摘要
目的探讨miR-146a对雌激素受体α阳性(ERα+)乳腺癌患者他莫昔芬(TAM)治疗敏感性的影响。方法选择2018年1月-2019年12月于我院行手术切除的乳腺癌患者48例,收集其肿瘤组织和相应的癌旁正常组织,并收集其年龄、病理分级、淋巴结转移状况、免疫组化结果、分子分型等临床病理特征资料;通过RT-qPCR实验检测48例乳腺肿瘤患者癌组织和癌旁正常组织、ERα+人乳腺癌细胞株(MCF-7)和人乳腺上皮细胞(MCF-10A)中的miR-146a水平。利用Kaplan Meier plotter在线数据库分析miR-146a表达水平与乳腺癌患者预后的关系。利用MCF-7细胞构建ERα+乳腺癌TAM耐药细胞株(TAM-R),将其分为未转染组(TAM-R组)、转染空白载体组(mimic-NC组)和转染hsa-miR-146a模拟物组(mimic组),通过CCK-8法检测各组细胞培养至第0、24、48、72和96小时时的增殖情况;通过流式细胞术检测各组细胞的凋亡率。使用在线预测工具预测miR-146a的靶基因,并通过双荧光素酶报告基因实验检测miR-146a与预测靶基因的相互作用。结果RT-qPCR实验检测结果显示,乳腺癌患者肿瘤组织和MCF-7细胞中miR-146a的表达明显低于相应癌旁正常组织和MCF-10A细胞(t=4.02、50.47,P<0.05);对临床病理特征资料分析结果显示,miR-146a水平在Luminal A、HER-2+、VEGF+及ERα+乳腺癌患者肿瘤组织中明显上调(F=10.79,t=2.23~11.08,P<0.05)。在总的ERα+乳腺癌患者和接受内分泌治疗的ERα+阳性乳腺癌患者中,miR-146a高表达者的生存率明显高于低表达者(HR=0.58、0.66,95%CI=0.41~0.84、0.41~0.89,P<0.05)。CCK-8法检测结果显示,培养第24小时到第96小时各时间点上mimic组细胞吸光度值均显著低于TAM-R组和mimic-NC组(F=25.09~92.17,q=5.09~12.17,P<0.05);流式细胞术检测结果显示,与mimic-NC组相比,mimic组的细胞凋亡率明显升高(F=38.24,q=5.60,P<0.05)。在线预测工具筛选结果显示,miR-146a的潜在靶基因为CARD10;双荧光素酶报告基因实验检测结果显示,CARD10-wt+hsa-miR-146a模拟物组细胞的荧光素酶活性显著低于CARD10-mut+hsa-miR-146a模拟物组、CARD10-wt+miR-146a模拟物空白载体组以及CARD10-mut+miR-146a模拟物空白载体组(F=51.93,q=4.16~5.41,P<0.05)。结论miR-146a的高表达能够增强ERα+乳腺癌患者TAM治疗敏感性,从而提高患者的生存率。
Objective To investigate the impact of miR-146a on tamoxifen treatment sensitivity in patients with estrogen receptor alpha-positive(ERα+)breast cancer.Methods A total of 48 patients with breast cancer who underwent surgical resection in our hospital from January 2018 to December 2019 were enrolled,and cancerous tissue samples and corresponding paracancerous tissue samples were collected,as well as clinicopathological features including age,pathological classification,lymph node metastasis,immunohistochemistry results,and molecular subtyping.RT-qPCR was used to measure the level of miR-146a in the cancerous and paracancerous tissue samples of 48 patients with breast cancer,the ERα+human breast cancer cell line(MCF-7),and the human breast epithelial cell line(MCF-10A),and the Kaplan-Meier plotter online database was used to analyze the association between the expression level of miR-146a and the prognosis of patients with breast cancer.MCF-7 cells were used to construct an ERα+breast cancer tamoxifen-resistant cell line(TAM-R),and then the cells were divided into untransfected group(TAM-R group),empty vector transfection group(mimic-NC group),and hsa-miR-146a mimic transfection group(mimic group).CCK-8 assay was used to measure cell proliferation at 0,24,48,72,and 96 hours of culture,and flow cytometry was used to measure cell apoptosis rate.The online prediction tools were used to predict the target genes of miR-146a,and dual-luciferase reporter assay was used to investigate the interaction between miR-146a and predicted target genes.Results RT-qPCR showed that the expression level of miR-146a in cancerous tissue of breast cancer patients and MCF-7 cells was significantly lower than that in the correspon-ding paracancerous tissue and MCF-10A cells(t=4.02,50.47,P<0.05).The analysis of clinicopathological features showed that the expression level of miR-146a was significantly upregulated in Lumi-nal A-type,HER-2+,VEGF+,and ERα+breast cancer tissue(F=10.79,t=2.23-11.08,P<0.05).In all patients with ERα+breast cancer and those receiving endocrine therapy,the patients with high miR-146a expression had a significantly lower survival rate than those with low miR-146a expression(HR=0.58,0.66,95%CI=0.41-0.84,0.41-0.89,P<0.05).CCK-8 assay showed that the mimic group had a significantly lower absorbance value than the TAM-R and mimic-NC groups at each time point from 24-96 hours of culture(F=25.09-92.17,q=5.09-12.17,P<0.05),and flow cytometry showed that compared with the mimic-NC group,the mimic group had a significant increase in cell apoptosis rate(F=38.24,q=5.60,P<0.05).The results of online prediction tools screening showed that CARD10 was the potential target gene of miR-146a,and dual-luciferase reporter assay showed that the CARD10-wt+hsa-miR-146a mimic group had a significantly lower luciferase activity than the CARD10-mut+hsa-miR-146a mimic group,the CARD10-wt+miR-146a mimic negative control vector group,and the CARD10-mut+miR-146a mimic negative control vector group(F=51.93,q=4.16-5.41,P<0.05).Conclusion The high expression of miR-146a enhances tamoxifen treatment sensitivity in ERα+breast cancer patients,thereby improving the survival rate of patients.
作者
方晓瑞
马耀先
孙桢
申晓燕
骆许静
FANG Xiaorui;MA Yaoxian;SUN Zhen;SHEN Xiaoyan;LUO Xujing(Department of Oncology,Xuchang Central Hospital Affiliated to Henan University of Science and Technology,Xuchang 461000,China)
出处
《精准医学杂志》
2025年第6期505-510,共6页
Journal of Precision Medicine
