小RNA干扰降低COX-2表达对乳腺癌细胞趋化和侵袭能力的影响

Influences of Disruption of Cyclooxygenase-2 by Using RNA Interference on Invasiveness and Chemotaxis of Breast Cancer Cells

目的探讨利用小RNA干扰技术降低COX-2表达对高度恶性乳腺癌MDA-MB-231细胞趋化和侵袭能力的影响。方法应用合成的小RNA干扰质粒转染MDA-MB-231细胞株,采用反转录-聚合酶链反应(RT-PCR)检测COX-2mRNA的表达。通过趋化运动实验检测细胞的运动能力;体外侵袭实验检测细胞的侵袭能力。结果限制性内切酶的酶切结果和DNA测序结果显示成功构建了干扰质粒pSUPER-siCOX-2,转染后的细胞株分别命名为MDA-MB-231/pSUPER-basic(对照组)和MDA-MB-231/pSUPER-siCOX-2(实验组)。转染后48h,与MDA-MB-231/pSUPER-basic细胞相比,MDA-MB-231/pSUPER-siCOX-2细胞的COX-2mRNA表达水平下降(P<0.01);COX-2减低的乳腺癌细胞的趋化运动能力比对照组细胞降低(P<0.01);COX-2减低的乳腺癌细胞侵袭并穿透Matrivgel膜基质的细胞数量比对照组细胞少(P<0.01)。结论利用siRNA干扰技术降低COX-2表达对乳腺癌MDA-MB-231细胞株的趋化和侵袭能力具有明显的抑制作用。

Objective To investigate the role of COX-2 in invasiveness and chemotaxis of the highly malignant breast cancer cell line MDA-MB-231 using RNA interference.Methods MDA-MB-231 cells were transfected with small RNA interference plasmids to disrupt COX-2 expression.COX-2 mRNA level was detected using RT-PCR.The in vitro invasion ability of MDA-MB-231 cells was examined using matrigel invasion assay.Results The results of restriction endonuclease digestion electrophoresis and DNA sequencing showed that the interferenced plasmid pSUPER-siCOX-2 was constructed successfully.The transfectants were named as MDA-MB-231/pSUPER-basic and MDA-MB-231/ pSUPER-siCOX-2,respectively.The results of RT-PCR showed that the levels of COX-2 mRNA of MDA-MB-231/pSUPER-siCOX-2 was obviously reduced at 48 hours after transfection,compared to MDA-MB-231/pSUPER-basic cells.The COX-2-reduced MDA-MB-231 cells showed decreased chemotaxis ability compared with the control cells（P0.01）.The invasion assay showed prominent differences between the COX-2-reduced MDA-MB-231 cells and the control cells（P 0.01）.Conclusion Using RNA interference,the reduction of COX-2 expression can obviously inhibit the invasion and chemotaxis of the highly malignant breast cancer cell line MDA-MB-231.