Department of General Surgery, The Second Affiliated Hospital of Fujian Medical University, Quanzhou, 362000, Fujian, China.
Cell Biol Int. 2020 May;44(5):1224-1236. doi: 10.1002/cbin.11323. Epub 2020 Mar 12.
This work aimed to investigate miR-93-5p expression in tumor tissue and its in vitro effects in colorectal cancer (CRC) by targeting programmed death ligand-1 (PD-L1). MiR-93-5p and PD-L1 expression was detected in CRC and adjacent normal tissues by quantitative real-time polymerase chain reaction and immunohistochemistry. The correlation between miR-93-5p and PD-L1 was validated by a dual-luciferase reporter assay. HCT116 and SW480 cells were divided into blank, miR-NC, miR-93-5p mimics, miR-93-5p inhibitor, PD-L1 small interfering RNA (siRNA) and miR-93-5p inhibitor + PD-L1 siRNA groups, and wound-healing and transwell assays were performed to detect cell migration and invasion, respectively. Protein expression was measured by western blotting. The secretion of cytokines was detected in the CRC cell/T coculture models. MiR-93-5p was downregulated in CRC tissues with upregulated PD-L1. In PD-L1-negative patients, miR-93-5p expression was increased compared with that in PD-L1-positive patients. MiR-93-5p and PD-L1 expression levels were associated with the tumor differentiation, lymphatic metastasis, TNM, Duke's stage, and prognosis of CRC. PD-L1 siRNA weakened the migration and invasion abilities via decreased expression of matrix metalloproteinase-1 (MMP-1), -2, and -9, and these effects were abolished by the miR-93-5p inhibitor. Additionally, anti-PD-L1 upregulated the expressions of interleukin-2 (IL-2), tumor necrosis factor-α (TNF-α), and interferon γ (IFN-γ) in the coculture of T cells with CRC cells, but downregulated the expressions of IL-1β, IL-10, and TGF-β. However, these changes were partially reversed by miR-93-5p inhibition. miR-93-5p is expected to be a novel target for CRC treatment since it decreases the migration and invasion, as well as the immune evasion, of CRC cells via targeting PD-L1.
本研究旨在通过靶向程序性死亡配体 1（PD-L1）来检测肿瘤组织中 miR-93-5p 的表达及其对结直肠癌（CRC）的体外作用。通过实时定量聚合酶链反应和免疫组织化学检测 CRC 组织和相邻正常组织中 miR-93-5p 和 PD-L1 的表达。通过双荧光素酶报告基因实验验证 miR-93-5p 与 PD-L1 的相关性。将 HCT116 和 SW480 细胞分为空白组、miR-NC 组、miR-93-5p 模拟物组、miR-93-5p 抑制剂组、PD-L1 小干扰 RNA（siRNA）组和 miR-93-5p 抑制剂+PD-L1 siRNA 组，分别通过划痕愈合和 Transwell 实验检测细胞迁移和侵袭，通过 Western blot 检测蛋白表达，通过 CRC 细胞/T 细胞共培养模型检测细胞因子分泌。miR-93-5p 在 CRC 组织中下调，同时 PD-L1 上调。在 PD-L1 阴性患者中，miR-93-5p 的表达水平高于 PD-L1 阳性患者。miR-93-5p 和 PD-L1 的表达水平与 CRC 的肿瘤分化、淋巴转移、TNM、杜克分期和预后相关。PD-L1 siRNA 通过降低基质金属蛋白酶-1（MMP-1）、-2 和 -9 的表达来减弱迁移和侵袭能力，而 miR-93-5p 抑制剂则消除了这些作用。此外，抗 PD-L1 在 T 细胞与 CRC 细胞共培养中上调白细胞介素-2（IL-2）、肿瘤坏死因子-α（TNF-α）和干扰素-γ（IFN-γ）的表达，但下调白细胞介素-1β（IL-1β）、白细胞介素-10（IL-10）和转化生长因子-β（TGF-β）的表达。然而，miR-93-5p 抑制部分逆转了这些变化。miR-93-5p 通过靶向 PD-L1 降低 CRC 细胞的迁移和侵袭以及免疫逃逸，有望成为 CRC 治疗的新靶点。
