Wang Lei, Wei Wan-Qing, Wu Zi-Yu, Wang Gong-Cheng
Department of Urology, The Affiliated Huai'an Hospital of Xuzhou Medical University and The Second People's Hospital of Huai'an, Huai'an 223200, China.
Mol Biosyst. 2017 Nov 21;13(12):2564-2573. doi: 10.1039/c7mb00406k.
Renal cell carcinoma (RCC) is the leading cause of death in renal malignancies. MicroRNA-590-5p (miR-590-5p) is of great importance in the processes of many cancers regarding regulation of cancer cell invasion and proliferation. In our study, alternation of miR-590-5p expression in RCC cell lines through transfection with pre-miR-590-5p (up-regulation) or anti-miR-590-5p (down-regulation) was performed. Apoptosis and viability of RCC cell lines were measured by flow cytometry and CCK-8 analysis, respectively. Cell invasion and migration were estimated by Transwell assay. The association of miR-590-5p with ARHGAP24 expression was evaluated using luciferase assays, real-time PCR and western blot assay. The expressions of apoptosis and migration-related protein were also measured by western blotting. We found that pre-miR-590-5p transfection in Caki-2 and 786-O cells showed significant increases in cell viability, invasion and migration, which were accompanied by decreased cell apoptosis, while anti-miR-590-5p transfection obviously inhibited the cell viability, migration and invasion of Caki-2 and 786-O cells as well as induced apoptosis, compared with the negative control group. Furthermore, bioinformatics combined with luciferase reporter assays indicated that ARHGAP24 is directly targeted by miR-590-5p. ARHGAP24 overexpression in 786-O and Caki-2 cells phenocopied the effects of anti-miR-590-5p transfection along with enhanced expression of active Caspase-3 and Bax/Bcl-2 ratio as well as decreased expression of MMP-2 and MMP-9. These findings suggested that miR-590-5p/ARHGAP24 seems to function as a potentially beneficial target for RCC treatment.
肾细胞癌(RCC)是肾恶性肿瘤的主要死因。微小RNA - 590 - 5p(miR - 590 - 5p)在许多癌症中对于调节癌细胞侵袭和增殖的过程具有重要意义。在我们的研究中,通过转染前体miR - 590 - 5p(上调)或抗miR - 590 - 5p(下调)来改变RCC细胞系中miR - 590 - 5p的表达。分别通过流式细胞术和CCK - 8分析来检测RCC细胞系的凋亡和活力。通过Transwell实验评估细胞侵袭和迁移能力。使用荧光素酶报告基因检测、实时定量PCR和蛋白质印迹法评估miR - 590 - 5p与ARHGAP24表达的相关性。还通过蛋白质印迹法检测凋亡和迁移相关蛋白的表达。我们发现,在Caki - 2和786 - O细胞中转染前体miR - 590 - 5p后,细胞活力、侵袭和迁移显著增加,同时细胞凋亡减少;而与阴性对照组相比,转染抗miR - 590 - 5p明显抑制了Caki - 2和786 - O细胞的活力、迁移和侵袭,并诱导细胞凋亡。此外,生物信息学结合荧光素酶报告基因检测表明ARHGAP24是miR - 590 - 5p的直接靶标。在786 - O和Caki - 2细胞中过表达ARHGAP24模拟了转染抗miR - 590 - 5p的效果,同时活性半胱天冬酶 - 3的表达增强,Bax/Bcl - 2比值升高,MMP - 2和MMP - 9的表达降低。这些发现表明,miR - 590 - 5p/ARHGAP24似乎是RCC治疗的一个潜在有益靶点。