中华肿瘤杂志
中華腫瘤雜誌
중화종류잡지
CHINESE JOURNAL OF ONCOLOGY
2010年
1期
22-28
,共7页
房新建%许文林%弓晋灵%陈琛%方莉莉%陈巧云
房新建%許文林%弓晉靈%陳琛%方莉莉%陳巧雲
방신건%허문림%궁진령%진침%방리리%진교운
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
乳腺腫瘤%CD44v17%基質金屬蛋白酶-2%基質金屬蛋白酶-9%腫瘤侵襲
유선종류%CD44v17%기질금속단백매-2%기질금속단백매-9%종류침습
Breast neoplasm%CD44 variant%Matrix metallopeptidase 2%Matrix metallopeptidase 9%Neoplasm invasiveness
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CD44v17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MMP-9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peDNA3.1-CD44v17后,CD44 mRNA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-2 mRNA表达量和蛋白活性分别为0.72±0.22和1.14.4-0.12,MMP-9 mRNA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-CDd4v17;HA与CD44v17受体结合,通过CD44→ras→MAPK信号传导通路调节MMP-2和MMP-9的表达,从而增加MCF-7细胞的侵袭力.
目的 探討一種新的CD44基因變異體(CD44v17)對人乳腺癌細胞株MCF-7侵襲能力的影響及其機製.方法 以人乳腺癌耐阿黴素細胞株(MCF-7/ADR)cDNA為模闆,採用聚閤酶鏈反應(PCR)擴增目的片段,將其T-A剋隆後,進行測序;構建CD44v17質粒真覈錶達載體(pcDNA3.1-CD44v17);應用脂質體轉染法將pcDNA3.1-CD44v17轉染入MCF-7細胞中,採用逆轉錄聚閤酶鏈反應(RT-PCR)及明膠酶譜法測定轉染細胞基質金屬蛋白酶(MMP)-2和MMP-9的錶達;Transwell法檢測CD44v17對MCF-7細胞的侵襲力;Western blot檢測胞外信號調節蛋白激酶(ERK)及燐痠化蛋白激酶(p-ERK)變化.結果 限製性內切酶消化證實,重組載體已正確剋隆;DNA序列分析顯示,CD44v17包含CD44基因1～4號外顯子、16～17號外顯子、18號外顯子1～205位堿基(GeneBank NO.FJ216964).MCF-7細胞轉染peDNA3.1-CD44v17後,CD44 mRNA錶達量和蛋白錶達率分彆為0.92±0.22和(70.0±2.5)%;透明質痠(HA)處理後,MMP-2 mRNA錶達量和蛋白活性分彆為0.72±0.22和1.14.4-0.12,MMP-9 mRNA錶達量和蛋白活性分彆為0.85±0.19和1.23±0.25,細胞侵襲能力明顯增加,侵襲細胞數目為352±33箇/視野,而CD44單抗可以阻斷這種作用;CD44單抗和促分裂原活化蛋白激酶(MAPK)通路抑製劑預處理轉染細胞後,顯著阻斷瞭P-ERK的錶達.結論 在MCF-7/ADR細胞中髮現CD44v17,併成功剋隆和建立pcDNA3.1-CDd4v17;HA與CD44v17受體結閤,通過CD44→ras→MAPK信號傳導通路調節MMP-2和MMP-9的錶達,從而增加MCF-7細胞的侵襲力.
목적 탐토일충신적CD44기인변이체(CD44v17)대인유선암세포주MCF-7침습능력적영향급기궤제.방법 이인유선암내아매소세포주(MCF-7/ADR)cDNA위모판,채용취합매련반응(PCR)확증목적편단,장기T-A극륭후,진행측서;구건CD44v17질립진핵표체재체(pcDNA3.1-CD44v17);응용지질체전염법장pcDNA3.1-CD44v17전염입MCF-7세포중,채용역전록취합매련반응(RT-PCR)급명효매보법측정전염세포기질금속단백매(MMP)-2화MMP-9적표체;Transwell법검측CD44v17대MCF-7세포적침습력;Western blot검측포외신호조절단백격매(ERK)급린산화단백격매(p-ERK)변화.결과 한제성내절매소화증실,중조재체이정학극륭;DNA서렬분석현시,CD44v17포함CD44기인1～4호외현자、16～17호외현자、18호외현자1～205위감기(GeneBank NO.FJ216964).MCF-7세포전염peDNA3.1-CD44v17후,CD44 mRNA표체량화단백표체솔분별위0.92±0.22화(70.0±2.5)%;투명질산(HA)처리후,MMP-2 mRNA표체량화단백활성분별위0.72±0.22화1.14.4-0.12,MMP-9 mRNA표체량화단백활성분별위0.85±0.19화1.23±0.25,세포침습능력명현증가,침습세포수목위352±33개/시야,이CD44단항가이조단저충작용;CD44단항화촉분렬원활화단백격매(MAPK)통로억제제예처리전염세포후,현저조단료P-ERK적표체.결론 재MCF-7/ADR세포중발현CD44v17,병성공극륭화건립pcDNA3.1-CDd4v17;HA여CD44v17수체결합,통과CD44→ras→MAPK신호전도통로조절MMP-2화MMP-9적표체,종이증가MCF-7세포적침습력.
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 CD44vl7 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 CD44vl7 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. l-CD44vl7 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 NCB1 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. l-CD44vl7.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/CD44vl7 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 cells. The expression vector pcDNA3. l-CD44vl7 has been cloned and constructed successfully. HA can be integrated with CD44 variant and then regulates the expression of MMP-2 and MMP-9, which increases the invasion ability of MCF-7 cells through the Ras /MAPK signaling pathway.
