摘要
为研究前动力蛋白1(PROK1)对结直肠癌细胞增殖与迁移的作用机制,设计合成3条针对PROK1基因的靶向干扰序列,采用荧光显微镜观察干扰片段的转染情况,实时荧光定量PCR分析靶向干扰PROK1基因后PROK1 mRNA下调的表达水平,采用MTT、平板克隆形成试验、transwell迁移实验检测敲除PROK1后对HT29细胞增殖和迁移的影响。结果显示:荧光标记的Cy3-siRNA成功转染到HT29细胞中;3条靶向PROK1基因siRNA-62、siRNA-341、siRNA-1121转染HT29细胞48 h后,沉默效率分别为35%、70%、50%;与阴性对照组相比,siRNA-341组细胞增殖明显被抑制,24 h抑制最显著(p<0.01);siRNA-341组的细胞克隆形成率(19.3±1.989 9)与空脂质体对照组(30.3±3.780 6)和阴性对照组(32.3±2.404 1)相比显著减少(p<0.05);siRNA-341组通过小室迁移的细胞数(76±9.539 3)与空脂质体对照组(212±2.645 7)和阴性对照组(193±7.539 8)相比明显减少(p<0.01)。本研究发现PROK1基因促进结直肠癌细胞的增殖和转移,PROK1基因有望成为治疗结直肠癌潜在的靶位点。
To study the cell proliferation and migration mechanism ofprokineticin 1 （PROK1） towards colorectal cancer, three interference sequences targeting PROK1 gene were designed and synthesized, and whether interference fragments were transfected or not was observed by fluorescence microscopy. The PROK1 mRNA expression levels after targeting interfering PROK1 were analyzed by Real-time PCR. The proliferation and migration of HT29 cells after knocking down PROK1 were detected by using MTT, colony formation assay and transwell migration assay. The results showed that fluorescently labeled Cy3-siRNA had been successfully transfected into HT29 cells. Meanwhile, 48 h after transfecting, the silencing efflcieny of three targeting PROK1 sequences including siRNA-62, siRNA-341, siRNA-1121 were 35%, 70% and 50%, respectively. Compared with the negative control group, cell proliferation in siRNA-341 group was significantly inhibited, whose suppression was the most significant after 24 h （P〈0.01）. Compared with cell colony formation rate in the control liposome g roup （30.3±3.780 6） and negative group （32.3±2.404 1）, colony formation rate in siRNA-341 group （19.3±1.989 9） had a significant reduction （P〈0.05）. Compared with control liposome group （212±2.645 7） and negative group （193±7.539 8）, migrate numbers through chambers in siRNA-341 group （76±9.539 3） had been significantly reduced （P〈0.01）. In conclusion, the study showed that PR OK1 gene had promoted the proliferation and metastasis ofcolorectal cancer cells, and PROK1 gene was expected to become a potential target sites for colorectal cancer therapy. Keywords PROK1, RNA interference, Colorectal cancer, Cell proliferation, Migration
出处
《基因组学与应用生物学》
CAS
CSCD
北大核心
2017年第4期1294-1300,共7页
Genomics and Applied Biology
基金
湖南省长沙市科技计划项目(k1501016-31)
长沙市科技项目(K1403158-61)
中南大学国家级大学生创新类项目(201610533027)共同资助
