LncRNA NBAT1通过调控miR-3664-5p/Caspase 9分子轴对人乳腺癌细胞增殖的影响

目的 探究lncRNA NBAT1通过调控miR-3664-5p/Caspase 9分子轴对人乳腺癌细胞增殖的影响。方法 通过在线生物软件分析TCGA(The Cancer Genome Atlas)数据库比较lncRNA NBAT1在乳腺癌和正常乳腺组织中的表达量。RT-qPCR方法比较正常细胞株MCF-10A和乳腺癌细胞株MCF-7、MDA-MB-231及MDA-MB-453中lncRNA NBAT1的表达水平。生物信息学预测lncRNA NBAT1、miR-3664-5p、Caspase 9三者之间关系,并进一步通过荧光素酶报告系统进行验证;lncRNA NBAT1过表达载体转染乳腺癌MCF-7细胞株,RT-qPCR检测转染后lncRNA NBAT1的表达,qPCR检测miR-3664-5的表达,Western blot检测Caspase 9的蛋白表达水平;CCK-8和克隆形成实验检测细胞的增殖能力;同时将lncRNA NBAT1过表达载体和mimics miR-3664-5p转染MCF-7细胞,观察mimics miR-3664-5p对lncRNA NBAT1抑制细胞增殖能力产生的缓解效应。结果 TCGA数据库分析发现,lncRNA NBAT1在乳腺癌组织中的表达量显著低于正常组织(P＜0.001);qPCR检测发现lncRNA NBAT1在正常细胞株MCF-10A细胞中表达量最高(P＜0.001),MDA-MB-231和MDA-MB-453细胞中表达量比MCF-10A细胞低(P＜0.001),MCF-7细胞表达量最低(P＜0.001);通过生物信息学预测lncRNA NBAT1可以吸附miR-3664-5p,Caspase 9是miR-3664-5p的靶基因,并进一步通过荧光素酶报告系统进行验证;lncRNA NBAT1过表达载体转染乳腺癌细胞株MCF-7,qRT-PCR检测发现pc-NBAT1组比pc-NC组LncRNA NBAT1的表达增高(P＜0.001),miR-3664-5的表达降低(P＜0.001),Western blot检测发现pc-NBAT1组比pc-NC组Caspase 9的表达增高(P＜0.001)。CCK-8和克隆形成实验发现pc-NBAT1组比pc-NC组细胞的增殖能力降低(P＜0.001);同时将lncRNA NBAT1过表达载体和mimics miR-3664-5p转染MCF-7细胞,发现mimics miR-3664-5p能够对lncRNA NBAT1抑制细胞增殖能力产生缓解效应(P＜0.001)。结论 lncRNA NBAT1通过调控miR-3664-5p/Caspase 9分子轴抑制乳腺癌细胞的增殖,为乳腺癌治疗提供新的潜在靶点。

Effect of lncRNA NBAT1 on proliferation of breast cancer cells by regulating the molecular axis of miR-3664-5p/Caspase 9

Objective The aim of this study was to investigate the effect of lncRNA NBAT1 on proliferation of human breast cancer cells by regulating the molecular axis of miR-3664-5p/Caspase 9.Methods The expression of lncRNA NBAT1 in breast cancer and normal breast tissues was compared by analyzing the TCGA(The Cancer Genome Atlas)database with online biological software.qRT-PCR was used to compare the expression of lncRNA NBAT1 in MCF-10A and MCF-7,MDA-MB-231 and MDA-MB-453 cells.Bioinformatics predicted the relationship between lncRNA NBAT1,miR-3664-5p,and Caspase 9,and further verified by the luciferase reporting system.lncRNA NBAT1 overexpression vectors were transfected into MCF-7 cells,RT-qPCR was used to detect the expression of lncRNA NBAT1,qPCR was used to detect the expression of miR-3664-5,and Western blot was used to detect the expression of Caspase 9 protein.The CCK-8 assay and clone formation test were used to detect the ability of cell proliferation.At the same time,lncRNA NBAT1 overexpression vector and mimics miR-3664-5p were transfected into MCF-7 cells,mimics miR-3664-5p was observed to alleviate lncRNA NBAT1′s ability to inhibit cell proliferation.Results The TCGA database analysis found that the expression of lncRNA NBAT1 in breast cancer tissues was significantly lower than that in normal tissues(P<0.001).lncRNA NBAT1 was highest expressed in normal control MCF-10A cells(P<0.001),low expressed in MDA-MB-231 and MDA-MB-453 cells(P<0.001)and lowest expressed in MCF-7 cells(P<0.001).It was predicted by bioinformatics that lncRNA NBAT1 could absorb miR-3664-5p,and Caspase 9 was the target gene of miR-3664-5p.These were further verified by the luciferase reporting system.LncRNA NBAT1 overexpression vectors were transfected into MCF-7 cells.The expression of lncRNA NBAT1 in the NBAT1 group was higher than that in the control group(P<0.001),and the expression of miR-3664-5 was reduced in MCF-7 cells(P<0.001).The expression of Caspase 9 protein in the NBAT1 group was higher than that in the control group(P<0.001).The proliferative ability of MCF-7 cells in the NBAT1 group was lower than that of the control group(P<0.001).At the same time,MCF-7 cells were transfected with lncRNA NBAT1 plasmids and mimics miR-3664-5p,mimics miR-3664-5p could alleviate the inhibitory effect of lncRNA NBAT1 on proliferation of MCF-7 cells(P<0.001).Conclusion LncRNA NBAT1 can inhibit the proliferation of breast cancer cells by regulating the molecular axis of miR-3664-5p/Caspase 9,providing a new potential target for breast cancer treatment.