唇腭裂相关基因IRF6基因沉默促进细胞增殖和迁移并抑制上皮间质转化

Cleft Lip and Palate Associated Gene IRF6 Gene Silencing Promotes Cell Proliferation and Migration and Inhibits EMT

干扰素调节因子6(interferon regulatory factor 6,IRF6)基因突变在单纯型和综合征型唇腭裂中均有报导。然而,其基因突变如何导致了唇腭裂的病理发生目前尚不清楚。本文以培养细胞为模型,研究了IRF6基因沉默对细胞增殖、迁移、凋亡以及上皮间质转化(epithelial-mesenchymal transition,EMT)的影响,从而探讨唇腭裂形成的可能的分子病理机制。采用分子克隆技术构建IRF6真核过表达载体;设计合成IRF6基因特异siRNA,成功构建IRF6基因沉默和过表达细胞模型;利用实时荧光定量PCR(qRT-PCR)、免疫印迹法(Western blot)检测转染siRNA-IRF6质粒48 h时,发现IRF6的mRNA和蛋白质表达均降低2倍;CCK8法检测转染siRNA-IRF6后,对细胞增殖能力提高1.98倍;划痕法观察转染siRNA-IRF6质粒72 h后检测细胞的迁移能力,比对照组增强2.36倍;利用Western印迹、qRT-PCR检测EMT标志性分子E-钙黏着蛋白(E-cadherin),发现过表达IRF6后EMT有显著降低。与对照相比,E-钙黏着蛋白表达下调3.57倍;流式细胞技术检测IRF6时未发现对细胞凋亡有影响。在体外培养细胞模型中,IRF6基因沉默显著促进了细胞的增殖和迁移,抑制EMT发生。提示IRF6这一唇腭裂相关基因有可能通过影响上述细胞事件,而导致唇腭裂的病理发生。

Interferon regulatory factor 6(IRF6) gene mutations have been reported in pure and syndrome cleft lip and palate, but how its gene mutation leads to the disease pathogenesis is still unclear. With cultured cells as the model in this paper, the effects of IRF6 silencing on cell proliferation, migration, apoptosis, and epithelial mesenchymal transformation(EMT) were studied. The possible molecular mechanism of cleft lip and palate formation was explored. The IRF6 eukaryotic overexpression plasmid was constructed by the molecular cloning technology. IRF6 gene specific siRNA was designed and synthesized, and the cell model of IRF6 silencing and overexpression was successfully constructed. Real-time fluorescence quantitative PCR(qRT-PCR) and Western blotting were used to detect the mRNA and protein expression of IRF6 after transfection of siRNA-IRF6 for 48 hours. CCK8 assays showed a 1.98-fold increase in cell proliferation after siRNA-IRF6. The migration ability of cells was observed after transfection for 72 hours with siRNA-IRF6 by the scratch assay, which revealed 2.36 times stronger than that of the control group. Western blotting and qRT-PCR were used to detect the EMT signature molecule E-cadherin. We found that EMT was significantly reduced after IRF6 overexpression, and the expression of E-cadherin was down-regulated by 3.57 fold. No effects of IRF6 on apoptosis was detected by flow cytometry. In the in vitro cultured cell model, IRF6 silencing significantly promoted the proliferation and migration of cells and inhibited the occurrence of EMT. Our results thus revealed the potential mechanism of IRF6 leading to cleft lip and palate through influencing related genes.