Department of Urology, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai 200233, P.R. China.
Int J Oncol. 2019 May;54(5):1676-1690. doi: 10.3892/ijo.2019.4726. Epub 2019 Feb 27.
The aim of the present study was to investigate the effects of lysyl oxidase‑like 2 (LOXL2) on the invasion, migration and epithelial‑to‑mesenchymal transition (EMT) of renal cell carcinoma (RCC) cells through the steroid receptor coactivator (Src)/focal adhesion kinase (FAK) signaling pathway. RCC tissues and adjacent normal tissues were collected from 80 patients with RCC. Immunohistochemistry was used to determine the positive expression rate of the LOXL2 protein. The expression levels of LOXL2 in the HK‑2, 786‑O, ACHN, Caki1 and A498 cell lines were detected by reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR). The high LOXL2‑expressing 786‑O cells were selected for gene silencing experiments, whereas Caki1 cells, which exhibited low LOXL2 expression, were used for overexpression experiments. RT‑qPCR and western blot analysis were applied to determine the expression of LOXL2, FAK, Src, matrix metalloproteinase (MMP)‑9, epithelial (E)‑cadherin, neuronal (N)‑cadherin and vimentin. A MTT assay, a Transwell assay, a wound healing assay and flow cytometry were performed to detect cell proliferation, invasion, migration, cell cycle distribution and apoptosis, respectively. The protein expression rate of LOXL2 in RCC tissues was higher compared with that in adjacent normal tissues. Compared with adjacent normal tissues, the mRNA and protein expression levels of LOXL2, FAK, Src, MMP‑9, N‑cadherin and vimentin and the levels of FAK and Src phosphorylation were increased, while the mRNA and protein expression levels of E‑cadherin were decreased in RCC tissues. Following the transfection of 786‑O cells with small interfering (si) RNA against LOXL2, the mRNA and protein expression levels of FAK, Src, MMP‑9, N‑cadherin and vimentin and the levels of phosphorylated FAK and Src were notably decreased in the si‑LOXL2 and PP2 inhibitor treated groups, while that of E‑cadherin was substantially increased. Additionally, cell proliferation, invasion, migration and the percentage of RCC cells in the G1 phase were reduced, and cell apoptosis was increased. Additionally, Caki1 cells transfected with LOXL2 exhibited an opposite trend. In summary, these results indicate that LOXL2 silencing inhibits the invasion, migration and EMT in RCC cells through inhibition of the Src/FAK signaling pathway.
本研究旨在通过类固醇受体共激活剂（Src）/黏着斑激酶（FAK）信号通路探讨赖氨酰氧化酶样 2（LOXL2）对肾细胞癌（RCC）细胞侵袭、迁移和上皮间质转化（EMT）的影响。收集 80 例 RCC 患者的 RCC 组织和相邻正常组织，采用免疫组织化学法检测 LOXL2 蛋白的阳性表达率。采用逆转录定量聚合酶链反应（RT-qPCR）检测 LOXL2 在 HK-2、786-O、ACHN、Caki1 和 A498 细胞系中的表达水平。选择 LOXL2 高表达的 786-O 细胞进行基因沉默实验，而选择 LOXL2 低表达的 Caki1 细胞进行过表达实验。采用 RT-qPCR 和 Western blot 分析检测 LOXL2、FAK、Src、基质金属蛋白酶（MMP）-9、上皮（E）-钙黏蛋白、神经元（N）-钙黏蛋白和波形蛋白的表达。采用 MTT 检测、Transwell 检测、划痕愈合检测和流式细胞术分别检测细胞增殖、侵袭、迁移、细胞周期分布和凋亡。与相邻正常组织相比，RCC 组织中 LOXL2 蛋白的表达率较高。与相邻正常组织相比，RCC 组织中 LOXL2、FAK、Src、MMP-9、N-钙黏蛋白和波形蛋白的 mRNA 和蛋白表达水平升高，E-钙黏蛋白的 mRNA 和蛋白表达水平降低，FAK 和 Src 磷酸化水平升高。转染 786-O 细胞的 LOXL2 小干扰（si）RNA 后，si-LOXL2 和 PP2 抑制剂处理组中 FAK、Src、MMP-9、N-钙黏蛋白和波形蛋白的 mRNA 和蛋白表达水平及磷酸化 FAK 和 Src 的水平明显降低，E-钙黏蛋白的水平显著升高。此外，细胞增殖、侵袭、迁移和 RCC 细胞 G1 期的比例降低，细胞凋亡增加。此外，转染 LOXL2 的 Caki1 细胞表现出相反的趋势。综上所述，这些结果表明 LOXL2 沉默通过抑制 Src/FAK 信号通路抑制 RCC 细胞的侵袭、迁移和 EMT。
