miRNA-32-5p对EZH2基因的靶向调控以及对口腔癌细胞CAL-27增殖的影响

miRNA-32-5p target regulating EZH2 and its effect on proliferation of human oral carcinoma cell line CAL-27

目的:构建miR-32-5p慢病毒载体,研究其对EZH2基因的靶向调控作用以及对CAL-27细胞增殖作用的影响。方法:分别构建p Sico R-miR-32-5p及p MIR-Report-EZH2过表达载体,应用双荧光素酶报告基因系统、Western免疫印迹及实时定量荧光PCR方法验证miR-32-5p与EZH2的靶向调控关系,应用MTT法检测过表达miR-32-5p后对CAL-27细胞的增殖调节作用,采用SPSS20.0软件包对数据进行统计学分析。结果:成功构建了p Sico R-miR-32-5p及p MIR-Report-EZH2重组质粒,并证实miR-32-5p过表达可明显抑制EZH2的蛋白及m RNA的表达水平(P<0.05);抑制miR-32-5p的表达,则明显上调EZH2的蛋白及m RNA的表达水平(P<0.05);MTT结果显示,miR-32-5p可显著抑制CAL-27的增殖。结论 :miR-32-5p可通过靶向作用于EZH2-3’UTR的特异序列,直接抑制EZH2基因的表达并调控CAL-27细胞的增殖。

PURPOSE： To construct a lentiviral vector of mi R- 32-5p and verify its targeted effect on EZH2 gene and its effect on the proliferation of CAL-27 cells. METHODS： p Sico R-miR-32-5p and p MIR-Report-EZH2 over-expression vector were constructed. The targeted effect of mi R-32-5p on EZH2 gene was verified by relative luciferase activity,Western blot and real-time PCR test. The effects on CAL-27 cells proliferation of over-expression mi R-32-5p were analyzed by MTT assay. SPSS 20.0 software package was used for statistical analysis. RESULTS： The recombinant plasmids of p Sico R-mi R- 32-5p and p MIR-Report-EZH2 3＇-UTR were constructed successfully. Over-expression of mi R-32-5p suppressed the m RNA and protein expression level of EZH2 significantly（P〈0.05）. Suppression of mi R-32-5p expression increased the expression level of EZH2 m RNA and protein significantly（P〈0.05）.MTT assay showed that the proliferation effect of lentivirus vector of mi R-32-5p on CAL-27 cells were significant（P〈0.05）. CONCLUSIONS： Mi R-32-5p can suppress EZH2 gene expression by targeting the specific sequence of EZH2-3＇UTR and affect proliferation of human oral carcinoma cell line CAL-27.