长链非编码RNA- - MIR17HG靶向TGFβ/BMP信号通路调控腭发育的分子机制

Cleft palate is a multi-factorial congenital malformation, whose genetic causes is unclear. The normal proliferation,extracellular matrix(ECM) secretion and osteogenesis of palatal mesenchymal cells(PMCs) play important roles during palatal development. Recent studies show that CMYC promote transcription of long noncoding RNA-MIR17HG; the splicing production of MIR17HG target components of SMAD dependent TGFB signaling pathway；SMAD dependent TGFB/BMP pathway regulate proliferation, apoptosis, ECM secretion and osteogenesis of PMCs. Here，we want to construct PMCs with increased/decreased expression of MIR17HG,and show that how could CMYC regulate transcription of MIR17HG, which splicing products of MIR17HG would target SMAD dependent TGFB/BMP pathway to regulate proliferation, apoptosis, ECM secretion and osteogenesis of PMCs. We'll finally indicate that whether "CMYC→MIR17HG→TGFB/BMP SMADs→SMAD target genes"pathway exists in PMCs during palatal development.

先天性腭裂是严重影响患儿身心健康的多基因遗传疾病，其致病基因还不明确。腭间充质细胞的正常增殖、胞外基质分泌和成骨细胞向分化是正常腭发育的保证。研究表明CMYC可促进长链非编码RNA- - MIR17HG的转录；MIR17HG的剪切产物可通过靶向作用于SMAD依赖的TGFB通路成员；SMAD依赖的TGFB/BMP通路成员可调控腭间充质细胞增殖、凋亡、胞外基质分泌和成骨细胞向分化。本项目拟构建MIR17HG基因高表达和沉默的小鼠腭间充质细胞，以此为模型，分析MIR17HG通过哪些剪切产物靶向调控TGFB/BMP信号通路中哪些成员从而影响腭间充质细胞增殖、凋亡、胞外基质分泌和成骨向分化等功能，并探索MIR17HG转录调控机制，最终阐明"CMYC→MIR17HG→TGFB/BMP SMADs→SMAD靶蛋白"这一关键调控通路在腭间充质细胞中的存在。

MIR17HG在E12、E13、E14、E15的腭部持续表达，并且其表达位置主要位于腭部生长发育中心，MIR17HG剪切产物miR-17、miR-18a、miR-19a、miR-19b、miR-20a、miR-92这6各miRNA在E12、E13、E14均有表达，其表达水平随着胚胎发育逐渐降低。MIR17H沉默（IN）显著抑制了腭间充质细胞的增殖活性，而模拟物（MI）显著增加了腭间充质细胞的增殖活性。MIR17H的沉默促进了腭间充质细胞外基质Col1a1, Col1a2, and Col3a1的分泌，而MIR17H模拟物显著抑制了Col1a1, Col1a2, and Col3a1的mRNA和蛋白水平的表达。MIR17H沉默显著抑制了TGFβR2、SMAD2和SMAD4的mRNA（图6）和蛋白水平，MIR17H模拟物促进了三者的表达。MIR17HG对TGFβ/BMP信号通路中TGFβR2、SMAD2和SMAD4的调控是通过miR-17和miR-20a对TGFβR2的调控实现，通过miR-18a调控SMAD2和SMAD4实现。采用GeneMANIA分析MIR17HG靶基因的信号通路和功能，绘制基因间相互作用图。结果发现MIR17HG靶基因的主要功能在于细胞周期的调控，尤其是G1期至S期的转化。通过荧光素酶报告系统，我们发现， miR-17-5p，miR-20a， miR-20b和miR-106a可以直接作用于E2F1的3’UTR区，直接靶向抑制其表达。.结论：长链非编码RNA——MIR17HG在腭间充质细胞中可以靶向TGFb通路成员和E2F1，从而在调控了腭发育过程中腭间充质细胞周期调控、增殖活性和细胞外基质合成等功能。