稳定过表达microRNA—96前列腺癌细胞株的建立及其对前列腺癌细胞增殖和迁移的影响

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[摘要] 目的 构建稳定过表达microRNA-96（miR-96）的前列腺癌DU-145细胞株并研究其对DU-145细胞增殖和迁移的影响。 方法 构建miR-96慢病毒表达载体并转染前列腺癌细胞DU-145，嘌呤霉素筛选出稳定表达miR-96（实验组）及阴性对照（对照组）的细胞株；通过实时荧光定量PCR（qRT-PCR）检测转染后两组细胞miR-96的表达；克隆形成实验和MTT实验检测两组的细胞增殖情况。transwell迁移实验检测两组细胞的迁移能力。 结果 经过筛选后各组转染细胞发光效率均> 90%；qRT-PCR结果显示实验组miR-96表达水平明显高于对照组（P < 0.05）；克隆形成实验显示实验组克隆形成率明显低于对照组（P < 0.05）；MTT实验显示实验组各个时间点吸光度值均低于对照组（P < 0.05）；transwell迁移实验表明实验组穿膜细胞数明显少于对照组（P < 0.05）。 结论 miR-96可抑制前列腺细胞的增殖和迁移能力，可能参与前列腺癌的发生、发展。

[关键词] 前列腺癌；microRNA-96；增殖；迁移

[中图分类号] R737.25 [文献标识码] A [文章编号] 1673-7210（2016）01（b）-0024-05

[Abstract] Objective To establish microRNA-96 （miR-96） stable overexpression cell lines of cervical cancer and detect its effect on proliferation of prostatic cancer cell lines DU-145. Methods Lentiviral vector stable overexpressing miR-96 was constructed and prostatic cancer cell lines DU-145 were transfected （experimental group）. The transfection cells were screened by using of puromycin and the stable transfection cell lines were obtained （control group）. Endogenous miR-96 levels in two groups were measured by quantitative RT-PCR（qRT-PCR）. The proliferation of DU-145 cells in two groups were detected by clonogenic proliferation assay and MTT assay. The migration of DU-145 cells in two groups were assessed by transwell assay. Results After screening， luminous efficiency rate of transfection cell in each group was over 90%. qRT-PCR showed that the expression level of miR-96 in experimental group was significantly higher than that in control group （P < 0.05）. Colony formation assay showed that the clonogenicity rate of experimental group was significantly lower than that of control group （P < 0.05）. MTT assay demonstrated that absorbance values all time points of experimental group were lower than those of control group （P < 0.05）. Transwell assay revealed that the number of cells through the well in experimental group were less than that in control group （P < 0.05）. Conclusion miR-96 can inhibit proliferation and migration of prostatic cancer cells and may be involved in the development of prostatic cancer.

[Key words] Prostatic cancer； MicroRNA-96； Proliferation； Migration

前列腺癌是一种常见恶性泌尿系统肿瘤，全球范围内每年约有20万例患者死于前列腺癌[1]。前列腺癌的发病原因迄今仍不清楚。微小RNA（microRNA，miRNA）是一类由内源性基因编码，长度为19～22个核苷酸的非编码小分子单链RNA。目前，研究发现，miRNA在多种肿瘤中异常表达[2-5]，与恶性肿瘤关系密切，大约有调控miRNA编码基因位的52%位于肿瘤相关染色体区域[6]。已有研究证明，miR-96在多种肿瘤中低表达，如：膀胱癌、乳腺癌、直肠癌等，是潜在的肿瘤抑制因子[7-11]。本实验通过构建过表达miR-96的慢病毒载体来感染前列腺癌DU-145细胞株，以构建过表达miR-96的前列腺癌细胞稳转细胞株，并分别进行克隆形成实验、MTT实验、transwell迁移实验，研究miR-96对前列腺癌细胞增殖和迁移的影响。