miR-338-3p靶向TCF4对人肾癌细胞增殖、凋亡和转移的调节

Effect of miR-338-3p Targeting TCF4 on Proliferation, Apoptosis and Metastasis of Human Kidney Cancer Cells

目的 探究miR-338-3p靶向T细胞生长因子4(T cell factor,TCF4)对肾癌细胞及肿瘤生长的影响.方法 将肾癌细胞786-O分为4组:对照组、miR-338-3p mimic组、pc-TCF4组、miR-338-3p+TCF4组.qRT-PCR检测miR-338-3p及TCF4 mRNA水平.荧光素酶实验验证miR-338-3p与TCF4靶向关系.免疫印记检测细胞中TCF4、Ki67、cleaved caspase-3、 血管内皮细胞生长因子(VEGF)及基质金属蛋白酶2(MMP-2)的蛋白水平.CCK-8检测细胞增殖.流式细胞术检测细胞凋亡.Transwell检测细胞侵袭.划痕实验检测细胞迁移.免疫组化检测肿瘤组织中Ki67,cleaved caspase-3,VEGF及MMP-2的表达.结果 ①细胞转染miR-338-3p mimic后miR-338-3p表达升高,TCF4 mRNA水平下降,miR-338-3p与TCF4存在靶向关系.②miR-338-3p mimic可降低TCF4蛋白水平,pc-TCF4可提高TCF4蛋白水平,并减弱miR-338-3p mimic对TCF4的抑制作用.③miR-338-3p mimic可降低细胞增殖和Ki67蛋白水平,提高细胞凋亡及cleaved caspase-3蛋白水平.pc-TCF4可提高细胞增殖和Ki67蛋白水平,降低细胞凋亡及cleaved caspase-3蛋白水平,并减弱miR-338-3p mimic的抑增殖促凋亡作用.④miR-338-3p mimic可降低侵袭细胞数、 划痕愈合率及VEGF和MMP-2表达,pc-TCF4可升高侵袭细胞数、 划痕愈合率及VEGF和MMP-2表达,并减弱miR-338-3p mimic对侵袭迁移的抑制作用.⑤miR-338-3p mimic可增加荷瘤小鼠存活率及cleaved caspase-3蛋白水平,减小移植瘤体积及Ki67,VEGF和MMP-2的蛋白水平.pc-TCF4可减少荷瘤小鼠存活率及cleaved caspase-3蛋白水平,增大移植瘤体积及Ki67,VEGF和MMP-2的蛋白水平,并减弱miR-338-3p mimic对小鼠的保护作用.结论 miR-338-3p靶向TCF4可减弱肾癌细胞增殖、 侵袭及迁移并诱导凋亡,抑制肿瘤生长.

Objective To explore the effects of miR-338-3p targeting T cell factor 4 (TCF4) on the cellular and tumor growth of renal cancer. Methods Renal cancer cells 786-O were divided into control group, miR-338-3p mimic group, pc-TCF4 group and miR-338-3p+TCF4 group. The mRNA levels of miR-338-3p and TCF4 were detected by qRT-PCR. The targeting relationship between miR-338-3p and TCF4 was verified by luciferase assay. The protein levels of TCF4, Ki67, cleaved caspase-3, vascular endothelial growth factor (VEGF) and matrix metalloprotein 2(MMP-2) were measured by Western blotting. Cell proliferaqtion was detected by CCK-8. Apoptosis was flow cytometrically measured. Cell invasion was examined by transwell assay. Cell migration was tested by wound healing. The protein levels of Ki67 , cleaved caspase-3, VEGF and MMP-2 in tumor tissuses were immumohistochemically detected. Results (1) After transfection with miR-338-3p mimic, the expression of miR-338-3p in 786-O cells was incrased;the mRNA level of TCF4 in 786-O cells was decreased, suggesting that TCF4 was a target of miR-338-3p.(2) miR-338-3p mimic reduced the TCF4 protein level, but pc-TCF4 enhanced it and weakened the inhibition of miR-338-3p mimic on TCF4.(3) miR-338-3p mimic decreased the proliferation and expression of Ki67, enhanced apoptosis and expression of cleaved caspase-3. pc-TCF4 increased the proliferation and expression of Ki67, reduced apoptosis and expression of cleaved caspase-3, and decreased the proliferation-inhibitory and appotosis-promoting effects of miR-338-3p mimic.(4) miR-338-3p mimic decreased the number of invasive cells, wound healing rate and expression of VEGF and MMP-2, but pc-TCF4 enhanced them and weakened the invasion-and migration-inhibi-ing effect of miR-338-3p mimic.(5) miR-338-3p mimic increased the survival rate of tumorbearing mice and expression of cleaved caspase-3 , decreased the tumor volume and expression of Ki67, VEGF and MMP-2. pc-TCF4 decreased the survival rate of the mice and expression of cleaved caspase-3, increased the tumor volume and expression of Ki67, VEGF and MMP-2, and weakened the protective effect of miR-338-3p mimic on the mice. Conclusion MiR-338-3p targeting TCF4 reduced proliferation, invasion, migration and apoptosis of renal cell carcinoma cells, thereby inhibiting the growth of the tumor.