Department of General Surgery, Beijing Chao-Yang Hospital, Capital Medical University, Beijing 100020, China.
Department of Colorectal and Anal Surgery, Beijing Rectum Hospital, Beijing 100120, China.
Dis Markers. 2022 Jul 21;2022:4433270. doi: 10.1155/2022/4433270. eCollection 2022.
Alternative splicing (AS) plays a crucial role in regulating the progression of colorectal cancer (CRC), but its distribution remains to be explored. Here, we aim to investigate the genes edited by AS which show differential expression in patients with mismatch repair deficiency (dMMR)/microsatellite instability (MSI).
We applied long-read nanopore sequencing to determine the mRNA profiles and screen AS genes using Oxford Nanopore Technologies (ONT) method in ten paired CRC tissues. CRC tissue and plasma samples were used to validate the differential genes with AS using real-time fluorescent quantitative PCR, immunohistochemistry, and enzyme-linked immunosorbent assay.
ONT sequencing identified 404 genes were downregulated, and 348 genes were upregulated in MSI cancer tissues compared with microsatellite stability (MSS) cancer tissues. In total, 6,200 AS events were identified in 2,728 mRNA transcripts. WGCNA revealed dMMR/MSI-correlated gene modules, including INHBA and RPL22L1, which were upregulated; conversely, HMGCS2 was downregulated in MSI cancer. Overexpression of RPL22L1, INHBA, and CAPZA1 was further confirmed in CRC tissues. INHBA was found to be associated with tumor lymphatic metastasis. Importantly, the levels of INHBA in CRC plasma were significantly increased compared with those in noncancer plasma. INHBA showed a higher level in dMMR/MSI CRC than in MSS CRC, indicating that INHBA is a useful biomarker.
Our results showed that ONT-identified genes provide a pool to explore AS-associated markers for dMMR/MSI CRC. We demonstrated INHBA as a promising signature for clinical application in predicting tumor lymphatic metastasis and screening dMMR/MSI candidates.
选择性剪接（AS）在调控结直肠癌（CRC）的进展中起着至关重要的作用，但它的分布仍有待探索。在这里，我们旨在研究在错配修复缺陷（dMMR）/微卫星不稳定（MSI）患者中差异表达的、由 AS 编辑的基因。
我们应用长读长纳米孔测序来确定 mRNA 图谱，并使用牛津纳米孔技术（ONT）方法在 10 对 CRC 组织中筛选 AS 基因。使用实时荧光定量 PCR、免疫组织化学和酶联免疫吸附试验，用 CRC 组织和血浆样本验证具有 AS 的差异基因。
ONT 测序确定，与微卫星稳定（MSS）癌症组织相比，MSI 癌症组织中 404 个基因下调，348 个基因上调。总共在 2728 个 mRNA 转录本中鉴定了 6200 个 AS 事件。WGCNA 揭示了 dMMR/MSI 相关的基因模块，包括 INHBA 和 RPL22L1 上调，而 HMGCS2 下调。在 CRC 组织中进一步证实了 RPL22L1、INHBA 和 CAPZA1 的过表达。发现 INHBA 与肿瘤淋巴转移有关。重要的是，CRC 血浆中的 INHBA 水平明显高于非癌血浆。INHBA 在 dMMR/MSI CRC 中的水平高于 MSS CRC，表明 INHBA 是一种有用的生物标志物。
我们的结果表明，ONT 鉴定的基因提供了一个探索用于 dMMR/MSI CRC 的 AS 相关标记的库。我们证明了 INHBA 作为预测肿瘤淋巴转移和筛选 dMMR/MSI 候选物的有前途的标志物。
