ING4/miR-940/Snail1途径调控结直肠癌EMT和转移的作用及机制

Previous studies have shown that inhibitor of growth 4 (ING4) as an important member of inhibitor of growth protein family is reduced in human colorectal cancer (CRC), which is closely associated with clinicopathological features. However, the role of ING4 in CRC metastasis and its mechanism is still poorly understood. Our preliminary studies have demonstrated that the lower expression of ING4 in CRC promotes its invasion and metastasis. ING4 inhibits CRC metastasis very probably via reversal of epithelial-mesenchymal transition (EMT) through downregulation of Snail1 EMT-inducing transcription factor (EMT-TF). We also screened and validated that ING4 is capable of upregulating miR-940 targeting Snail1 in CRC cells. To this end, we propose the hypothesis that ING4 would downregulate Snail1 in CRC cells via the miR-940/Snail1 pathway, which may be an important regulatory mechanism for ING4-triggered suppression of EMT and metastasis in CRC. To verify the above hypothesis, we established the ING4-overexpressed and the ING4-knocked down CRC transgenic cells by using lentivirus-mediated transfection. We then examined the effect of ING4 overexpression or knockdown on CRC EMT and metastasis in in vitro cell model and in vivo tumor xenograft model in athymic BALB/c nude mouse by using the assays of wounding healing, Transwell chamber migration/invasion and in vivo bioluminescence image. We sought to elucidate the underlying mechanism, whether ING4-mediated regulation of miR-940/Snail1 signaling axis is a vital mechanism for ING4-invovled modulation the invasion and metastasis of CRC, by using the assays of Western blot, immunohistochemistry (IHC), in situ hybridization, qRT-PCR, luciferase reporter, agomir/antagomir, siRNA and so on. Furthermore, we analyzed the relationship of ING4, miR-940 and Snail1 in CRC clinical tissue specimens and then evaluated the correlation between their expression and clinicopathological characteristics or prognosis of CRC patients. This project is likely to provide the new ideas for the prevention and therapy of CRC metastasis.

研究显示ING4在结直肠癌（CRC）中表达降低，与临床病理因素密切相关。然而ING4对CRC转移的作用及其机制仍知之甚少。课题组前期研究发现：ING4在CRC中低表达促进CRC的侵袭转移，ING4可能通过下调Snail1参与抑制CRC EMT、转移，筛选验证ING4能上调靶基因Snail1的miR-940。为此我们提出假设：ING4通过miR-940/Snail1途径下调Snail1可能是ING4抑制CRC EMT、转移的重要分子调控机制。为验证该假说，本项目将构建慢病毒介导的ING4过表达、沉默CRC细胞株，采用迁移、侵袭、Western blot、免疫组化、原位杂交、qRT-PCR、荧光素酶报告基因、miRNA激动/拮抗、siRNA、动物活体成像等方法，在细胞、动物、临床标本水平探讨ING4/miR-940/Snail1途径调控CRC EMT、转移的作用，为CRC转移的防治提供新思路。

本研究我们发现，生长抑制因子4（ING4）ING4在结直肠癌（CRC）组织和细胞株中的表达水平降低。CRC组织中ING4的表达水平与CRC患者的肿瘤大小、淋巴结转移和TNM分期等恶性临床病理特征呈负相关。低的ING4表达水平与CRC患者的不良预后也密切相关。建立ING4过表达、敲减的CRC转基因细胞株后，通过体外细胞模型和体内肿瘤异种移植裸鼠模型的生物学功能实验发现，ING4过表达抑制了CRC细胞的迁移、侵袭和远处肺转移能力；而ING4敲减则促进了CRC细胞的上述生物学行为。基因表达谱芯片高通量筛选和RT-qPCR、Western blot验证发现，ING4能下调CRC细胞中Snail1的表达和逆转CRC的EMT表型。重要的是，敲减Snail1在很大程度上抵消了ING4缺失而促进CRC EMT和转移的作用。结果表明，ING4抑制CRC EMT、转移的作用依赖于ING4介导的Snail1下调。miRNA表达谱芯片高通量筛选、数据库TargetScan、PITA、microRNAorg预测靶基因为Snail1相关miRNA、RT-qPCR验证及Snail1 3’UTR荧光素酶报告基因实验发现，ING4能上调CRC细胞中靶基因为Snail1的miR-940，参与ING4对Snail1的负性调控。CRC组织中ING4、miR-940、Snail1三者表达的临床相关性分析发现，CRC组织中ING4与Snail1的表达水平呈负相关，而与miR-940的表达呈正相关；miR-940与Snail1的表达水平呈负相关。因而，本研究揭示了ING4 通过miR-940/Snail1途径下调 Snail1抑制 CRC EMT、转移的新机制，为CRC转移的防治提供了新视角。