CXCL10基因对乳腺癌细胞株MCF-7肿瘤相关基因表达的影响

The effects of transfection of CXCL10 gene into breast cancer cell line MCF-7 on expression of tumor related genes

目的构建CXCL10-pcDNA3．1（＋）重组质粒及稳定转染乳腺癌细胞系MCF-7细胞株，探讨CXCL10对MCF-7细胞株肿瘤相关基因表达的影响。方法从1干扰素（IFN-1）刺激的人外周血单个核细胞中克隆CXCL10基因，构建CXCL10-pcDNA3．1（＋）重组质粒，用CXCL10-pcDNA3．1（＋）和对照质粒pcDNA3．1（＋）分别以脂质体转染MCF-7细胞株，经G418筛选后获得稳定转染细胞株，以逆转录（RT）-PCR鉴定CXCL10基因表达，免疫印迹（WB）及ELISA鉴定CXCL10蛋白表达及分泌。以琼脂克隆形成实验分别计算实验组[转染CXCL10-pcDNA3．1（＋）的MCF-7细胞株]、对照组[pcDNA3．1（＋）的MCF-7细胞株]及未转染组（未转染的MCF-7细胞株）的细胞增殖活性；RT—PCR鉴定肿瘤相关基因基质金属蛋白酶9（MMP9）、血管内皮细胞生长因子（VEGF）及信号转导和转录激活因子3（STAT3）在各细胞株中的表达。结果重组质粒CXCL10-pcDNA3．1（＋）及质粒pcDNA3．1（＋）稳定转染MCF-7细胞株构建成功；克隆形成率在实验组、对照组及未转染组分别为78．2％、79．9％及87．8％，3组间比较差异无统计学意义（χ2=3．8，P〉0．05）；实验组细胞能高效表达CXCL10mRNA，3组间比较差异有统计学意义（F=50．5，P〈0．01）；ELISA结果显示细胞培养上清液中CXCL10的表达量3组间比较，差异有统计学意义（F=54．8，P〈0．01）；MMP9基因转录水平在实验组、对照组及未转染组分别为0．067±0．003、0．103±0．007及0．085±0．026，均很低，且组间差异无统计学意义（F=4．2，P〉0．05）；VEGF在实验组、对照组及未转染组灰度值分别为0．183±0．003、0．787±0．019及0．789±0．011，3组间比较差异有统计学意义（F=2232，P〈0．01）；STAT3在实验组、对照组及未转染组灰度值分别为0．573±0．016、0．707±0．008及0．711±0．013，3组间比较差异有统计学意义（F=115，P〈0．01）。结论CXCL10能在MCF-7细胞株稳定表达，且CXCL10能抑制MCF-7细胞VEGF及STAT3表达，从而发挥抗肿瘤作用。

Objective To construct stable transfected MCF-7 cell line by recombinant plasmid CXCL10-pcDNA3.1 （ ＋ ） and explore the effects of CXCL10 on expression of tumor related genes. Methods CXCL10 gene, cloned from treated PBMCs by IFN-5,,was used to construct recombinant plasmid CXCL10- peDNA3.1 （ ＋ ）. Recombinant plasmid CXCL10-pcDNA3.1 （ ＋ ） and control plasmid pcDNA3.1 （＋ ） were separately transfeeted into MCF-7 cells by lipifeetion-2000. The stable transfected cell lines were screened by G418. The transcription, expression and secretion of transfected CXCL10 gene was assayed by RT-PCR, Western blot and ELISA in turn. The cell growth activity of stable transfected cell lines was detected by soft agar cloning formation in experimental group [ transfected by CXCL10-pcDNA 3. 1 （ ＋ ） ] , control group [transfected by pcDNA3.1 （ ＋ ）] and blank group （nothing transfected）. Furthermore, the reverse transcriptions of three tumor related genes, included MMP9, VEGF and STAT3, were assayed by RT-PCR. Results The stable MCF-7 cell lines transfected by combined plasmid CXCL10-pcDNA3. 1 （ ＋ ） and plasmid pcDNA3.1 （ ＋ ） were constructed successfully. There was no significant difference in cell growth activity rate between the experimental group and the control group （χ2 = 3. 8, P 〉 0. 05, the rates of the 3 groups were 78.2% ,79.9% and 87.8% in turn）. There was significant difference in CXCL10 mRNA expression level between the experimental group and the control group （ F = 50. 5, P 〈 0. 01 ）. The ELISA result showed that CXCL10 had significant difference between the experimental group and the control group （ F = 54. 8,P 〈 0.01 ）. MMP9, showed stable low expression level in the 3 groups simutaneously （ F = 4. 2, P〉0.05, the gray values of the 3 groups were seperately 0.067 ±0.003, 0. 103 ±0.007 and 0.085 ± 0. 026）. Comparing with the control group, the experimental group showed significant lower expression level of VEGF （F=2 232,P 〈0. 01, the gray values of the 3 groups were 0. 183 ±0. 003, 0. 787 ±0. 019 and 0.789±0.011） and STAT3 （F=II5,P〈0.01, the gray values of the 3 groups were 0.573 ±0.016, 0. 707± 0. 008 and 0. 711 ± 0. 013 ）. Conclusions CXCL10 can express stablely in MCF-7 cell lines, which resulted in down-regulation of expression of VEGF and STAT3 gene. CXCL10 played an important role in anti-tumor effect.