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

摘要:

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。

Abstract:

Objective：To detect the expression of microRNA-496（miR-496） in ovarian serous carcinoma，so as to observe its expres－sion 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 in－volvement（1.60±0.44 vs. 1.35±0.43，t=5.11，P=0.011），bilat－eral 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（ χ2=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 regu－late cell proliferation and apoptosis by negatively regulating the expression of SIX1.