LncRNA CRNDE靶向miR-384影响结直肠癌细胞放射敏感性的研究

Effect of lncRNA CRNDE targeting miR-384 on radiosensitivity of colorectal cancer cells

目的研究长链非编码RNA(Lnc RNA)结直肠肿瘤差异表达基因(CRNDE)对结直肠癌细胞放射敏感性的影响及其机制。方法以结直肠癌HT-29细胞作为体外研究对象,转染CRNDE shRNA,实时定量PCR测定干扰效果。以8 Gy X射线照射转染CRNDE shRNA后的HT-29细胞,四甲基偶氮唑盐(MTT)比色法和流式细胞术分别检测细胞增殖和凋亡水平。平板克隆实验检测放射敏感性。生物信息学软件预测CRNDE与miR-384有互补结合位点,荧光素酶报告系统鉴定靶向关系。将CRNDE shRNA和miR-384 inhibitor共转染至HT-29细胞中,以8 Gy剂量照射处理,MTT和流式细胞术检测细胞增殖和凋亡变化。结果CRNDE shRNA能够降低HT-29细胞中CRNDE表达水平(1.00±0.08 vs.0.42±0.06,t=10.051,P<0.05)。CRNDE shRNA和放射均可以抑制HT-29细胞增殖并诱导细胞凋亡,并且二者联合具有协同作用[凋亡率:(2.27±0.13)%、(23.58±2.35)%、(26.91±2.81)%、(36.84±3.24)%,F=24.66,P<0.05;吸光度(A)值:0.45±0.06、0.30±0.02、0.28±0.03、0.20±0.02,F=106.21,P<0.05]。CRNDE shRNA转染后可以提高HT-29细胞放射敏感性,放射增敏比为1.374。CRNDE靶向负调控miR-384表达。miR-384 inhibitor能够拮抗CRNDE shRNA对放射处理的结直肠癌细胞增殖抑制和凋亡促进的作用。结论下调LncRNA CRNDE表达可增强结直肠癌细胞的放射敏感性,其作用机制与靶向负调控miR-384表达有关。

Objective To study the effect of LncRNA CRNDE on radiosensitivity of colorectal cells and underlying mechanism.Methods Colorectal cancer HT-29 cells were transfected with CRNDE shRNA and the interference efficiency was determined by Real time PCR.HT-29 cells transfected with CRNDE shRNA or co-transfected with CRNDE shRNA and miR-384 inhibitor were irradiated at 8 Gy dose,then cell proliferation and apoptosis were detected by MTT and flow cytometry assay,respectively,and cell radiosensitivity was evaluated by cloning assay.It was predicted by a bioinformatics software that CRNDE and miR-384 have complementary binding sites,and this was identified by a luciferase reporting system.Results CRNDE shRNA reduced the expression of CRNDE in HT-29 cells(1.00±0.08 vs.0.42±0.06,t=10.051,P<0.05).Both CRNDE shRNA and radiation inhibited the proliferation and induced apoptosis of HT-29 cells,and their combination treatment had synergistic effect in apoptosis induction[Apoptosis rates:(2.27±0.13)%,(23.58±2.35)%,(26.91±2.81)%,(36.84±3.24)%,F=24.660,P<0.05;A values:0.45±0.06,0.30±0.02,0.28±0.03,0.20±0.02,F=106.207,P<0.05].Transfection of CRNDE shRNA increased the radiosensitivity of HT-29 cells with a radiosensitization ratio of 1.374.CRNDE negatively regulated the expression of its target miR-384.The miR-384 inhibitor antagonized the effect of CRNDE shRNA on proliferation inhibition and apoptosis promotion of radiation-treated colorectal cancer cells.Conclusions Down-regulation of LncRNA expression enhances the radiosensitivity of colorectal cancer cells by regulating miR-384 expression.