【摘要】 目的对影响乳腺癌(BRCA)预后的微小RNA(miRNA)进行探索挖掘,并探讨hsa-mir-301b作用机制。方法使用miRNA抑制剂干扰BRCA细胞中hsa-mir-301b的表达,qRT-PCR检测hsa-mir-301b的表达水平;CCK-8实验检测细胞增殖能力;miRDB数据库预测hsa-mir-301b靶基因,同时通过GEPIA网站进一步筛选出BRCA中低表达,提示预后不良的候选靶基因2个,应用ENCORI及RNAhybrid在线预测靶基因NR3C2与hsa-mir-301b的靶向关系;Western blot检测NR3C2蛋白表达水平。结果与癌旁组织相比,hsa-mir-301b在BRCA组织中显著高表达,且高表达患者预后不良;hsa-mir-301b在乳腺癌细胞系MCF-7细胞系中表达量低于正常乳腺上皮细胞MCF-10A的表达(P<0.0001),抑制hsa-mir-301b表达后,细胞活力下降。NR3C2为hsa-mir-301b的靶基因。NR3C2在BRCA患者组织中低表达,且低表达提示预后不良,并与hsa-mir-301b存在负相关关系;其3’非编码区域与hsa-mir-301b存在结合位点;Western blot实验证明hsa-mir-301b抑制剂能够促进NR3C2蛋白水平表达。结论 hsa-mir-301b在BRCA患者组织中及MCF-7细胞中高表达,发挥癌基因作用,其抑制剂能够减弱BRCA细胞MCF-7的活力,干扰NR3C2的表达。更多还原

【Abstract】 Objective To explore the miRNAs that affect the prognosis of BRCA using patients’ data,and preliminarily explore that suggested mechanism of hsa-mir-301 b. Methods The expression of hsa-mir-301 b in BRCA cells was interfered by miRNA inhibitors and detected by qRT PCR. CCK-8 assay was performed to detect cell proliferation. We used miRDB database to predict target genes of hsa-mir-301 b,and GEPIA website was used to screen out two target genes,with low expression and poor prognosis in cancer tissues. The targeting relationship between hsa-mir-301 b and NR3 C2 was predicted by online ENCORI and RNAhybrid,and the expressions of NR3 C2 were detected by Western blot. Results The expression of hsa-mir-301 b was significantly higher in BRCA tissues than in normal tissues,and patients with high expression had poor prognosis. Further hsa-mir-301 b was significantly higher in bRCA MCF-7 cells than in MCF-10 A cells of normal tissues(P<0.0001),and the cell viability decreased after inhibiting hsa-mir-301 b MR3 C2 was the target gene of hsa-mir-301 b. The expression of NR3 C2 was down-regulated in BRCA tissues,and the decreased expression was associated with poorer prognosis of BRCA patients(P<0.05). ENCORI revealed that the expression of hsa-mir-301 b was negatively correlated with the expression of NR3 C2,and the 3’noncoding region of NR3 C2 had binding sites with hsa-mir-301 b. Western blotting showed hsa-mir-301 b inhibitor could promote NR3 C2 protein expression.Conclusion Hsa-mir-301 b expression is increased in BRCA tissues and MCF-7 cells and acts as an oncogene. Its inhibitors can reduce the viability of BRCA MCF-7 cells and interfere with the expression of NR3 C2.更多还原