miR-496在卵巢浆液性癌中的表达及与SIX1的靶向关系研究

Expression of miR-496 in ovarian serous carcinoma and its targeting correlation with SIX1

目的:检测卵巢浆液性癌中微小RNA-496(microRNA-496,miR-496)的表达,关注其表达意义及与同源异型蛋白SIX1(homologous heteroprotein SIX1,SIX1)的靶向关系。方法:选择75例卵巢浆液性癌作为观察组,选择75例卵巢浆液性囊腺瘤作为对照组,应用实时荧光定量PCR法检测2组中miR-496的表达,应用免疫组化法检测观察组中增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)和B细胞淋巴瘤-2相关X蛋白(Bcl-2 associated X protein,BAX)的表达,应用Western blot检测观察组中SIX1的表达。选择人卵巢浆液性癌细胞系SKOV-3,设置空白对照组、miR-496转染组、miR-496和SIX1共转染组,采用双荧光素酶基因实验验证miR-496与靶基因SIX1的关系。结果:miR-496在观察组的表达量明显低于对照组(1.52±0.36 vs.2.03±0.25,t=7.56,P=0.001),miR-496的表达在不同肿瘤最大径(1.65±0.36 vs. 1.42±0.33,t=5.32,P=0.012)、病理分级(1.64±0.35 vs. 1.43±0.40,t=5.11,P=0.010)、有无脉管累犯(1.60±0.44 vs. 1.35±0.43,t=5.11,P=0.011)、是否双侧发生(1.61±0.36 vs.1.40±0.32,t=5.11,P=0.010)、有无淋巴结转移(1.62±0.42 vs. 1.35±0.41,t=5.66,P=0.008)和不同TNM分期(1.70±0.37 vs.1.42±0.39,t=5.65,P=0.009)的分组中有统计学差异。miR-496与生存时间有关(χ2=4.13,P=0.010),miR-496与PCNA(r=-0.54,P=0.0186)、miR-496与SIX1(r=-0.58,P=0.0130)均具有负相关性,miR-496与BAX(r=0.52,P=0.0110)具有正相关性。双荧光素酶基因实验显示,miR-496能引起转染pGL3-SIX1-WT的细胞系中荧光素酶活性明显降低。结论:卵巢浆液性癌中miR-496的表达下降,是促进肿瘤形成和进展的重要分子因素,检测miR-496对判断预后可能有一定价值。miR-496可能通过负向调节靶基因SIX1调控肿瘤细胞的增殖和凋亡。

Objective:To detect the expression of microRNA-496(miR-496)in ovarian serous carcinoma,so as to observe its expression significance,and to analyze its correlation with homologous heteroprotein sine oculis homeobox homolog 1(SIX1).Methods:A total of 75 patients with ovarian serous carcinoma were selected as the observation group,and another 75 patients with ovarian serous cystic adenoma were selected as the control group.Expression of miR-496 in two groups was detected by real-time fluorescence quantitative PCR.Expression of proliferating cell nuclear antigen(PCNA)and Bcl-2 associated X protein(BAX)in the observation group was detected by immunohistochemical method.Expression of SIX1 in the observation group was detected by Western blot.Ovarian serous cystic adenoma cell lines were selected;blank control group,micR-496 transfection group,and miR-496 and SIX1 co-transfection group were set up.Relationship between miR-496 and target gene SIX1 was verified by applying double luciferase gene experiment.Results:Expression of miR-496 in the observation group was significantly lower than that in the control group(1.52±0.36 vs.2.03±0.25,t=7.56,P=0.001).The expression of miR-496 was statistically significant in different groups of tumor maximum diameter(1.65±0.36 vs.1.42±0.33,t=5.32,P=0.012),histological grade(1.64±0.35 vs.1.43±0.40,t=5.11,P=0.010),vascular involvement(1.60±0.44 vs.1.35±0.43,t=5.11,P=0.011),bilateral occurrence(1.61±0.36 vs.1.40±0.32,t=5.11,P=0.010),lymph node metastasis(1.62±0.42 vs.1.35±0.41,t=5.66,P=0.008)and different TNM stages(1.70±0.37 vs.1.42±0.39,t=5.65,P=0.009).Expression of miR-496 was related with survival time(χ^2=4.13,P=0.010);it had negative correlation with PCNA(r=-0.54,P=0.019)and SIX1(r=-0.58,P=0.013);it had positive correlation with BAX(r=0.52,P=0.011).Double luciferase gene experiment results showed that miR-496 was able to significantly reduce the luciferase activity in pGL3-SIX1-WT transfected cell lines.Conclusion:Expression of miR-496 in ovarian serous carcinoma is decreased,which is the molecular factor for accelerating tumors’formation and development.Detection of miR-496 has a certain significance for judging the prognosis.MiR-496 may regulate cell proliferation and apoptosis by negatively regulating the expression of SIX1.