针对uPA的siRNA对人乳腺癌细胞侵袭的抑制作用

Inhibition of Invasiveness of Human Breast Cancer Cells by siRNA Against Urokinase-type Plasminogen Activator

目的:通过RNA干涉的方法抑制乳腺癌细胞中uPA的表达,观察uPA的表达抑制后对肿瘤细胞的体外侵袭能力的影响。方法:(1)构建可以表达针对uPA的siRNA的干涉载体,转染高侵袭性人乳腺癌细胞系MDA-MB 231,G418抗性筛选,挑选单克隆株;(2)分别通过RT-PCR和Western Blot的方法检测uPA的表达;(3)平板克隆形成试验检测转染前后肿瘤细胞的克隆形成能力;4Bovden chamber Assay检测肿瘤细胞体外侵袭能力。结果:(1)可以稳定表达针对uPA的siRNA的单克隆株,uPA的表达水平显著下降;(2)转染了针对uPA的siRNA的单克隆株的克隆形成能力降低;(3)转染了针对uPA的siRNA的单克隆株体外侵袭能力与原代细胞MDA-MB 231相比明显受到抑制。结论:uPA在人乳腺癌侵袭行为中发挥重要的作用,针对uPA的siRNA可以显著降低uPA的表达,从而抑制肿瘤细胞的侵袭,可望成为抗肿瘤侵袭治疗的一种有效手段。

Objective:To inhibit the metastasis of breast cancer cell line MDA-MB 231 by means of silencing the expression of uPA. Methods: The vectors expressing siRNA against uPA were contructed and introduced into highly invasive breast cancer cell line MDA-MB 231 with lipofectin and positive single colonies were screened out with G418. RT-PCR and Western Blot were performed to detect the expression of uPA.Colony forming test was applied to detect the ability of cancer cell to form colony.The in vitro invasive ability of cancer cells was defined with Boyden Chamber Assay.Results:One of the colonies Al was found to express siRNA against uPA effectively because the expression of uPA in this colony was significantly decreased. The result from colony forming test indicated that the ability of Al to form colony is inhibited compared with that of untransfected MDA-MB 231 cell. The in vitro invasive ability of the clone A1 was significantly decreased. Conclusion: The siRNA against uPA can inhibit the expression of uPA and siginificantly decrease the invasive ability of at least a kind of cancer cell lines.RNAi is expected to become an effective method in anti-invasion therapy of cancers.