Yin Juan, Ye Yu-Lan, Hu Tong, Xu Li-Juan, Zhang Li-Ping, Ji Ru-Ning, Li Ping, Chen Qian, Zhu Jian-Yun, Pang Zhi
Department of Digestive Disease and Nutrition Research Center, the Affiliated Suzhou Hospital of Nanjing Medical University, Suzhou 215008, Jiangsu Province, China.
Department of Gastroenterology, the Affiliated Suzhou Hospital of Nanjing Medical University, Suzhou 215008, Jiangsu Province, China.
World J Gastroenterol. 2020 Jun 14;26(22):3034-3055. doi: 10.3748/wjg.v26.i22.3034.
The incidence of inflammatory bowel disease, a chronic intestinal inflammatory disorder that includes Crohn's disease (CD) and ulcerative colitis, is rising. Circular RNAs are considered valuable diagnostic biomarkers for CD. Current evidence supports the views that epithelial-mesenchymal transition (EMT) plays an important role in CD pathogenesis, and that hsa-miR-130a-3p can inhibit transforming growth factor-β1 (TGF-β1)-induced EMT. Our previous study revealed that hsa_circRNA_102610 was upregulated in CD patients. Moreover, we predicted an interaction between hsa_circRNA_102610 and hsa-miR-130a-3p. Thus, we hypothesized that hsa_circRNA_102610 may play roles in the proliferation and EMT of intestinal epithelial cells by sponging hsa-miR-130a-3p to participate in the pathogenesis of CD.
To explore the mechanism of hsa_circRNA_102610 in the pathogenesis of CD.
The relative expression levels of hsa_circRNA_102610 and hsa-miR-130a-3p in patients were detected by quantitative reverse transcription-polymerase chain reaction. The proliferation of human intestinal epithelial cells (HIECs) and normal-derived colon mucosa cell line 460 (NCM460) cells was detected by cell counting kit-8, 5-ethynyl-2'-deoxyuridine staining and cell cycle assays following overexpression or downregulation of hsa_circRNA_102610. Cell proliferation assays were performed as described above in a rescue experiment with hsa-miR-130a-3p mimics. The interaction of hsa_circRNA_102610 and hsa-miR-130a-3p was verified by fluorescence in situ hybridization and dual luciferase reporter assays. The relative expression levels of CyclinD1, mothers against decapentaplegic homolog 4 (SMAD4), E-cadherin, N-cadherin and Vimentin were detected by western blotting following hsa_circRNA_102610 overexpression, TGF-β1-induced EMT or hsa-miR-130a-3p mimic transfection (in rescue experiments).
Upregulation of hsa_circRNA_102610 was determined to be positively correlated with elevated fecal calprotectin levels in CD ( = 0.359, = 0.007) by Pearson correlation analysis. Hsa_circRNA_102610 promoted the proliferation of HIECs and NCM460 cells, while hsa-miR-130a-3p reversed the cell proliferation-promoting effects of hsa_circRNA_102610. Fluorescence in situ hybridization and dual luciferase reporter assays showed that hsa_circRNA_102610 directly bound hsa-miR-130a-3p in NCM460 and 293T cells. An inverse correlation between downregulation of hsa-miR-130a-3p and upregulation of hsa_circRNA_102610 in CD patients was observed ( = -0.290, = 0.024) by Pearson correlation analysis. Moreover, overexpression of hsa_circRNA_102610 promoted SMAD4 and CyclinD1 protein expression validated by western-blotting. Furthermore, over-expression of hsa_circRNA_102610 promoted TGF-β1 induced EMT in HIECs and NCM460 cells targeting of hsa-miR-130a-3p, with increased expression of Vimentin and N-cadherin and decreased expression of E-cadherin.
Hsa_circRNA_102610 upregulation in CD patients could promote the proliferation and EMT of intestinal epithelial cells sponging of hsa-miR-130a-3p.
炎症性肠病是一种慢性肠道炎症性疾病，包括克罗恩病（CD）和溃疡性结肠炎，其发病率正在上升。环状RNA被认为是CD有价值的诊断生物标志物。目前的证据支持上皮-间质转化（EMT）在CD发病机制中起重要作用的观点，以及hsa-miR-130a-3p可抑制转化生长因子-β1（TGF-β1）诱导的EMT的观点。我们之前的研究表明，hsa_circRNA_102610在CD患者中上调。此外，我们预测了hsa_circRNA_102610与hsa-miR-130a-3p之间的相互作用。因此，我们假设hsa_circRNA_102610可能通过海绵吸附hsa-miR-130a-3p在肠上皮细胞的增殖和EMT中发挥作用，从而参与CD的发病机制。
探讨hsa_circRNA_102610在CD发病机制中的作用机制。
采用定量逆转录-聚合酶链反应检测患者中hsa_circRNA_102610和hsa-miR-130a-3p的相对表达水平。在过表达或下调hsa_circRNA_102610后，通过细胞计数试剂盒-8、5-乙炔基-2'-脱氧尿苷染色和细胞周期分析检测人肠上皮细胞（HIECs）和正常来源的结肠黏膜细胞系460（NCM460）细胞的增殖情况。在使用hsa-miR-130a-3p模拟物的拯救实验中，按照上述方法进行细胞增殖分析。通过荧光原位杂交和双荧光素酶报告基因检测验证hsa_circRNA_102610与hsa-miR-130a-3p的相互作用。在hsa_circRNA_102610过表达、TGF-β1诱导的EMT或hsa-miR-130a-3p模拟物转染后（在拯救实验中），通过蛋白质印迹法检测细胞周期蛋白D1、抗五聚体蛋白同源物4（SMAD4）、E-钙黏蛋白、N-钙黏蛋白和波形蛋白的相对表达水平。
通过Pearson相关分析确定，hsa_circRNA_102610的上调与CD患者粪便钙卫蛋白水平升高呈正相关（r = 0.359，P = 0.007）。hsa_circRNA_102610促进了HIECs和NCM460细胞的增殖，而hsa-miR-130a-3p逆转了hsa_circRNA_102610促进细胞增殖的作用。荧光原位杂交和双荧光素酶报告基因检测表明，hsa_circRNA_102610在NCM460和293T细胞中直接与hsa-miR-130a-3p结合。通过Pearson相关分析观察到，CD患者中hsa-miR-130a-3p的下调与hsa_circRNA_102610的上调呈负相关（r = -0.290，P = 0.024）。此外，蛋白质印迹法验证了hsa_circRNA_102610的过表达促进了SMAD4和细胞周期蛋白D1的蛋白表达。此外，hsa_circRNA_102610的过表达通过靶向hsa-miR-130a-3p促进了HIECs和NCM460细胞中TGF-β1诱导的EMT，波形蛋白和N-钙黏蛋白表达增加，E-钙黏蛋白表达减少。
CD患者中hsa_circRNA_102610的上调可通过海绵吸附hsa-miR-130a-3p促进肠上皮细胞的增殖和EMT。
