| 李胜男,陈必良,朱媛媛,武 雁,赵丽莎.LMTK2基因沉默抑制人上皮性卵巢癌细胞生长和转移的作用机制[J].,2022,(11):2034-2041 |
| LMTK2基因沉默抑制人上皮性卵巢癌细胞生长和转移的作用机制 |
| The Mechanism of Silencing LMTK2 Gene on the Growth and Metastasis of Ovarian Cancer Cells |
| 投稿时间：2021-12-05 修订日期：2021-12-28 |
| DOI：10.13241/j.cnki.pmb.2022.11.006 |
| 中文关键词: 上皮性卵巢癌（EOC） 狐猴酪氨酸激酶2（LMTK2） 细胞增殖 转移 PI3K/Akt信号通路 |
| 英文关键词: Ovarian cancer Lemur tyrosine kinase 2 Cell proliferation Metastasis PI3K/Akt signaling pathway |
| 基金项目:国家自然科学基金面上项目（81672583） |
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| 中文摘要: |
| 摘要 目的：探讨狐猴酪氨酸激酶2（LMTK2）基因沉默对人上皮性卵巢癌(EOC)细胞生长和转移的抑制作用及其可能的机制。方法：通过RT-qPCR和Western-blot检测了人正常卵巢上皮细胞IOSE80和人上皮性卵巢癌细胞系（SKOV3、ES2、OVCAR-3和HEY）中LMTK2的表达，使用Lipofectamine 3000转染试剂将LMTK2的短发夹RNA（shRNA）、阴性对照shRNA、LMTK2过表达重组pcDNA3.1质粒或阴性对照质粒转染到SKOV3细胞中，并分为LMTK2-shRNA组、NC-shRNA组、LMTK2-pcDNA3.1组或NC-pcDNA3.1组。另外，使用PI3K/Akt抑制剂LY294002处理SKOV3细胞1 h。通过CCK-8法测定细胞增殖，Annexin V-FITC/PI染色法测定细胞凋亡，划痕实验评价细胞迁移，Transwell实验评价细胞侵袭。对BALB/c雌性裸鼠皮下注射转染NC-shRNA或LMTK2-shRNA的SKOV3细胞建立体内移植瘤模型，并记录接种28 d内的肿瘤体积。结果：与人正常卵巢上皮细胞IOSE80相比，卵巢癌细胞系（SKOV3、ES2、OVCAR-3和HEY）中LMTK2的mRNA和蛋白表达水平均显著升高，其中SKOV3的LMTK2 mRNA和蛋白表达水平最高（P<0.05）。与NC-shRNA组相比，LMTK2-shRNA组SKOV3细胞活力、相对迁移面积、侵袭细胞数均显著降低，而细胞凋亡率显著升高（P<0.05）。此外，与NC-shRNA组相比，LMTK2-shRNA组SKOV3细胞中Bax的蛋白表达水平显著升高，而Bcl-2、MMP2、MMP9、p-Akt的蛋白表达水平显著降低（P<0.05）。LY294002处理逆转了上调LMTK2对SKOV3细胞生长和转移的影响（P<0.05）。在接种第21天和28天时，与NC-shRNA组相比，LMTK2-shRNA组裸鼠的肿瘤体积显著降低（P<0.05）。结论：LMTK2基因沉默通过抑制PI3K/Akt信号通路降低了人上皮性卵巢癌细胞的生长和转移能力。 |
| 英文摘要: |
| ABSTRACT Objective: The aim of this study is to reveal the role of lemur tyrosine kinase 2 (LMTK2) in the growth and metastasis of ovarian cancer cells. Methods: The expression of LMTK2 in human normal ovarian epithelial cells IOSE80 and ovarian cancer cell lines (SKOV3, ES2, OVCAR-3 and HEY) were detected by RT-qPCR and Western blot. Short hairpin RNA (shRNA), negative control shRNA, LMTK2 overexpression recombinant pcDNA3.1 plasmid or negative control plasmid were transfected into SKOV3 cells by Lipofectamine 3000 transfection reagent, and were divided into LMTK2 shRNA group, NC- shRNA group, LMTK2-pcDNA3.1 group or NC-pcDNA3.1group. In addition, SKOV3 cells were treated with PI3K/Akt inhibitor LY294002 for 1 h. Cell proliferation was measured by CCK-8 method, cell apoptosis was measured by Annexin V-FITC/PI staining method, cell migration was evaluated by wound healing experiment, and cell invasion was evaluated by Transwell experiment. SKOV3 cells transfected with NC-shRNA or LMTK2-shRNA were subcutaneously injected into female BALB/c nude mice to establish an in vivo transplantation tumor model, and the tumor volume within 28 days of inoculation was recorded. Results: Compared with human normal ovarian epithelial cells IOSE80, the mRNA and protein expression levels of LMTK2 in ovarian cancer cell lines (SKOV3, ES2, OVCAR-3 and HEY) were significantly increased, of which the LMTK2 mRNA and protein expression levels in SKOV3 were the highest(P<0.05). Compared with NC-shRNA group, the SKOV3 cell viability, relative migration area and number of invaded cells in LMTK2-shRNA group were significantly reduced, while the apoptosis rate was significantly increased(P<0.05). In addition, compared with NC-shRNA group, the protein expression level of Bax in SKOV3 cells in LMTK2-shRNA group was significantly increased, while the protein expression levels of Bcl-2, MMP2, MMP9, and p-Akt were significantly reduced(P<0.05). LY294002 treatment reversed the effect of up-regulation of LMTK2 on the growth and metastasis of SKOV3 cells(P<0.05). On the 21st and 28th day of inoculation, compared with the NC-shRNA group, the tumor volume of nude mice in the LMTK2-shRNA group was significantly reduced(P<0.05). Conclusion: Silencing LMTK2 reduces the growth and metastasis ability of ovarian cancer cells by inhibiting the PI3K/Akt signaling pathway. |
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