摘要
目的通过构建CXCR7短发夹状RNA(short hairpin RNA,shRNA)序列的真核表达载体,探讨趋化因子受体CXCR7对人乳腺癌细胞MDA-MB-435s增殖、凋亡、周期的影响。方法针对CXCR7构建编码shRNA序列的重组表达载体;分别用RT-PCR和Westernblot检测CXCR7mRNA及蛋白表达水平;MTT法检测细胞增殖;TUNEL法检测细胞凋亡;流式细胞术检测细胞周期。结果构建针对CXCR7短发夹状RNA序列的表达载体,经酶切及测序证实与设计完全一致;将2个CXCR7shRNA载体(CXCR7-shRNA1;CXCR7-shRNA2)转染MDA-MB-435s细胞后,CXCR7mRNA及蛋白平均降低;抑制CXCR7的表达后,CXCR7-shRNA组细胞增殖率明显下降(P<0.05),细胞凋亡增多(P<0.05),细胞周期无明显改变。结论重组表达载体CXCR7-shRNA1、CXCR7-shRNA2能有效抑制靶基因CXCR7的表达,进而可抑制人乳腺癌MDA-MB-435s细胞的增殖,促进细胞凋亡,而对细胞周期无明显影响。
Objective To construct an expressing vector encoding short hairpin RNA （shRNA） targeting chemokine receptor 7 （CXCR7） and explore its effect on the proliferation, apoptosis, and cycle of the human breast cancer cell line MDA-MB-435s. Methods Expressing vector of CXCR7 shRNA was constructed and transfected into MDA-MB-435s cells. Expression levels of CXCR7 mRNA and CXCR7 protein were assayed by RT-PCR and Western blot analysis respectively. Cell proliferation was measured by MTT method, cell apoptosis by TUNEL assay, and cell cycle by flow cytometry. Results Recombinant expressing vector CXCR7 shRNAs was successfully constructed. Enzyme digesting and sequencing showed that the insertion sequence was correct. After transfection of 2 CXCR7 shRNA vectors （CXCR7-shRNA1 and CXCR7-shRNA2）, the expression levels of CXCR7 protein and CXCR7mRNA in the MDA-MB-435s cells were significantly decreased compared with those of control shRNA and untreated cells （P0.05）. The proliferation of cancer cells was decreased significantly （P0.05） while the apoptosis was increased significantly after CXCR7-shRNA1 and CXCR7-shRNA2 transfection （P0.05）. However, the cell cycle had no significant difference compared with those of control shRNA and untreated cells. Conclusion Recombinant expressing vector CXCR7 shRNAs effectively inhibits the expression of CXCR7, and suppresses the proliferation of MDA-MB-435s cells to promote cell apoptosis, but has no obvious influence on the cell cycle after the inhibition of the CXCR7.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2009年第20期2005-2008,共4页
Journal of Third Military Medical University
