摘要
目的探讨lncRNA CCAT1和miR-130b-3p对体外培养的胰腺癌细胞PANC-1放射敏感性的影响。方法采用Real-time PCR检测胰腺癌组织及其细胞系和2 Gy X射线照射后PANC-1细胞中CCAT1和miR-130b-3p的相对表达水平。沉默CCAT1表达、抑制miR-130b-3p表达后,应用流式细胞仪、Caspase 3活性检测试剂盒及克隆形成实验检测细胞凋亡率、Caspase 3活性和细胞存活分数,并绘制单击多靶模型拟合曲线;利用starBase v2.0在线预测、荧光素酶报告基因、RNA结合蛋白免疫沉淀实验(RIP)及Real-time PCR实验,验证CCAT1和miR-130b-3p的靶向关系。结果在放射抵抗的胰腺癌组织、胰腺癌细胞系和2 Gy照射的PANC-1细胞中,CCAT1表达均上调(t=6.322~8.555,P<0.05),miR-130b-3p表达下调(t=3.950~18.795,P<0.05)。2 Gy照射并沉默CCAT1,PANC-1细胞存活分数降低(t=2.929、5.047、5.234、5.125,P<0.05),细胞凋亡率增加(t=6.953, P<0.05),Caspase 3活性升高(t=6.836, P<0.05)。发现CCAT1能靶向调控miR-130b-3p表达,抑制miR-130b-3p表达,PANC-1细胞存活分数增大(t=4.564、6.736、8.656,P<0.05),细胞凋亡减少(t=5.234,P<0.05),Caspase 3活性降低(t=10.440,P<0.05)。结论沉默CCAT1表达能够促进miR-130b-3p表达,从而增加PANC-1细胞放射敏感性。
Objective To investigate the effect of lncRNA CCAT1 and miR-130b-3p on the radiosensitivity of human pancreatic cancer cells PANC-1. Methods Real-time PCR was used to detect the relative expression levels of CCAT1 and miR-130b-3p in pancreatic cancer tissues and cell lines including PANC-1 cells irradiated with 2 Gy X-rays. After silencing CCAT1 and/or inhibiting miR-130b-3p expression, cell apoptosis rate, Caspase 3 activity and cell survival were detected by flow cytometry, Caspase 3 activity detection kit and colony formation assay, respectively. Cell survival curve was stimulated by the multi-target single-hit model. Based on the starBase v2.0 online analysis, the luciferase reporter gene assay, RNA-binding protein immunoprecipitation assay (RIP) and Real-time PCR assay were applied to verify the relationship between CCAT1 and miR-130b-3p. Results CCAT1 expression was up-regulated (t=6.322-8.555, P<0.05), but miR-130b-3p expression was down-regulated (t=3.950-18.795, P<0.05) in the radiation-resistant pancreatic cancer tissues, pancreatic cancer cell lines and 2 Gy-irradiated PANC-1 cells. When the CCAT1 silenced PANC-1 cells were irradiated with 2 Gy, cell survival fraction decreased (t=2.929, 5.047, 5.234, 5.125, P<0.05), apoptosis rate and Caspase 3 activity increased (t=6.953, 6.836, P<0.05). CCAT1 could selectively regulate miR-130b-3p expression. Inhibition of miR-130b-3p expression could enhance PANC-1 cell survival (t=4.564, 6.736, 8.656, P<0.05), but reduced apoptosis rate (t=5.234, P<0.05) and Caspase 3 activity (t=10.440, P<0.05). Conclusions Silencing CCAT1 promotes the expression of miR-130b-3p and enhances radiosensitivity of PANC-1 cells.
作者
宋锐
袁金金
侯歌
杨军
陈晓娟
柴婷
王成
刘宗文
Song Rui;Yuan Jinjin;Hou Ge;Yang Jun;Chen Xiaojuan;Chai Ting;Wang Cheng;Liu Zongwen(Department of Tumor Radiotherapy,The Second Affiliated Hospital of Zhengzhou University,Zhengzhou 450014,China)
出处
《中华放射医学与防护杂志》
CAS
CSCD
北大核心
2019年第4期247-254,共8页
Chinese Journal of Radiological Medicine and Protection
基金
国家重点研发计划项目(2016YFC0105713).
