2nd Department of Internal Medicine, Semmelweis University, Szentkirályi str. 46, Budapest, 1088, Hungary.
Molecular Medicine Research Unit, Hungarian Academy of Sciences, Budapest, Hungary.
BMC Cancer. 2019 Nov 6;19(1):1059. doi: 10.1186/s12885-019-6180-5.
Long non-coding RNAs (lncRNAs) play a fundamental role in colorectal cancer (CRC) development, however, lncRNA expression profiles in CRC and its precancerous stages remain to be explored. We aimed to study whole genomic lncRNA expression patterns in colorectal adenoma-carcinoma transition and to analyze the underlying functional interactions of aberrantly expressed lncRNAs.
LncRNA expression levels of colonic biopsy samples (20 CRCs, 20 adenomas (Ad), 20 healthy controls (N)) were analyzed with Human Transcriptome Array (HTA) 2.0. Expression of a subset of candidates was verified by qRT-PCR and in situ hybridization (ISH) analyses. Furthermore, in silico validation was performed on an independent HTA 2.0, on HGU133Plus 2.0 array data and on the TCGA COAD dataset. MiRNA targets of lncRNAs were predicted with miRCODE and lncBase v2 algorithms and miRNA expression was analyzed on miRNA3.0 Array data. MiRNA-mRNA target prediction was performed using miRWALK and c-Met protein levels were analyzed by immunohistochemistry. Comprehensive lncRNA-mRNA-miRNA co-expression pattern analysis was also performed.
Based on our HTA results, a subset of literature-based CRC-associated lncRNAs showed remarkable expression changes already in precancerous colonic lesions. In both Ad vs. normal and CRC vs. normal comparisons 16 lncRNAs, including downregulated LINC02023, MEG8, AC092834.1, and upregulated CCAT1, CASC19 were identified showing differential expression during early carcinogenesis that persisted until CRC formation (FDR-adjusted p < 0.05). The intersection of CRC vs. N and CRC vs. Ad comparisons defines lncRNAs characteristic of malignancy in colonic tumors, where significant downregulation of LINC01752 and overexpression of UCA1 and PCAT1 were found. Two candidates with the greatest increase in expression in the adenoma-carcinoma transition were further confirmed by qRT-PCR (UCA1, CCAT1) and by ISH (UCA1). In line with aberrant expression of certain lncRNAs in tumors, the expression of miRNA and mRNA targets showed systematic alterations. For example, UCA1 upregulation in CRC samples occurred in parallel with hsa-miR-1 downregulation, accompanied by c-Met target mRNA overexpression (p < 0.05).
The defined lncRNA sets may have a regulatory role in the colorectal adenoma-carcinoma transition. A subset of CRC-associated lncRNAs showed significantly differential expression in precancerous samples, raising the possibility of developing adenoma-specific markers for early detection of colonic lesions.
长非编码 RNA（lncRNA）在结直肠癌（CRC）的发展中起着至关重要的作用，然而，CRC 及其癌前阶段的 lncRNA 表达谱仍有待探索。我们旨在研究结直肠腺瘤-癌转变过程中的全基因组 lncRNA 表达模式，并分析异常表达的 lncRNA 的潜在功能相互作用。
使用人类转录组阵列（HTA）2.0 分析结肠活检样本（20 例 CRC、20 例腺瘤（Ad）、20 例健康对照（N））的 lncRNA 表达水平。通过 qRT-PCR 和原位杂交（ISH）分析验证候选物的表达。此外，还在独立的 HTA 2.0、HGU133Plus 2.0 阵列数据和 TCGA COAD 数据集上进行了计算验证。使用 miRCODE 和 lncBase v2 算法预测 lncRNA 的 miRNA 靶标，并在 miRNA3.0 阵列数据上分析 miRNA 的表达。使用 miRWALK 进行 miRNA-mRNA 靶标预测，并通过免疫组织化学分析 c-Met 蛋白水平。还进行了全面的 lncRNA-mRNA-miRNA 共表达模式分析。
基于我们的 HTA 结果，一组基于文献的 CRC 相关 lncRNA 在癌前结肠病变中已经表现出显著的表达变化。在 Ad 与正常和 CRC 与正常的比较中，包括下调的 LINC02023、MEG8、AC092834.1 和上调的 CCAT1、CASC19，在内的 16 个 lncRNA 被鉴定为在早期癌变过程中表现出差异表达，这种表达模式一直持续到 CRC 形成（经 FDR 调整的 p<0.05）。CRC 与 N 和 CRC 与 Ad 比较的交集定义了在结肠肿瘤中具有恶性特征的 lncRNA，其中 LINC01752 的显著下调和 UCA1 和 PCAT1 的过表达被发现。在腺瘤-癌转变中表达增加最大的两个候选物通过 qRT-PCR（UCA1、CCAT1）和 ISH（UCA1）进一步得到证实。与肿瘤中某些 lncRNA 的异常表达一致，miRNA 和 mRNA 靶标的表达显示出系统的改变。例如，CRC 样本中 UCA1 的上调与 hsa-miR-1 的下调平行，伴有 c-Met 靶 mRNA 的过表达（p<0.05）。
所定义的 lncRNA 集可能在结直肠腺瘤-癌转变中具有调节作用。一组与 CRC 相关的 lncRNA 在癌前样本中表现出显著的差异表达，这为开发用于早期检测结肠病变的腺瘤特异性标志物提供了可能性。
