背景:结直肠癌(CRC)是全球第三大致命和第四大诊断癌症。尽管在早期诊断和先进的治疗选择方面取得了进展,CRC显示预后不良,5年生存率为〜45%。PRDM2/RIZ,PR/SET域家族(PRDM)的成员,表达两个主要的分子变异,PR+同工型(RIZ1)和PR-负型(RIZ2)。在许多癌症类型中观察到它们的表达水平的不平衡有利于RIZ2。全长RIZ1已经在几种癌症中被广泛研究,它作为肿瘤抑制因子,而很少有研究探索RIZ2致癌特性。PRDM2通常是CRC中移码突变和异常DNA甲基化的靶标。然而,对其在CRC中的作用知之甚少。
方法:我们结合了癌症基因组图谱(TCGA)CRC数据集的计算机调查,细胞和分子分析,转录组测序和功能注释分析评估RIZ2在人类CRC中的作用。
结果:我们对TCGA数据集的计算机分析证实,PRDM2基因在CRC中经常发生突变和转录失调,并揭示RIZ2增加与RIZ1显著下调高度相关。然后,我们通过qRT-PCR分析了几种CRC细胞系的主要PRDM2转录本和选择的DLD1细胞系,显示最低的RIZ2水平。因此,我们在这些细胞中过表达RIZ2,以模拟TCGA数据集分析结果,从而评估PRDM2/RIZ2在CRC中的作用.来自RNA-seq的数据显示,RIZ2过表达通过EGF途径失调诱导CRC细胞转录组的深刻变化,提示RIZ2参与EGF自分泌调节DLD1细胞行为。值得注意的是,强制RIZ2表达增加了细胞活力,增长,菌落形成,迁移和类器官形成。这些作用可以通过过表达RIZ2的DLD1细胞释放高EGF水平来介导。
结论:我们的发现为CRC中推定的RIZ2肿瘤促进功能增加了新的见解,尽管需要额外的努力来定义潜在的分子机制。
Colorectal cancer (CRC) is the third most deadly and fourth most diagnosed cancer worldwide. Despite the progress in early diagnosis and advanced therapeutic options, CRC shows a poor prognosis with a 5 year survival rate of ~ 45%. PRDM2/RIZ, a member of PR/SET domain family (PRDM), expresses two main molecular variants, the PR-plus isoform (RIZ1) and the PR-minus (RIZ2). The imbalance in their expression levels in favor of RIZ2 is observed in many cancer types. The full length RIZ1 has been extensively investigated in several cancers where it acts as a tumor suppressor, whereas few studies have explored the RIZ2 oncogenic properties. PRDM2 is often target of frameshift mutations and aberrant DNA methylation in CRC. However, little is known about its role in CRC.
We combined in-silico investigation of The Cancer Genome Atlas (TCGA) CRC datasets, cellular and molecular assays, transcriptome sequencing and functional annotation analysis to assess the role of RIZ2 in human CRC.
Our in-silico analysis on TCGA datasets confirmed that PRDM2 gene is frequently mutated and transcriptionally deregulated in CRC and revealed that a RIZ2 increase is highly correlated with a significant RIZ1 downregulation. Then, we assayed several CRC cell lines by qRT-PCR analysis for the main PRDM2 transcripts and selected DLD1 cell line, which showed the lowest RIZ2 levels. Therefore, we overexpressed RIZ2 in these cells to mimic TCGA datasets analysis results and consequently to assess the PRDM2/RIZ2 role in CRC. Data from RNA-seq disclosed that RIZ2 overexpression induced profound changes in CRC cell transcriptome via EGF pathway deregulation, suggesting that RIZ2 is involved in the EGF autocrine regulation of DLD1 cell behavior. Noteworthy, the forced RIZ2 expression increased cell viability, growth, colony formation, migration and organoid formation. These effects could be mediated by the release of high EGF levels by RIZ2 overexpressing DLD1 cells.
Our findings add novel insights on the putative RIZ2 tumor-promoting functions in CRC, although additional efforts are warranted to define the underlying molecular mechanism.