G.Tissue inhibitors of metalloproteinases[J].Genome Biol,2011,12(11): 233-236.

[11] 陈培生,邓三花,王伟飞,等.重组质粒pcDNA3.1/SurP/Trail 联合吉西他滨诱导胰腺癌细胞凋亡的实验研究[J].胃肠病和肝病学杂志,2015,24(1):72-74.

[12] 陶国权.TIMP在结直肠癌中的作用及其调控机制研究[J].中国普通外科杂志,2013,12(5):1341-1345.

[13] Guan Z,Zhang J,Song S,et al.Promoter methylation and expression of TIMP3 gene in gastric cancer[J].Diagn Pathol,2013,8:110.

[14] Hassan ZK,Elamin MH,Daghestani MH,et al.Oleuropein induces anti-metastatic effects in breast cancer[J].Cancer Prev,2012,13(9):4555-4559.

[15] Babykutty S,Suboj P,Srinivas P,et al.Insidious role of nitric oxide in migration /invasion of colon cancer cells by upregulating MMP-2/9 via activation of cGMP-PKG-ERK signaling pathways[J].Clin Exp Metastasis,2012,29(5):471-492.

[16] Das AM,Seynhaeve AL,Rens JA,et al.Differential TIMP3 expression affects tumor progression and angiogenesis in melanomas through regulation of directionally persistent endothelial cell migration[J].Angiogenesis,2014,17(1):163-177.

[17] 张莹,李青艳,崔泽实,等.TSG101与HIF-1α在乳腺癌组织中的表达及意义[J].中国实用内科杂志,2014,34(增1):96-98.