CD44基因变异体对人乳腺癌细胞株MCF-7的侵袭作用  

CD44 variant increases the invasive ability of human breast cancer cell line MCF-7 cells

作　　者：房新建[1,2] 许文林[1] 弓晋灵[1] 陈琛[1] 方莉莉[1] 陈巧云[1] 

机构地区：[1]江苏大学附属人民医院中心实验室,镇江市212002 [2]蚌埠医学院附属连云港市第二人民医院肿瘤内科,222002

出　　处：《中华肿瘤杂志》2010年第1期22-28,共7页Chinese Journal of Oncology

基　　金：江苏省卫生重大课题基金资助项目（K2005017）

摘　　要：目的探讨一种新的CD44基因变异体（CD44v17）对人乳腺癌细胞株MCF-7侵袭能力的影响及其机制。方法以人乳腺癌耐阿霉素细胞株（MCF-7／ADR）cDNA为模板，采用聚合酶链反应（PCR）扩增目的片段，将其T．A克隆后，进行测序；构建CIM4v17质粒真核表达载体（pcDNA3．1-CD44v17）；应用脂质体转染法将pcDNA3．1-CD44v17转染入MCF-7细胞中，采用逆转录聚合酶链反应（RT—PCR）及明胶酶谱法测定转染细胞基质金属蛋白酶（MMP）-2和MMPO的表达；Transwell法检测CD44v17对MCF-7细胞的侵袭力；Western blot检测胞外信号调节蛋白激酶（ERK）及磷酸化蛋白激酶（p-ERK）变化。结果限制性内切酶消化证实，重组载体已正确克隆；DNA序列分析显示，CD44v17包含CD44基因1～4号外显子、16～17号外显子、18号外显子1～205位碱基（GeneBank NO．FJ216964）。MCF-7细胞转染pcDNA3．1-CD44v17后，CD44mRNA表达量和蛋白表达率分别为0．92±0．22和（70．0±2．5）％；透明质酸（HA）处理后，MMP-2mRNA表达量和蛋白活性分别为0．72±0．22和1．14±0．12，MMPOmRNA表达量和蛋白活性分别为0．85±0．19和1．23±0．25，细胞侵袭能力明显增加，侵袭细胞数目为352±33个／视野，而CD44单抗可以阻断这种作用；CD44单抗和促分裂原活化蛋白激酶（MAPK）通路抑制剂预处理转染细胞后，显著阻断了p-ERK的表达。结论在MCF-7／ADR细胞中发现CD44v17，并成功克隆和建立pcDNA3．1-CD44v17；HA与CD44v17受体结合，通过CD44→ras→MAPK信号传导通路调节MMP-2和MMPO的表达，从而增加MCF-7细胞的侵袭力。Objective To evaluate the impact of a new CD44 variant on invasion of human breast cancer cell line MCF-7, and its possible mechanisms. Methods The full length cDNA encoding CD44v17 was obtained from the total RNA isolated from the MCF-7/ADR cells by reverse transcript-polymerase chain reaction （RT-PCR） and subcloned into pMD19-T vector. The CDd4vl7 gene sequence and reading frame were confirmed by two restriction enzymes and nucleotide sequencing, and then inserted into the eukaryotic expression vector pcDNA3.1. The pcDNA3.1-CD44v17 was transfected into MCF-7 cells by Lipofectamine. The changes of MMP-2 and MMP-9 expression at gene and protein levels were detected by RT-PCR and gelatin zymography, respectively. The number of the cells through the artificial matrix membrane in every group was counted to compare the change of the invasive ability regulated by CD44 variant. The ERK and p-ERK were investigated by Western blotting to approach the molecular mechanisms of MMP-2 and MMP-9 expression regulated by CD44 variant. Results The new gene sequence was successfully cloned into recombinant vector pcDNA3. 1 and identified by the two restriction enzymes. It was confirmed that the reconstructed plasmid contained the sequence of CD44 gene variant which was composed of 1 to 4 exons, 16 to 17 exons, and 1 to 205 bases of 18 exons. The new gene sequence was sent to NCBI for publication and obtained the registered number FJ216964. The up-regulated levels of the CD44 gene mRNA and protein were respectively detected by RT-PCR and flow cytometry in MCF-7 cells transfected with pcDNA3.1-CD44v17.The invasiveness of the cells and the activity of MMP-2 and MMP-9 were clearly activated by hyaluronic acid （HA） treatment and blocked by CD44 neutralizing antibody. Pretreated MCF-7/CD44v17 cells with the neutralizing antibody against CD44 and the inhibitor of MAPKs signaling pathway strongly block the expression of p-ERK. Conclusion A new CD44 gene variant has been found in adriamycin-resistant human breast cancer MCF-7/ADR c

关 键 词：乳腺肿瘤 CD44v17 基质金属蛋白酶-2 基质金属蛋白酶-9 肿瘤侵袭 

分 类 号：R737.9[医药卫生—肿瘤]