溶酶体组织蛋白酶L对人卵巢癌SKOV3细胞迁移及侵袭的作用

Cathepsin L Promotes Invasion and Migration of Human Ovarian Cancer Cell Line SKOV3

目的探讨溶酶体组织蛋白酶L(Cathepsin L)是否通过上皮-间充质转化(EMT)影响卵巢癌细胞的侵袭及迁移能力。方法荧光定量PCR法检测人卵巢癌细胞ES-2、SKOV3、OV1以及OV2中Cathepsin L的表达水平;设计并合成靶向Cathepsin L的特异性sh RNA,通过脂质体转染法转染Cathepsin L表达最高的卵巢癌细胞SKOV3以构建稳定低表达Cathepsin L细胞株,Western blot和定量PCR法验证sh RNA的干扰效率;划痕实验及Transwell法检测干扰后细胞的迁移及侵袭能力,Western blot法检测EMT相关指标E-cadherin和N-cadherin以及其上游信号分子Snail、p-AKT的变化。结果四株卵巢癌细胞中,SKOV3的Cathepsin L表达水平最高(以此为参照),而ES-2、OV1以及OV2细胞的表达水平分别为0.72±0.04、0.34±0.03和0.55±0.05。Cathepsin L-sh RNA转染SKOV3细胞后,SKOV3/sh RNA中的Cathepsin L的表达水平较空白组和对照组显著下降;划痕12 h和24 h后,SKOV3/sh RNA组的细胞迁移能力明显受到抑制;SKOV3、SKOV3/Con和SKOV3/sh RNA组的穿膜细胞数分别为(93.67±8.62)、(90.33±12.22)、(35.67±4.73),与对照组相比,SKOV3/sh RNA组细胞侵袭能力受到显著抑制(P<0.01);Cathepsin L干扰组细胞的E-cadherin表达增加,N-cadherin的表达降低;此外,Cathepsin L干扰组细胞的Snail、p-AKT表达较对照组显著下降。结论 Cathepsin L可以促进卵巢癌细胞的迁移和侵袭;其机制可能与调节EMT的上游信号分子Snail、p-AKT有关。提示Cathepsin L在卵巢癌的发生发展中起重要作用。

Objective To investigate whether Cathepsin L affects the invasion and migration of ovarian cancer cells SKOV-3 via regulating epithelial mesenchymal transition(EMT).Methods The expression of Cathepsin L in ES-2,SKOV3,OV1 and OV2 cell lines were analyzed by Real-time PCR.sh RNA targeting Cathepsin L was designed and synthesized,and then transfected into SKOV-3 cells which expressed the highest level Cathepsin L in four cell lines via Lipofectamine 2000 mediation.Western blot and Real-time PCR were used to detect the expression of Cathepsin L in SKOV-3 cells which were transfected with sh RNA.The migration ability of SKOV-3 was analyzed by Wound scratch assay.The invasion ability was detected by Transwell assay.The expression of E-cadherin,N-cadherin,p-AKT and Snail were detected by Western blot.Results The expression of Cathepsin L m RNA and protein were the highest in SKOV3 cells,while those in ES-2,OV1 and OV2 were(0.72±0.04),(0.34±0.03) and(0.55±0.05).Capthesin L-sh RNA could significantly down-regulate Cathepsin L expression in SKOV-3/sh RNA.Cell migration ability was significantly inhibited in sh RNA interference group at 12 and 24 h time points.The migration cells of SKOV3/sh RNA was(35.67±4.73),while those of the SKOV3 and SKOV3/Con groups were(93.67±8.62) and(90.33±12.22)(P<0.001).In addition,the expression of E-cadherin was increased,while those of N-cadherin,p-AKT and Snail were decreased in sh RNA interference group.Conclusion Cathepsin L gene could promote the invasion and migration of ovarian cancer cells SKOV-3 via regulating upstream signaling pathway of EMT,suggesting that Cathepsin L plays an important role in the development of ovarian cancer.