长链非编码RNA FOXD2-AS1在乳腺癌发展中的作用及其临床意义

Effect of long non-coding RNA FOXD2-AS1 in development of breast cancer and its clinical significance

目的研究长链非编码RNA FOXD2-AS1在乳腺癌发展中的作用,明确FOXD2-AS1在乳腺癌中表达的临床意义。方法收集2014年1月至2014年5月广州医科大学附属肿瘤医院乳腺外科34例乳腺癌患者的乳腺癌组织和相应癌旁正常组织,应用RT-PCR检测乳腺癌组织、相应癌旁正常组织、乳腺癌细胞系(MCF-7、MDA-MB-231、MDA-MB-453、MDA-MB-468)和人正常乳腺上皮细胞(MCF-10A)中FOXD2-AS1的表达,分析乳腺癌患者FOXD2-AS1表达与临床病例特征的关系。使用Kaplan-Meier曲线和Log-rank检验比较乳腺癌患者FOXD2-AS1高表达或低表达组间总生存率的差异。人乳腺癌细胞系(MCF-7、MDA-MB-468)转染慢病毒介导的靶向FOXD2-AS1的干涉寡核苷酸下调FOXD2-AS1的水平,每种细胞均分为对照组(LV-NC)和实验组(LV-shFOXD2-AS1),对照组使用对照小干扰RNA(siRNA),实验组使用靶向FOXD2-AS1的短发夹RNA(shRNA)。荧光显微镜下观察绿色荧光蛋白的表达并计算各组细胞的转染效率。RT-PCR检测转染后各组细胞FOXD2-AS1的表达。通过CCK-8试验、划痕愈合实验和Transwell实验检测各组细胞的增殖活性、迁移和侵袭能力。结果与癌旁正常组织比较,FOXD2-AS1的表达在乳腺癌组织中明显升高(P<0.05),与MCF-10A细胞比较,乳腺癌细胞系中FOXD2-AS1的表达明显升高(均P<0.05)。FOXD2-AS1 mRNA表达与乳腺癌患者雌激素受体(ER)、人表皮生长因子受体2(Her-2)、远处转移、淋巴结转移及TNM分期有关(均P<0.05),而与年龄、孕激素受体(PR)、表皮生长因子受体(EGFR)无关(均P>0.05)。根据乳腺癌组织FOXD2-AS1mRNA表达的中位数(6.9),将上述乳腺癌组织样本分为FOXD2-AS1高表达组(≥6.9,n=15)和FOXD2-AS1低表达组(<6.9,n=19),FOXD2-AS1的高表达组乳腺癌患者的生存率低于低表达组(P<0.05)。各组慢病毒的介导的靶向FOXD2-AS1转染效率达到85%以上。与对照组比较,实验组MCF-7和MDA-MB-468细胞FOXD2-AS1的表达明显降低(均P<0.05)。转染72 h后,MCF-7和MDA-MB-468细胞对照组细胞吸光度开始明显高于实验组(均P<0.05)。划痕24 h后,MCF-7和MDA-MB-468细胞实验组分别较对照组细胞的迁移距离变短(0.54±0.12比1±0.01,0.61±0.15比1±0.01,均P<0.05)。MCF-7和MDA-MB-468细胞实验组分别较对照组侵袭细胞的数目减少[(52±13)个比(89±17)个,(48±16)个比(92±14)个,均P<0.05]。下调FOXD2-AS1表达可抑制MCF-7和MDA-MB-468细胞增殖活性、迁移和侵袭能力。结论FOXD2-AS1在乳腺癌发展中具有促进作用,其高表达与患者预后密切相关。

Objective To determine the effect of long-chain non-coding RNA FOXD2-AS1 in the development of breast cancer,and to clarify the clinical significance of FOXD2-AS1 expression in breast cancer.Methods The breast cancer tissues and adjacent normal tissues were collected from 34 patients with breast cancer,who underwent breast surgery in the Department of Breast Surgery,Affiliated Tumor Hospital of Guangzhou Medical University between January 2014 and May 2014.The expression level of FOXD2-AS1 in breast cancer tissues,adjacent normal tissues,breast cancer cell lines(CF-7,MDA-MB-231,MDA-MB-453 and MDA-MB-468)and human mammary epithelial cell line(MCF-10A)was determined by RT-PCR.The correlation between FOXD2-AS1 expression and clinical characteristics in the breast cancer patients was determined.Kaplan-Meier curve and Log-rank test were used to compare the difference in overall survival rate between the high and low expression groups of FOXD2-AS1 in breast cancer patients.Human breast cancer cell lines(MCF-7 and MDA-MB-468)transfected with lentiviral-mediated interference oligonucleotide targeting FOXD2-AS1 down-regulated FOXD2-AS1 level,and each cell line was divided into the control group and(LV-NC)and study group(LV-shFOXD2-AS1).The control siRNA was included as the control group,and the shRNA targeting FOXD2-AS1 was included as the study group.The expression of green fluorescent protein was examined under fluorescence microscope,and the transfection efficiency in each group of cells was calculated.The expression of FOXD2-AS1 in each group was measured by RT-PCR after the transfection.The proliferation activity,migration and invasion in each group were examined by CCK-8 test,scratch test and Transwell assay.Results Compared with normal adjacent tissues,the expression level of FOXD2-AS1 significantly increased in breast cancer tissues(P<0.05).Compared with MCF-10A cells,the expression level of FOXD2-AS1 significantly increased in breast cancer cell lines(all P<0.05).The mRNA expression of FOXD2-AS1 was correlated with estrogen receptor(ER),human epidermal growth factor receptor 2(Her-2),distant metastasis,lymph node metastasis and TNM staging in breast cancer patients(all P<0.05),and was not correlated with age,progesterone receptor(PR)and epidermal growth factor receptor(EGFR)(all P>0.05).According to the median mRNA expression level(6.9)of FOXD2-AS1 in breast cancer tissues,the above breast cancer tissue samples were divided into FOXD2-AS1 high expression group(≥6.9,n=15)and FOXD2-AS1 low expression group(<6.9,n=19).The survival rate of breast cancer patients in the FOXD2-AS1 high expression group was lower than that in the low expression group(P<0.05).The transfection efficiency of lentivirus-mediated interference oligonucleotide targeting FOXD2-AS1 reached more than 85%.Compared with the control group,the expression level of FOXD2-AS1 in MCF-7 and MDA-MB-468 cells in the study groups significantly decreased(both P<0.05).At 72 h after the transfection,the absorbance of MCF-7 and MDA-MB-468 cells in the control group began to be significantly higher than that in the study group(both P<0.05).At 24 h after the scratch test,the migration distance of MCF-7 and MDA-MB-468 cells in the study group was shorter than that in the control group(0.54±0.12 vs 1±0.01,0.61±0.15 vs 1±0.01,both P<0.05).The number of invasive MCF-7 and MDA-MB-468 cells in the study group was lower than that in the control group(52±13 vs 89±17,48±16 vs 92±14,both P<0.05).Down-regulation of FOXD2-AS1 expression inhibited the proliferation activity,migration and invasion of MCF-7 and MDA-MB-468 cells.Conclusion FOXD2-AS1 may promote the development of breast cancer,and its high expression is closely related to the prognosis of patients.