我们先前报道了B细胞特异性莫洛尼鼠白血病病毒整合位点1(Bmi-1)和Raf激酶抑制蛋白(RKIP)之间的反比关系,这与胃癌(GC)的预后有关。在这项研究中,我们进一步探索了微小RNA(miRNA)在Bmi-1和RKIP之间的调控机制。
首先进行微阵列分析以鉴定在过表达Bmi-1的细胞中差异表达的miRNA谱。然后,鉴定了可以调节RKIP的miRNA。进行定量实时PCR(qRT-PCR)和Western印迹以测量Bmi-1、miR-155、miR-27a和RKIP的表达。RKIP通过荧光素酶报告基因测定被确认为miR-27a和miR-155的靶标,qRT-PCR和Western印迹。Bmi-1/miR-27a/RKIP和Bmi-1/miR-155/RKIP轴对肿瘤生长的影响,扩散,迁移,入侵,集落形成能力,在体外和体内研究了转移和化学耐药性。
Bmi-1对RKIP的下调发生在蛋白质水平而不是mRNA水平。这表明可能的转录后调控。分析具有Bmi-1异位表达的细胞的miRNA表达谱,并通过微阵列分析与对照细胞的miRNA表达谱进行比较。鉴定了总共51个上调的miRNA和72个下调的miRNA。基于公开可用的算法,预测miR-27a和miR-155,选择并展示目标RKIP。Bmi-1、miR-27a和miR-155在人GC中升高,并与GC的不良预后相关。而RKIP在GC中表达水平较低,与预后良好相关。然后,体外试验表明,除了通过miR-27a和miR-155调节RKIP表达外,Bmi-1还能够调节迁移,入侵,扩散,GC细胞的集落形成能力和化学敏感性通过相同的途径。最后,体内试验结果相似,Bmi-1基因的敲除导致肿瘤生长的抑制,通过miR-27a和miR-155的转移和化学抗性。
Bmi-1被证明通过在GC中靶向RKIP来诱导miR-27a和miR-155的表达,从而促进肿瘤转移和化学抗性。总的来说,miR-27a和miR-155可能是筛选的有希望的靶标,诊断,预后,GC的治疗和疾病监测。
We previously reported an inverse relationship between B cell-specific Moloney murine leukemia virus integration site 1 (Bmi-1) and Raf kinase inhibitory protein (
RKIP), which is associated with the prognosis of gastric cancer (GC). In this study, we further explored the microRNA (miRNA) regulatory mechanism between Bmi-1 and
RKIP.
Microarray analysis was first carried out to identify miRNA profiles that were differentially expressed in cells overexpressing Bmi-1. Then, miRNAs that could regulate
RKIP were identified. Quantitative real-time PCR (qRT-PCR) and Western blotting were performed to measure the expression of Bmi-1, miR-155, miR-27a and RKIP. RKIP was confirmed as a target of miR-27a and miR-155 through luciferase reporter assays, qRT-PCR and Western blotting. The effects of the Bmi-1/miR-27a/
RKIP and Bmi-1/miR-155/
RKIP axes on tumor growth, proliferation, migration, invasion, colony-formation ability, metastasis and chemoresistance were investigated both in vitro and in vivo.
The downregulation of
RKIP by Bmi-1 occurred at the protein but not mRNA level. This indicates probable posttranscriptional regulation. miRNA expression profiles of cells with ectopic expression of Bmi-1 were analyzed and compared to those of control cells by microarray analysis. A total of 51 upregulated and 72 downregulated miRNAs were identified. Based on publicly available algorithms, miR-27a and miR-155 were predicted, selected and demonstrated to target RKIP. Bmi-1, miR-27a and miR-155 are elevated in human GC and associated with poor prognosis of GC, while RKIP is expressed at lower levels in GC and correlated with good prognosis. Then, in vitro tests shown that in addition to regulating RKIP expression via miR-27a and miR-155, Bmi-1 was also able to regulate the migration, invasion, proliferation, colony-formation ability and chemosensitivity of GC cells through the same pathway. Finally, the in vivo test showed similar results, whereby the knockdown of the Bmi-1 gene led to the inhibition of tumor growth, metastasis and chemoresistance through miR-27a and miR-155.
Bmi-1 was proven to induce the expression of miR-27a and miR-155 and thus promote tumor metastasis and chemoresistance by targeting RKIP in GC. Overall, miR-27a and miR-155 might be promising targets for the screening, diagnosis, prognosis, treatment and disease monitoring of GC.