摘要
目的探讨miR-26a对涎腺腺样囊性癌ACC-M细胞增殖能力的影响及其与EZH2的靶向关系。方法构建pcDNATM6.2-pre-miR-26a、pmirGLO-EZH2野生型(pmirGLO-EZH2-WT)和突变型(pmirGLO-EZH2-MT)重组质粒,并采用荧光实时定量PCR(qRT-PCR)检测pre-miR-26a在ACC-M细胞中的转染效率,并与未处理组(control组)进行比较。同时采用MTT法分析miR-26a过表达对ACC-M细胞增殖活性的影响,并采用双荧光素酶报告基因系统、qRT-PCR和Western blot检测miR-26a与EZH2的靶向关系。结果将测序结果采用NCBI BLAST进行序列比对,结果显示pre-miR-26a、EZH2-WT和EZH2-MT重组质粒均构建成功,无碱基缺失或突变。qRT-PCR显示转染pre-miR-26a的ACC-M细胞其miR-26a的表达显著高于未处理组(P<0.001)。MTT分析显示miR-26过表达可以明显抑制ACC-M细胞的增殖活性(P<0.05)。此外,双荧光素酶报告基因系统(P=0.001)、qRT-PCR和Western blot均证实miR-26a能够显著下调EZH2的mRNA(P=0.001)和蛋白的表达(P=0.006)。结论 miR-26a能够显著抑制涎腺腺样囊性癌ACC-M细胞的增殖活性,并且与EZH2之间存在良好的靶向关系。
Objective To investigate the effect of miR-26a on proliferation of salivary adenoid cystic carcinoma cells （SACC） and its relationship with EZH2.Methods The pre-miR-126,EZH2-WT and EZH2-MT recombinant plasmid were synthesized and cloned into pcDNATM6.2 and pmirGLO plasmids respectively.The transfection efficiency of pre-miR-26a was identified by qRT-PCR.In addition,the effect of miR-26a on the proliferation of ACC-M cells was analyzed by MTT.The target relationship between miR-26a and EZH2 was identified by Dual-Luciferase Assay System,qRT-PCR and Western blot.Results The recombinant plasmids were successfully constructed,including pcDNATM6.2-pre-miR-26a,pmirGLO-EZH2-WT and pmirGLO-EZH2-MT.The miR-26a expression was increased in ACC-M cells after transfected with pre-miR-26a（P ＜0.001）.MTT assay showed that miR-26 overexpression significantly inhibited the proliferation of ACC-M cells （P ＜ 0.05）.In addition,the target relationship between miR-26a and EZH2 was identified by Dual-Luciferase Assay System（P =0.001）,and miR-26a decreased the mRNA（P =0.001） and protein expression levels of EZH2 （P =0.006）.Conclusion The miR-26 overexpression could inhibit cell proliferation of salivary adenoid cystic carcinoma cells through targeting EZH2.
出处
《山西医科大学学报》
CAS
2014年第2期101-105,共5页
Journal of Shanxi Medical University
