Department of Gastroenterology, The Fourth Affiliated Hospital of Kunming Medical University, Kunming 650021, Yunnan Province, China.
Department of Clinical Laboratory, The Geriatrics Hospital of Yunnan Province, Kunming 650011, Yunnan Province, China.
World J Gastroenterol. 2020 Feb 14;26(6):627-644. doi: 10.3748/wjg.v26.i6.627.
Colorectal cancer (CRC) is one of the most common malignancies worldwide.
To explore the expression of microRNA miR-19a-3p and Forkhead box F2 (FOXF2) in patients with CRC and the relevant mechanisms.
Sixty-two CRC patients admitted to the hospital were enrolled into the study group, and sixty healthy people from the same period were assigned to the control group. Elbow venous blood was sampled from the patients and healthy individuals, and blood serum was saved for later analysis. MiR-19a-3p mimics, miR-19a-3p inhibitor, miR-negative control, small interfering-FOXF2, and short hairpin-FOXF2 were transfected into HT29 and HCT116 cells. Then quantitative polymerase chain reaction was performed to quantify the expression of miR-19a-3p and FOXF2 in HT29 and HCT116 cells, and western blot (WB) analysis was conducted to evaluate the levels of FOXF2, glycogen synthase kinase 3 beta (GSK-3β), phosphorylated GSK-3β (p-GSK-3β), β-catenin, p-β-catenin, α-catenin, N-cadherin, E-cadherin, and vimentin. The MTT, Transwell, and wound healing assays were applied to analyze cell proliferation, invasion, and migration, respectively, and the dual luciferase reporter assay was used to determine the correlation of miR-19a-3p with FOXF2.
The patients showed high serum levels of miR-19a-3p and low levels of FOXF2, and the area under the curves of miR-19a-3p and FOXF2 were larger than 0.8. MiR-19a-3p and FOXF2 were related to sex, tumor size, age, tumor-node-metastasis staging, lymph node metastasis, and differentiation of CRC patients. Silencing of miR-19a-3p and overexpression of FOXF2 suppressed the epithelial-mesenchymal transition, invasion, migration, and proliferation of cells. WB analysis revealed that silencing of miR-19a-3p and FOXF2 overexpression significantly suppressed the expression of p-GSK-3β, β-catenin, N-cadherin, and vimentin; and increased the levels of GSK-3β, p-β-catenin, α-catenin, and E-cadherin. The dual luciferase reporter assay confirmed that there was a targeted correlation of miR-19a-3p with FOXF2. In addition, a rescue experiment revealed that there were no differences in cell proliferation, invasion, and migration in HT29 and HCT116 cells co-transfected with miR-19a-3p-mimics+sh-FOXF2 and miR-19a-3p-inhibitor+si-FOXF2 compared to the miR-negative control group.
Inhibiting miR-19a-3p expression can upregulate the FOXF2-mediated Wnt/β-catenin signaling pathway, thereby affecting the epithelial-mesenchymal transition, proliferation, invasion, and migration of cells. Thus, miR-19a-3p is likely to be a therapeutic target in CRC.
结直肠癌（CRC）是全球最常见的恶性肿瘤之一。
探讨微小 RNA miR-19a-3p 和叉头框 F2（FOXF2）在 CRC 患者中的表达及其相关机制。
纳入 62 例医院收治的 CRC 患者作为研究组，同期纳入 60 名健康人作为对照组。采集患者和健康人的肘部静脉血样，并保存血清以备后续分析。将 miR-19a-3p 模拟物、miR-19a-3p 抑制剂、miR 阴性对照、小干扰 RNA-FOXF2 和短发夹 RNA-FOXF2 转染至 HT29 和 HCT116 细胞中。然后进行定量聚合酶链反应以量化 HT29 和 HCT116 细胞中 miR-19a-3p 和 FOXF2 的表达，并进行 Western blot（WB）分析以评估 FOXF2、糖原合成酶激酶 3β（GSK-3β）、磷酸化 GSK-3β（p-GSK-3β）、β-连环蛋白、p-β-连环蛋白、α-连环蛋白、N-钙黏蛋白、E-钙黏蛋白和波形蛋白的水平。分别采用 MTT、Transwell 和划痕愈合实验分析细胞增殖、侵袭和迁移，双荧光素酶报告基因实验用于确定 miR-19a-3p 与 FOXF2 的相关性。
患者血清中 miR-19a-3p 水平升高，FOXF2 水平降低，miR-19a-3p 和 FOXF2 的曲线下面积均大于 0.8。miR-19a-3p 和 FOXF2 与 CRC 患者的性别、肿瘤大小、年龄、肿瘤-淋巴结-转移分期、淋巴结转移和分化有关。沉默 miR-19a-3p 和过表达 FOXF2 抑制了细胞的上皮-间充质转化、侵袭、迁移和增殖。WB 分析显示，沉默 miR-19a-3p 和过表达 FOXF2 显著抑制了 p-GSK-3β、β-连环蛋白、N-钙黏蛋白和波形蛋白的表达，并增加了 GSK-3β、p-β-连环蛋白、α-连环蛋白和 E-钙黏蛋白的水平。双荧光素酶报告基因实验证实 miR-19a-3p 与 FOXF2 之间存在靶向相关性。此外，在 HT29 和 HCT116 细胞中转染 miR-19a-3p 模拟物+sh-FOXF2 和 miR-19a-3p 抑制剂+si-FOXF2 后，细胞增殖、侵袭和迁移与 miR-阴性对照组相比没有差异。
抑制 miR-19a-3p 的表达可以上调 FOXF2 介导的 Wnt/β-连环蛋白信号通路，从而影响细胞的上皮-间充质转化、增殖、侵袭和迁移。因此，miR-19a-3p 可能是 CRC 的治疗靶点。
