miR-640通过抑制SLIT1介导Wnt/β-catenin通路促进脑胶质瘤对替莫唑胺的耐药

miR-640 Promote Proliferation, Invasion and Resistance to Temozolomide in Glioma by Inhibiting SLIT1-mediated Wnt/β-catenin Pathway

[目的]评价miR-640介导Wnt/β-catenin通路对脑胶质瘤耐药细胞增殖、侵袭及替莫唑胺(temazolamide,TMZ)敏感性的影响。[方法]利用实时荧光定量聚合酶链反应(RT-qPCR)测定脑胶质瘤细胞内miR-640的表达。U87/TMZ细胞分为NC inhibitor组、 miR-640 inhibitor组和miR-640 inhibitor+si-SLIT1组。用不同浓度(15、30、60、120、240、480和960μmol/L)TMZ处理细胞,检测细胞对TMZ的耐药性;运用细胞计数试剂盒8(CCK-8)、Transwell实验检测U87/TMZ细胞增殖活力和侵袭力;用双荧光素酶报告基因实验验证miR-640与SLIT1的靶向关系。采用Western blot检测Wnt/β-Catenin信号通路相关蛋白表达水平。[结果] miR-640在脑胶质瘤细胞中高表达,转染后miR-640 inhibitor组的miR-640表达水平、侵袭细胞数目(57±23 vs 153±31)、IC_(50)值(131.61±8.97 vs 293.33±11.28)、增殖率(18.48±4.23vs 43.84±8.94)均比NC inhibitor组低(P<0.05)。miR-640靶向调控SLIT1的表达,miR-640 inhibitor+siSLIT1组β-catenin、c-myc、Cyclin D1蛋白表达水平均高于miR-640 inhibitor组(P<0.05)。[结论]miR-640通过SLIT1介导Wnt/β-catenin通路促进脑胶质瘤增殖、侵袭及对替莫唑胺的耐药性。

[Objective] To investigate the effects of mi R-640-mediated Wnt/β-catenin pathway on proliferation, invasion and sensitivity to temazolamide(TMZ) in glioma cells. [Methods] The expression of mi R-640 in glioma cells as determined by real-time fluorescence quantitative polymerase chain reaction(RT-q PCR).TMZ-resistant human glioma U87/TMZ cells were transfected with NC inhibitor, mi R-640 inhibitor and mi R-640 inhibitor+ si-SLIT1, respectively. U87/TMZ cells were treated with TMZ(15, 30, 60, 120, 240, 480 and 960 μmol/L). Cell counting Kit 8(CCK-8) and Transwell assay were used to detect the proliferation and invasion of U87/TMZ cells. Dual luciferase reporter gene assay was used to verify the targeting relationship between mi R-640 and SLIT1. Western blot was used to detect the expression levels of Wnt/β-catenin signaling pathway related proteins. [Results] mi R-640 was highly expressed in glioma cells. The mi R-640 expression level, number of invaded cells(57±23 vs 153±31), IC_(50) value(131.61±8.97 vs 293.33±11.28) and proliferation rate(18.48±4.23 vs 43.84±8.94) in mi R-640 inhibitor group were significantly lower than those in NC inhibitor group(P<0.05). The mi R-640 specifically regulated the expression of SLIT1, and the protein expression levels of β-catenin, c-myc and Cyclin D1 in mi R-640 inhibitor+ si-SLIT1 group were higher than those in mi R-640 inhibitor group(P<0.05). [Conclusion] mi R-640 may promote glioma proliferation, invasion and resistance to TMZ through SLIT1-mediated Wnt/β-catenin pathway.