微小RNA-21靶向调控肿瘤易感性候选基因2在肾癌细胞中的表达和作用

MicroRNA-21 can regulate the expression and roles of cancer susceptibility candidate 2 in renal cell carcinoma cells

目的观察调控肿瘤易感性候选基因2（CASC2）在。肾癌细胞中的表达和作用，微小RNA-21（miR-21）能否调控CASC2表达和作用。方法采用实时定量聚合酶链反应（Real-time PCR）法检测肾癌细胞株（786-O和A498）和人胚胎肾细胞株（HEK293）的CASC2表达，构建高表达质粒pcDNA3.1（＋）-CASC2并转染上调CASC2表达，通过噻唑蓝（MTT）比色分析法和细胞划痕实验观察786-O和A498细胞株的增殖、迁移是否受影响；构建野生型CASC2质粒（CASC2-WT）和突变体CASC2质粒（CASC2-MUT），分别与miR-21 mimic共转染，通过双荧光素酶报告分析确认miR-21对CASC2直接靶向作用，并用qPCR法检测CASC2表达变化；通过MTT和细胞划痕实验观察共转染对。肾癌细胞增殖、迁移能力的影响。结果786-O和A498细胞株的CASC2表达水平明显低于HEK293细胞（P=0．017、0．033）；pcDNA3．1（＋）-CASC2过表达质粒转染后，786-O和A498细胞的增殖和迁移能力明显受到抑制（P=0．001、0．008、0．007、0．009）、miR-21高表达可直接靶向作用于CASC2，降低786-O和A498细胞株的CASC2表达水平下降（P=0．000、0．005），CASC2对786-O和A498细胞株的增殖与迁移的抑制作用也被部分消除（P=0．017、0．011、0．042、0．025）。结论CASC2在肾癌细胞中表达明显降低，起到肿瘤抑制基因的作用，miR-21直接靶向作用CASC2并抑制其表达，从而部分消除CASC2对肿瘤细胞的抑制作用。

Objective To investigate the expression of cancer susceptibility candidate 2 （CASC2） in renal cell carcinoma （RCC） cells lines, to explore whether CASC2 plays an important role in RCC, to explore whether microRNA-21 regulates the expression and role of CASC2. Methods CASC2 expression was measured in three cell lines （786-O, A498, HEK 293 ） by real-time quantitative polymerase chain reaction （Real-time PCR）. Overexpression vector pcDNA3.1（＋）-CASC2 was constructed by pcD-NA3.1, then up-regulation of CASC2 expression levels were induced by pcDNA3.1 （＋） - CASC2 vector in RCC cell lines （ 786-O and A498 ） , MTF and wound scratch assays were used to observe the change of cell proliferation and migration. After co-transfeetion of miR-21 mimics and CASC2-WT or CASC2-MUT recombinant vectors, dual-lueiferase reporter assays were used to confirm whether CASC2 was a direct target gene of miR-21, CASC2 expression was measured by qPCR, MTT and wound scratch assays were used to confirm whether miR-21 can interfere CASC2-mediated inhibition in RCC cell lines. Results CASC2 expression in cell lines 786-O, A498 were statistically significantly lower than HEK 293 cell lines （P =0.017, 0.033）. Overexpression of CASC2 by pcDNA3.1（＋）-CASC2 inhibits the pro-liferation and migration of RCC cells （P=0.001, 0. 008, 0. 007, 0. 009） , miR-21 could target CASC2 directly and decrease the expression of CASC2 in 786-O and A498 cells （P=0.000, 0.005 ）. Moreover, over-expression of miR-21 partly abrogated CASC2-mediated inhibition of 786-O and A498 cells pro-liferation and migration （ P=0. 017, 0.011, 0.042, 0.025 ）. Conclusion CASC2 expressions in RCC cell lines are decreased obviously, CASC2 play tumor suppressor in RCC, miR-21 can target CASC2 and down-regulate the expression of CASC2, then partly abrogated CASC2-mediated inhibition in renal cellcarcinoma.