miR-449a对胰腺癌细胞放射敏感性影响

Effect of miR-449a on radiosensitivity of pancreatic cancer cells

目的 研究miR-449a对胰腺癌SW1990细胞放射敏感性的影响方法 qRT-PCR检测放疗前后胰腺癌SW1990细胞系中miR-449a的表达变化,利用Lipofectamine 2000转染试剂盒将miR-449a mimics及miR-NC转染到SW1990细胞中,流式细胞术、克隆形成实验检测放射处理后细胞放射敏感性变化。使用TargetScan预测及双荧光素酶报告基因实验证明miR-449a与Cyclin D1的靶向作用,免疫组织化学法观察Cyclin D1在胰腺癌组织和胰腺癌旁5 cm的正常胰腺组织的分布,基因敲除Cyclin D1验证其对胰腺癌细胞放射敏感性的影响。结果 经放射处理后,miR-449a在胰腺癌细胞中的表达量明显降低;过表达miR-449a增加了放射后胰腺癌细胞的凋亡率,并使胰腺癌细胞克隆形成率也明显降低;TargetScan预测及双荧光素酶报告基因实验证实了Cyclin D1是miR-449a的靶标;Cyclin D1蛋白在胰腺癌患者组织中的阳性染色率（70%,35/50）明显高于正常胰腺组织（20%,2/10）,Cyclin D1增加了胰腺癌细胞的放射敏感性。结论 miR-449a通过靶向干扰Cyclin D1的表达,促进胰腺癌细胞的放射敏感性。

Objective The purpose of this study is to investigate the effect of miR-449a on pancreatic cancer cells and the molecular mechanism. Methods The expression levels of miR-449a in pancreatic cancer cells treated or untreated with radiation was detected by qRT-PCR.High expression of miR-449a was achieved by transfecting miR-449a mimics into SW1990 cells. The cell growth,apoptosis and colony formation ability was assessed by MTT assay,flow cytometry and colony formation assay,respectively. The relationship of miR-449a and Cyclin D1 was determined by the TargetScan and dual luciferase reporter. Immunohistochemistry was used to examine protein levels of Cyclin D1 in pancreatic cancer and normal pancreas tissues. Si-Cyclin D1 was used to detecte the effect of Cyclin D1 on radiosensitivity of pancreatic cancer cells. Resutls The expression levels of miR-449a in pancreatic cancer cells with radiation treatment were decreased significantly. Mir-449a mimics increased the cell proliferation rates and apoptosis rates obviously,and decreased the colony formation ability in SW1990 cells treated with radiation. Resutls from the TargetScan and dual luciferase reporter showed that Cyclin D1 was the target of miR-449a. The positive staining rates of Cyclin D1 in pancreatic cancer tissue （85.7%,30/35） was higher than those in normal pancreas tissue （20%,2/10）.Knockdown of Cyclin D1 enhanced the radiosensitivity of pancreatic cancer cells. Conclusion MiR-449a enhances the radiosensitivity of pancreatic cancer cells by targeting Cyclin D1.