联合沉默转化生长因子受体Ⅰ及结缔组织生长因子基因对胶质母细胞瘤细胞血管拟态及其干细胞形成的影响

Effects of combined silencing of transforming growth factor receptorⅠand connective tissue growth factor on vasculogenic mimicry and stem cells formation of human glioma cells

目的研究siRNA单独或联合沉默胶质母细胞瘤细胞中的转化生长因子受体Ⅰ(TGFβR1)和结缔组织生长因子(CTGF)表达对其血管拟态生成和肿瘤干细胞成球生长的影响。方法以U87MG为模式细胞,空白组不给予转染,阴性对照组转染siNC,siTGFβR1组、siCTGF组和双靶点组分别转染siTGFβR1、siCTGF、siTGFβR1联合siCTGF。以实时荧光定量聚合酶链反应和蛋白质印迹法检测siRNA的沉默效率,以血管拟态生成和肿瘤干细胞成球实验检测各组的抑制效果。结果siTGFβR1组和siCTGF组的mRNA沉默效率分别为(83.83±2.93)%和(84.42±5.68)%,蛋白沉默效率分别为(69.65±6.82)%和(64.41±3.88)%,与空白组及阴性对照组比较,差异均有统计学意义(均P<0.01)。siTGFβR1组、siCTGF组和双靶点组的拟态血管分支点比值分别为(54.15±3.96)%、(62.87±4.91)%和(37.36±1.34)%,成球实验肿瘤球数量比值分别为(57.00±10.95)%、(68.50±14.32)%和(32.00±8.91)%;与空白组及阴性对照组比较,差异均有统计学意义(均P<0.05)。结论单靶点siTGFβR1和siCTGF均可有效抑制U87MG的血管拟态生成和肿瘤干细胞形成,而同时沉默双靶点基因的表达其抑制功效更强。

Objective To investigate the effects of siRNA alone or in combination with silencing the expression of transforming growth factor receptorⅠ(TGFβR1)and connective tissue growth factor(CTGF)on vasculogenic mimicry and sphere forming growth of cancer stem cells in human glioblastomam cells.Methods U87MG is a model cell,the control group was not transfected,the negative control group was transfected with siNC,and the siTGFβR1,siCTGF and dual targeting groups were transfected with siTGFβR1,siCTGF or siTGFβR1 combined with siCTGF,respectively.The silencing efficiencies of siRNA were determined by reverse transcription-polymerase chain reaction and Western Blot.The inhibitory efficiencies were determined by vasculogenic mimicry and tumor stem cell formation experiments.Results The efficiencies of mRNA silencing in siTGFβR1 and siCTGF groups were(83.83±2.93)%and(84.42±5.68)%;the efficiencies of silencing protein expression were(69.65±6.82)%and(64.41±3.88)%.Compared with those of control and negative control groups,they showed statistical differences(all P<0.01).The ratios of branching points of the mimicry vessels in the siTGFβR1,siCTGF and dual targeting groups were(54.15±3.96)%,(62.87±4.91)%and(37.36±1.34)%;the ratios of the number of tumor sphere in sphere forming experiment were(57.00±10.95)%,(68.50±14.32)%and(32.00±8.91)%,respectively.Compared with those of control and negative control groups,they showed statistical differences(all P<0.05).Conclusion Both siTGFβR1 and siCTGF can effectively inhibit vasculogenic mimicry and tumor stem cell formation of U87MG,while silencing the expression of the dual targets simultaneously has a stronger inhibitory effect.