中华肿瘤杂志
中華腫瘤雜誌
중화종류잡지
CHINESE JOURNAL OF ONCOLOGY
2008年
5期
325-329
,共5页
张孟贤%韩娜%于世英%冷彦
張孟賢%韓娜%于世英%冷彥
장맹현%한나%우세영%랭언
CXCR4基因%短发卡状RNA%乳腺肿瘤%肿瘤转移
CXCR4基因%短髮卡狀RNA%乳腺腫瘤%腫瘤轉移
CXCR4기인%단발잡상RNA%유선종류%종류전이
CXCR4%shRNA%Breast neoplasms%Neoplasm metastasis
目的 利用RNA干扰技术下调趋化性细胞因子受体CXCR4基因的表达,探讨其沉默对乳腺癌细胞体外侵袭及肺转移潜能的影响.方法 设计合成CXCR4的特异性短发卡状RNA(shRNA),将其插入至pSilencer载体中,并将重组后的pSilencer质粒载体经脂质体包裹转染乳腺癌MDA-MB-231细胞株,抗性筛选稳定抑制CXCR4表达的永久细胞克隆.应用逆转录聚合酶链反应(RT-PCR)和Western blot法,检测shRNA对细胞内CXCR4基因表达的影响.利用Boyden小室模型检测细胞体外侵袭能力.二苯基溴化四氮唑蓝(MTT)法检测细胞的增殖状况.通过裸鼠尾静脉瘤细胞注射方法,构建肺转移模型,检测CXCR4基因沉默对乳腺癌细胞肺转移能力的影响.结果 成功构建和筛选出CXCR4特异性的shRNA质粒载体,稳定转染CXCR4-shRNA的乳腺癌细胞的CXCR4 mRNA和蛋白的表达较空白对照组明显下调(29.5%±3.8%比69.7%±2.6%,15.4%±1.1%比39.0%±2.4%;均P<0.01),体外侵袭能力减弱,增殖速度减慢,肺转移能力下降.结论 以CXCR4为靶向的shRNA能够有效下调CXCR4基因的表达,降低人乳腺癌细胞体外侵袭、增殖以及肺转移的能力.CXCR4是乳腺癌侵袭和转移过程中的重要调控因子.
目的 利用RNA榦擾技術下調趨化性細胞因子受體CXCR4基因的錶達,探討其沉默對乳腺癌細胞體外侵襲及肺轉移潛能的影響.方法 設計閤成CXCR4的特異性短髮卡狀RNA(shRNA),將其插入至pSilencer載體中,併將重組後的pSilencer質粒載體經脂質體包裹轉染乳腺癌MDA-MB-231細胞株,抗性篩選穩定抑製CXCR4錶達的永久細胞剋隆.應用逆轉錄聚閤酶鏈反應(RT-PCR)和Western blot法,檢測shRNA對細胞內CXCR4基因錶達的影響.利用Boyden小室模型檢測細胞體外侵襲能力.二苯基溴化四氮唑藍(MTT)法檢測細胞的增殖狀況.通過裸鼠尾靜脈瘤細胞註射方法,構建肺轉移模型,檢測CXCR4基因沉默對乳腺癌細胞肺轉移能力的影響.結果 成功構建和篩選齣CXCR4特異性的shRNA質粒載體,穩定轉染CXCR4-shRNA的乳腺癌細胞的CXCR4 mRNA和蛋白的錶達較空白對照組明顯下調(29.5%±3.8%比69.7%±2.6%,15.4%±1.1%比39.0%±2.4%;均P<0.01),體外侵襲能力減弱,增殖速度減慢,肺轉移能力下降.結論 以CXCR4為靶嚮的shRNA能夠有效下調CXCR4基因的錶達,降低人乳腺癌細胞體外侵襲、增殖以及肺轉移的能力.CXCR4是乳腺癌侵襲和轉移過程中的重要調控因子.
목적 이용RNA간우기술하조추화성세포인자수체CXCR4기인적표체,탐토기침묵대유선암세포체외침습급폐전이잠능적영향.방법 설계합성CXCR4적특이성단발잡상RNA(shRNA),장기삽입지pSilencer재체중,병장중조후적pSilencer질립재체경지질체포과전염유선암MDA-MB-231세포주,항성사선은정억제CXCR4표체적영구세포극륭.응용역전록취합매련반응(RT-PCR)화Western blot법,검측shRNA대세포내CXCR4기인표체적영향.이용Boyden소실모형검측세포체외침습능력.이분기추화사담서람(MTT)법검측세포적증식상황.통과라서미정맥류세포주사방법,구건폐전이모형,검측CXCR4기인침묵대유선암세포폐전이능력적영향.결과 성공구건화사선출CXCR4특이성적shRNA질립재체,은정전염CXCR4-shRNA적유선암세포적CXCR4 mRNA화단백적표체교공백대조조명현하조(29.5%±3.8%비69.7%±2.6%,15.4%±1.1%비39.0%±2.4%;균P<0.01),체외침습능력감약,증식속도감만,폐전이능력하강.결론 이CXCR4위파향적shRNA능구유효하조CXCR4기인적표체,강저인유선암세포체외침습、증식이급폐전이적능력.CXCR4시유선암침습화전이과정중적중요조공인자.
Objective To construct a CXCR4 specific recombinant plasmid vector and study its inhibiting effect on invasion capacity in vitro of human breast cancer MDA-MB-231 cell line and its metastatic potential to the lung in nude mice. Methods A CXCR4 specific recombinant plasmid vector was constructed and transfected into the cultured MDA-MB-231 cell line with lipofectamine 2000. RT-PCR and Western blot were used to detect the mRNA and protein expression of CXCR4, respectively. Invasion capability in vitro of the cells was evaluated by Boyden chamber. The cell proliferation capacity was detected by MTT method. The nude mouse model of lung metastasis was established by injection of MDA-MB-231 cells into the tail vein. The animals were sacrificed at 6 weeks after the tumor cells injection. Whole lung tissues were harvested, embedded in paraffin, sectioned serially, and the HE-stained paraffin sections were examined pathologically to evaluate the presence and number of metastatic tumors. Results The CXCR4 mRNA expression rate was 29.5%±3.8% in the CXCR4-shRNA group, significantly lower than that of the control group (69.7%±2.6%, P<0.01) and mock-control group (67.8%±3.5%, P<0.01). The CXCR4 protein expression rate was 15.4%±1.1% in the CXCR4-shRNA group, significantly lower than that of the control group (39.0%±2.4%, P<0.01) and mock-control group (35.9%±3.9%, P<0.01). Silencing of CXCR4 by shRNA lead to a significant decrease in breast cancer cell invasion and proliferation capacity in vitro. Furthermore, tumor cells with CXCR4 shRNA permanent transfcetion had a much lower lung metastatic potential in nude mice than control cells and mock control cells in vivo. Conclusion CXCR4 shRNA can inhibit the expression of CXCR4 and decrease the invasion and lung metastatic potential of human breast cancer cells.
