Department of Pediatric Surgery, Shengjing Hospital of China Medical University, Shenyang, Liaoning, 110004, PR China.
The Key Laboratory of Health Ministry for Congenital Malformation, Shengjing Hospital of China Medical University, Shenyang, Liaoning, 110004, PR China.
Biomed Pharmacother. 2020 Jul;127:110117. doi: 10.1016/j.biopha.2020.110117. Epub 2020 Mar 31.
Anorectal malformations (ARMs) is one of the most common gastrointestinal anomalies. Previous research revealed that miR-92a-2-5p was upregulated in ARMs. However, the underlying roles remains unknown. The current study was to further investigate the spatiotemporal expression patterns of miR-92a-2-5p and its target gene protein kinase C alpha (PRKCA) predicted by bioinformatic method, and to explore their potential functions in anorectal malformations (ARMs). Rat models with ethylenethiourea-induced ARMs were made for subsequent experiments. Direct target relationship between miR-92a-2-5p and PRKCA was validated using a luciferase reporter assay. The spatiotemporal expression pattern of miR-92a-2-5p was evaluated using fluorescence in situ hybridization (FISH), while the expression of PRKCA was revealed by immunohistochemical staining and western blotting. IEC-6 cells were transfected with mimics/mimics NC (Negative control)/inhibitor/inhibitor NC of miR-92a-2-5p or si-PRKCA/si-PRKCA NC, respectively. Then the downstream molecules of miR-92a-2-5p, PRKCA and β-catenin, were subsequently detected. Meanwhile, apoptosis and viability assays were measured. Dual luciferase assay confirmed the direct regulatory relationship between miR-92a-2-5p and PRKCA. FISH revealed that miR-92a-2-5p was expressed with a higher level in ARMs fetuses. Further analyses of PRKCA showed lower protein expression level in ARMs group, which was opposite to miR-92a-2-5p. In vitro experiments revealed that overexpression of miR-92a-2-5p or knockdown of PRKCA can down-regulate PRKCA, up-regulate and facilitate nuclear localization of β-catenin, increase apoptosis and decrease proliferation of IEC-6. Taken together, these findings suggest that aberrantly high expression of miR-92a-2-5p potentially contribute to ARMs by inhibiting proliferation and enhancing apoptosis of intestinal cells via negatively regulating PRKCA/β-catenin.
肛门直肠畸形（ARMs）是最常见的胃肠道异常之一。先前的研究表明，miR-92a-2-5p 在 ARMs 中上调。然而，其潜在作用尚不清楚。本研究旨在进一步探讨生物信息学方法预测的 miR-92a-2-5p 及其靶基因蛋白激酶 Cα（PRKCA）的时空表达模式，并探讨其在肛门直肠畸形（ARMs）中的潜在功能。随后进行了乙基硫脲诱导的 ARMs 大鼠模型实验。利用荧光原位杂交（FISH）评估 miR-92a-2-5p 的时空表达模式，通过免疫组织化学染色和 Western blot 揭示 PRKCA 的表达。采用荧光原位杂交（FISH）检测 miR-92a-2-5p 的时空表达模式，采用免疫组织化学染色和 Western blot 检测 PRKCA 的表达。用 miR-92a-2-5p 的 mimics/mimics NC（阴性对照）/inhibitor/inhibitor NC 或 si-PRKCA/si-PRKCA NC 转染 IEC-6 细胞，然后检测下游分子 miR-92a-2-5p、PRKCA 和β-catenin。同时，进行了细胞凋亡和活力测定。双荧光素酶报告基因实验证实了 miR-92a-2-5p 与 PRKCA 的直接调控关系。FISH 显示 miR-92a-2-5p 在 ARMs 胎鼠中表达水平较高。进一步分析 PRKCA 显示 ARMs 组蛋白表达水平较低，与 miR-92a-2-5p 相反。体外实验表明，miR-92a-2-5p 过表达或 PRKCA 敲低均可下调 PRKCA，上调并促进β-catenin核定位，增加 IEC-6 细胞凋亡，减少增殖。综上所述，这些发现表明，miR-92a-2-5p 的异常高表达可能通过负调控 PRKCA/β-catenin 抑制肠细胞增殖和增强凋亡，从而导致 ARMs。
