miR-149调控猪滋养层细胞增殖与迁移的分子机制

Proliferation and migration of trophoblast cells (Tr) impair placental efficiency directly, which would affect the growth and development of embryos. But the mechanism of the proliferation and migration of Tr cells is still unclear. Our previous study revealed that miR-149 is down regulated in IUGR placentas, and miR-149 was also reported to be dis-regulated in porcine trophoblast cells (pTr). Target gene prediction and IPA analysis showed that cytokines TGFB2, IL1A and FASLG are important target genes of miR-149 and also the signaling molecules of p38 MAPK. Thus, we propose the hypothesis that miR-149 regulates p38 MAPK by targeting TGFB2, IL1A and FASLG, which would further affect the proliferation and migration of pTr cells. To validate that, this project would study the targeting relationship between miR-149 and its target genes, to explore how miR-149 regulates p38 MAPK, as well as proliferation and migration of pTr, by targeting TGFB2, IL1A and FASLG, in porcine IUGR model, and reveal the molecular mechanism of the proliferation and migration of pTr regulated by miR-149.

滋养层细胞（Tr）增殖与迁移直接影响胎盘效率，进而影响胚胎生长发育，但调控Tr增殖与迁移的机制尚不明确。前期研究显示miR-149在宫内发育迟缓（IUGR）胎盘显著下调表达，且在猪滋养层细胞（pTr）表达异常。靶基因预测及IPA分析得知，细胞因子TGFB2、IL1A、FASLG均为miR-149的重要靶标且同时为p38 MAPK的信号分子。据此假设：miR-149靶向TGFB2、IL1A、FASLG，调控p38 MAPK，进而影响pTr增殖与迁移。为证实假设，项目拟以猪IUGR为模型，从miR-149调控靶基因表达水平为切入点，采用双荧光素酶报告系统、miRNA过表达/抑制表达、流式细胞术、细胞增殖与迁移实验、蛋白亚细胞定位、qPCR、Western等技术探究miR-149介导其靶基因对p38 MAPK的调控以及对pTr增殖与迁移的影响，揭示miR-149调控pTr增殖与迁移的分子机制。

滋养层细胞（Tr）增殖与迁移直接影响胎盘效率，进而影响胚胎生长发育，但调控Tr增殖与迁移的机制尚不明确。通过靶标预测及IPA通路分析，确定了miR-149候选靶标基因TGFB2及其可能参与的信号通路 p38 MAPK。本项目围绕“miR-149调控猪滋养层细胞迁移”这一科学问题，首先验证了miR-149及其候选靶标TGFB2在IUGR子宫内膜中的表达模式；接着利用双荧光素酶报告系统鉴定了miR-149与其候选靶标TGFB2之间的直接靶向关系；通过过表达或抑制表达猪滋养层细胞miR-149，发现miR-149对其靶标TGFB2具有负调控作用，且miR-149对猪滋养层细胞迁移有抑制作用。推测miR-149可能通过靶向抑制猪滋养层细胞内TGFB2的表达，从而抑制猪滋养层细胞迁移。最终揭示miR-149调控猪滋养层细胞迁移的分子机制，为将来miRNA作为分子靶点调控猪滋养层细胞及胚胎发育奠定一定理论基础。