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

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CathepsinL的表达水平；设计并合成靶向Cathepsin L的特异性shRNA，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shRNA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-shRNA转染SKOV3细胞后，SKOV3/shRNA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/shRNA组的细胞迁移能力明显受到抑制；SKOV3、SKOV3/Con和SKOV3/shRNA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/shRNA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。

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

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. shRNA 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 shRNA. 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 mRNA 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-shRNA could significantly down-regulate Cathepsin L expression in SKOV-3/shRNA. Cell migration ability was significantly inhibited in shRNA interference group at 12 and 24h time points. The migration cells of SKOV3/shRNA 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 shRNA 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.