miR-365a-3p靶向调控JAK-STAT信号通路抑制三阴性乳腺癌细胞的恶性生物行为研究

miR-365a-3p suppresses malignant biological behaviors of triple negative breast cancer cells by targeted-regulating JAK-STAT signal pathway

目的针对三阴性乳腺癌(TNBC)探究微小RNA(miRNA)-365a-3p对其恶性生物行为的影响并分析潜在的分子调控机制。方法选取2018年6月至2019年12月该院收治的行外科手术切除治疗的TNBC患者50例作为研究对象,收集TNBC组织和癌旁组织,并统计TNBC患者的肿瘤相关临床资料。采用实时荧光定量聚合酶链反应检测miR-365a-3p在组织和乳腺癌细胞中的表达水平,将miR-365a-3p模拟物(mimic)及阴性对照(NC)mimic转染至MDA-MB-453细胞后分别通过CCK-8实验、克隆平板形成、Transwell和划痕实验检测TNBC细胞的恶性生物行为。采用双荧光素酶报告基因实验预测miR-365a-3p靶通路,并对细胞质双面蛋白酪氨酸激酶(JAK)和信号传导及转录激活因子(STAT)蛋白进行定量检测。结果TNBC组织及乳腺癌细胞系中miR-365a-3p表达均明显降低,差异有统计学意义(P<0.05);且肿瘤越大、Ki67表达水平越高、浸润范围越深、存在淋巴结转移及TNM分期越差者miR-365a-3p表达水平越低,差异均有统计学意义(P<0.05)。同时转染miR-365a-3p mimic后能显著抑制MDA-MB-468细胞株增殖、集落形成、迁移及侵袭能力。JAK与miR-365a-3p存在相同位点,且JAK-WT+miR-365a-3p共转染组荧光素酶活性与JAK-MUT+miR-365a-3p共转染组比较,差异有统计学意义(P<0.05);此外miR-365a-3p mimics明显抑制JAK、STAT蛋白水平,差异有统计学意义(P<0.05)。结论miR-365a-3p与TNBC肿瘤不良进展有关,并通过抑制JAK-STAT信号通路抑制TNBC细胞的恶性生物行为。

Objective To explore the effect of microRNA(miR)-365a-3p on the malignant biological behavior of triple negative breast cancer(TNBC)cells,and to analyze its potential molecular regulatory mechanisms.Methods Fifty cases of TNBC treated by operation in this hospital from June 2018 to December 2019 were selected as the study subjects.The TNBC tissues and paracancerous tissues were collected,and the tumor related clinical data were statistically analyzed.The expression level of miR-365a-3p in tissues and TNBC cells was detected by real-time fluorescent quantitative PCR.After transfection of miR-365a-3p mimics and negative control(NC)mimics into MDA-MB-468 cells,the malignant biological behaviors of TNBC cells were detected by the CCK-8 test,clone plate formation,Transwell and scratch test,respectively.The double luciferase reporter gene assay was adopted to predict miR-365a-3p target pathway,and the Janus Kinase(JAK)and signal transducers and activators of transcription(STAT)protein were quantitatively detected.Results The expression of miR-365a-3p in the TNBC tissues and breast cancer cell lines was significantly reduced with statistical difference(P<0.01),moreover the bigger the tumor,the higher the Ki67 expression level,the deeper the invasion range,the lower the miR-365a-3p lever,in the patients with lymph node metastasis and poor TNM stage,and the differences were statistically significant(P<0.05).At the same time,the transfection with miR-365a-3p mimic could significantly inhibit the proliferation,colony formation,migration and invasion abilityof MDA-MB-468 cell lines.The same site between JAK and miR-365a-3p existed,moreover the luciferase activity of the JAK-WT+miR-365a-3p co-transfection group was statistically different from that of the JAK-MUT+miR-365a-3p co-transfection group,and the difference was statistically significnt(P<0.01).In addition,miR-365a-3p mimics significantly inhibited the levels of JAK and STAT proteins,and the difference was statistically significant(P<0.01).Conclusion miR-365a-3p is related to the adverse tumor progression of TNBC and inhibits the malignant biological behavior of TNBC cells by inhibiting the JAK-STAT signaling pathway.