目的:本研究将集中于4T1细胞,小鼠乳腺腺癌细胞系,作为主要研究课题。目的探讨普萘洛尔对乳腺癌细胞上皮间质转化(EMT)的抑制作用及其机制。旨在在miRNA水平上阐明这一现象。
方法:在本研究中,通过体外和动物实验观察普萘洛尔对EMT的抑制作用。为了筛选潜在的靶miRNA和下游靶基因,进行了第二代测序(SGS)和生物信息学分析。在筛选过程之后,使用各种实验方法确认鉴定的靶miRNA及其各自的靶基因。为了确认靶miRNA和靶基因,西部印迹(WB),逆转录聚合酶链反应(RT-PCR),和免疫荧光实验。
结果:在这项研究中,我们发现普萘洛尔显著降低4T1小鼠乳腺癌细胞的肺转移(p<0.05)。体外和体内实验表明,普萘洛尔抑制上皮-间质转化(EMT),如WesternBlot分析所证明的(p<0.05)。通过下一代测序(SGS),随后的生物信息学分析,和PCR验证,我们发现miR-499-5p显著下调(p<0.05),提示其可能参与介导普萘洛尔对EMT的抑制作用。miR-499-5p过表达促进EMT,迁移,和4T1细胞的侵袭,普萘洛尔没有逆转或减弱这些作用(通过WesternBlot验证,伤口愈合试验,Transwell迁移,和入侵检测,p<0.05)。Sox6被鉴定为miR-499-5p的功能靶标,其下调与观察到的EMT变化相关(p<0.05)。沉默Sox6或过表达miR-499-5p抑制Sox6表达,进一步推进EMT进程,入侵,并在4T1细胞中迁移。值得注意的是,普萘洛尔不能缓解这些影响(通过WesternBlot验证,伤口愈合试验,Transwell迁移,和入侵检测,p<0.05)。miR-499-5p与Sox6mRNA之间的直接相互作用通过双荧光素酶报告基因测定得到证实。
结论:这些结果表明,普萘洛尔通过靶向EMT及其调节机制,可能具有作为乳腺癌治疗药物的潜力。
OBJECTIVE: This study will focus on 4T1 cells, a murine mammary adenocarcinoma cell line, as the primary research subject. We aim to investigate the inhibitory effects and mechanisms of propranolol on epithelial-mesenchymal transition (EMT) in breast cancer cells, aiming to elucidate this phenomenon at the miRNA level.
METHODS: In this study, the EMT inhibitory effect of propranolol was observed through in vitro and animal experiments. For the screening of potential target miRNAs and downstream target genes, second-generation sequencing (SGS) and bioinformatics analysis were conducted. Following the screening process, the identified target miRNAs and their respective target genes were confirmed using various experimental methods. To confirm the target miRNAs and target genes, Western Blot (WB), reverse transcription polymerase chain reaction (RT-PCR), and immunofluorescence experiments were performed.
RESULTS: In this study, we found that propranolol significantly reduced lung metastasis in 4T1 murine breast cancer cells (p < 0.05). In vitro and in vivo experiments demonstrated that propranolol inhibited the epithelial-mesenchymal transition (EMT) as evidenced by Western Blot analysis (p < 0.05). Through next-generation sequencing (SGS), subsequent bioinformatics analysis, and PCR validation, we identified a marked downregulation of miR-499-5p (p < 0.05), suggesting its potential involvement in mediating the suppressive effects of propranolol on EMT. Overexpression of miR-499-5p promoted EMT, migration, and invasion of 4T1 cells, and these effects were not reversed or attenuated by propranolol (Validated via Western Blot, wound healing assay, transwell migration, and invasion assays, p < 0.05). Sox6 was identified as a functional target of miR-499-5p, with its downregulation correlating with the observed EMT changes (p < 0.05). Silencing Sox6 or overexpressing miR-499-5p inhibited Sox6 expression, further promoting the processes of EMT, invasion, and migration in 4T1 cells. Notably, these effects were not alleviated by propranolol (validated via Western Blot, wound healing assay, transwell migration, and invasion assays, p < 0.05). The direct interaction between miR-499-5p and Sox6 mRNA was confirmed by dual-luciferase reporter gene assay.
CONCLUSIONS: These results suggest that propranolol may have potential as a therapeutic agent for breast cancer treatment by targeting EMT and its regulatory mechanisms.