Tang JingTong, Gao Wei, Liu Gang, Sheng WeiWei, Zhou JianPing, Dong Qi, Dong Ming
Department of Gastrointestinal Surgery & Hernia and Abdominal Wall Surgery, The First Hospital, China Medical University, Shenyang, 110001, Liaoning, People's Republic of China.
Department of General Surgery, The People's Hospital of China Medical University, Shenyang, People's Republic of China.
Onco Targets Ther. 2021 Mar 31;14:2311-2325. doi: 10.2147/OTT.S290567. eCollection 2021.
miR-944 belongs to the MicroRNAs family, as shown in our previous study, and is essential in the colorectal cancer (CRC) progression. It is negatively associated with invasion depth and lymph node status. Epithelial-mesenchymal transition (EMT) is essential in tumor invasion and metastasis. However, the relationship between miR-944 and EMT in CRC is unknown and should be further investigated.
Epithelial-mesenchymal transition (EMT) progression in CRC cell lines was detected with Cell morphology and Western blotting. CRC cell migration and invasion were examined using Transwell assays. Transcriptome and clinical data were obtained from The Cancer Genome Atlas (TCGA) database. The potential pathway of miR-944 and GATA6 were predicted using KEGG analysis. Colocalization was validated using immunofluorescence and Immunohistochemistry. Nuclear and Cytoplasmic Protein Extraction assays were conducted to determine the effects of miR-944 on Wnt/β-catenin signaling.
We found that miR‑944 influences EGF-induced EMT malignant phenotype in vitro. KEGG analyses showed that miR-944 and GATA6 are associated with EMT related pathways, wnt signaling pathways. On the other hand, Western Blot analyses showed that miR-944 can regulate EMT and wnt-β-catenin pathway-related protein, including β-catenin, ZEB1, snail1 via GATA6 regulation. miR-944 also abrogates E-ca after EGF induction. Immunohistochemistry (IHC) and Immunofluorescence (IF) co-expression showed that GATA6 expression is positively associated with β-catenin and ZEB1. GATA6 silencing can reverse EMT malignant phenotype and alterations of related protein induced by miR-944. Quantitative polymerase chain reaction analysis results showed that miR-944 is negatively associated with the UICC stage (P= 0.02), lymph nodes (p=0.04), and liver metastasis (p=0.03). Moreover, patients with high miR-944 expression have better survival (p=0.045). We finally combined miR-944 and GATA6 and found that miR-944/GATA6 ratio could be a novel prognostic biomarker in the TCGA dataset and it is an independent risk prognosis factor (p=0.045).
Our results suggest that miR-944 suppresses the aggressive biological processes by directly repressing GATA6 expression and could be a potential candidate for therapeutic applications in CRC.
如我们之前的研究所表明，miR-944属于微小RNA家族，在结直肠癌（CRC）进展中至关重要。它与浸润深度和淋巴结状态呈负相关。上皮-间质转化（EMT）在肿瘤侵袭和转移中至关重要。然而，miR-944与CRC中EMT的关系尚不清楚，应进一步研究。
通过细胞形态学和蛋白质印迹法检测CRC细胞系中的上皮-间质转化（EMT）进展。使用Transwell试验检测CRC细胞的迁移和侵袭。从癌症基因组图谱（TCGA）数据库获取转录组和临床数据。使用KEGG分析预测miR-944和GATA6的潜在途径。使用免疫荧光和免疫组织化学验证共定位。进行细胞核和细胞质蛋白提取试验以确定miR-944对Wnt/β-连环蛋白信号传导的影响。
我们发现miR-944在体外影响表皮生长因子（EGF）诱导的EMT恶性表型。KEGG分析表明，miR-944和GATA6与EMT相关途径、Wnt信号通路相关。另一方面，蛋白质印迹分析表明，miR-944可通过GATA6调节来调控EMT和Wnt-β-连环蛋白途径相关蛋白，包括β-连环蛋白、锌指E盒结合蛋白1（ZEB1）、蜗牛蛋白1（snail1）。miR-944在EGF诱导后也可消除E-钙黏蛋白。免疫组织化学（IHC）和免疫荧光（IF）共表达表明，GATA6表达与β-连环蛋白和ZEB1呈正相关。GATA6沉默可逆转miR-944诱导的EMT恶性表型和相关蛋白的改变。定量聚合酶链反应分析结果表明，miR-944与国际抗癌联盟（UICC）分期（P = 0.02）、淋巴结（P = 0.04）和肝转移（P = 0.03）呈负相关。此外，miR-944高表达的患者生存情况更好（P = 0.045）。我们最终将miR-944和GATA6结合起来，发现miR-944/GATA6比值可能是TCGA数据集中一种新的预后生物标志物，并且它是一个独立的风险预后因素（P = 0.045）。
我们的结果表明，miR-944通过直接抑制GATA6表达来抑制侵袭性生物学过程，可能是CRC治疗应用的潜在候选物。
