Liu M B, Shi X F, Liu K, Song W Y, Wang L, Wu H Y, Wu G Y
Department of Cancer Center, Henan Provincial People's Hospital, Zhengzhou 450003, China.
Department of Obstetrics, Henan Provincial People's Hospital, Zhengzhou 450003, China.
Zhonghua Zhong Liu Za Zhi. 2021 Mar 23;43(3):299-305. doi: 10.3760/cma.j.cn112152-20200408-00322.
To explore the role and molecular mechanism of trophoblastic cell surface antigen 2 (Trop2) in the invasion and migration of ovarian cancer. Through the data mining of Cancer Cell Line Encyclopedia and TCGA database, the clinical significance of Trop2 expression was analyzed. Western blot was used to detect Trop2 protein expression in ovarian cancer cell lines including A3O, A1780 and SKOV3. SKOV3 cells were used to construct Trop2-short hairpin RNA (shRNA) cell model. Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) was used to detect the SKOV3 mRNA expression in SKOV3-shRNA and SKOV3-NC cells. Cell counting kit-8 (CCK8) was used to detect the proliferation of SKOV3-shRNA cells and SKOV3-NC cells. Flow cytometry was used to detect cell cycle and apoptosis in two groups of cells. Transwell array was used to detecte the invasion and migration of SKOV3-shRNA cells and SKOV3-NC cells. Western blot was used to detect the protein expressions of AKT, p-AKT, β-catenin, caspase3, bcl-2, E-cadherin and vimentin. Trop2 mRNA highly expressed in ovarian cancer, and was related to the tumor stage and patient survival. Compared with A3O cells, Trop2 overexpressed in A1780 and SKOV3 cells (<0.05). The relative expression levels of Trop2 mRNA in SKOV3-NC group and SKOV3-shRNA group were 1.18±0.24 and 0.42±0.08, with statistically significant difference (<0.05). The results of CCK-8 array showed that the cell viability of SKOV3-NC group was significantly higher than that of SKOV3-shRNA group (<0.05). The proportion of G(0)/G(1) cells in SKOV3-NC and SKOV3-shRNA groups were (38.67±4.22)% and (60.24±8.17)%, respectively. G(0)/G(1) arrest was observed in SKOV3-shRNA cells (<0.05). The apoptosis rate of SKOV3-shRNA group was (26.32±1.81)%, significantly higher than (6.54±1.32)% of SKOV3-NC group (<0.05). The number of migrating SKOV3 cells in the SKOV3-shRNA and SkOV3-NC groups were 1 255.83±108.44 and 1 679.71±213.92, while the number of invading cells were 242.49±52.09 and 473.54±73.11, respectively. Compared with the SKOV3-NC group, the number of migrating and invading SKOV3-shRNA group was significantly reduced (all <0.05). The expressions of p-AKT2, Bcl-2, vimentin and β-catenin were down-regulated, and the expressions of caspase 3 and E-cadherin were up-regulated in SKOV3-shRNA cells. There was no significant change in the total protein level of AKT. Trop2 expression is related to ovarian cancer stage and postoperative survival. Trop2 can promote ovarian cancer cell proliferation and metastasis by activating the AKT/β-catenin signaling pathway and knockdown of Trop2 inhibits the progression of ovarian cancer.
探讨滋养层细胞表面抗原2（Trop2）在卵巢癌侵袭和迁移中的作用及分子机制。通过对癌症细胞系百科全书和TCGA数据库的数据挖掘，分析Trop2表达的临床意义。采用蛋白质免疫印迹法检测包括A3O、A1780和SKOV3在内的卵巢癌细胞系中Trop2蛋白的表达。利用SKOV3细胞构建Trop2短发夹RNA（shRNA）细胞模型。采用定量逆转录-聚合酶链反应（qRT-PCR）检测SKOV3-shRNA和SKOV3-NC细胞中SKOV3的mRNA表达。使用细胞计数试剂盒-8（CCK8）检测SKOV3-shRNA细胞和SKOV3-NC细胞的增殖情况。采用流式细胞术检测两组细胞的细胞周期和凋亡情况。使用Transwell小室检测SKOV3-shRNA细胞和SKOV3-NC细胞的侵袭和迁移能力。采用蛋白质免疫印迹法检测AKT、p-AKT、β-连环蛋白、半胱天冬酶3、bcl-2、E-钙黏蛋白和波形蛋白的蛋白表达。Trop2 mRNA在卵巢癌中高表达，且与肿瘤分期及患者生存相关。与A3O细胞相比，Trop2在A1780和SKOV3细胞中过表达（<0.05）。SKOV3-NC组和SKOV3-shRNA组中Trop2 mRNA的相对表达水平分别为1.18±0.24和0.42±0.08，差异具有统计学意义（<0.05）。CCK-8检测结果显示，SKOV3-NC组细胞活力显著高于SKOV3-shRNA组（<0.05）。SKOV3-NC组和SKOV3-shRNA组中G(0)/G(1)期细胞比例分别为(38.67±4.22)%和(60.24±8.17)%。SKOV3-shRNA细胞出现G(0)/G(1)期阻滞（<0.05）。SKOV3-shRNA组的凋亡率为(26.32±1.81)%，显著高于SKOV3-NC组的(6.54±1.32)%（<0.05）。SKOV3-shRNA组和SKOV3-NC组迁移的SKOV3细胞数分别为1 255.83±108.44和1 679.71±213.92，侵袭细胞数分别为242.49±52.09和473.54±73.11。与SKOV3-NC组相比，SKOV3-shRNA组迁移和侵袭的细胞数均显著减少（均<0.05）。SKOV3-shRNA细胞中p-AKT2、Bcl-2、波形蛋白和β-连环蛋白的表达下调，半胱天冬酶3和E-钙黏蛋白的表达上调。AKT的总蛋白水平无显著变化。Trop2表达与卵巢癌分期及术后生存相关。Trop通过激活AKT/β-连环蛋白信号通路促进卵巢癌细胞增殖和转移，敲低Trop2可抑制卵巢癌进展。
Zotero 插件