LncRNA CRNDE靶向miR-384影响结直肠癌细胞放射敏感性的研究

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。

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

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.