miR-424靶向VEGF/VEGFR-2调控牙髓干细胞促血管生成潜能的研究

miRNAs are recently discovered class of small noncoding RNAs that regulate gene expression at post-transcriptional level. miRNAs play a critical role in mesenchymal stem cells self-renewal and differentiation, which were recognized as a switch during cell differentiation. Our previous study showed for the first time that miR-424 was significantly downregulated during dental pulp stem cells (DPSCs) differentiated to endothelial cells, and its target genes were predicted and verified to vascular endothelial growth factor (VEGF) and VEGF receptor-2 (VEGFR-2) using bioinformatic analysis and rea-time PCR. Based on the above results, luciferase reporter assay was performed to validate the putative binding sites of target genes of miR-424. Then, we observed the role of miR-424 overexpression or underexpression during DPSCs differentiate into endothelialocytes and the downstream signaling pathways. Furthermore, the angiogenic potential of miR-424 in DPSCs was investigated in vivo experiment. This study will provide new insight into the underling mechanisms that can guide DPSCs toward the desired differentiation paths, and presents a prospectus for the angiogenic potential of miR-424 in dental pulp tissue engineering with vascularization .

miRNAs是近年发现的一类非编码小分子RNAs，参与基因转录后调控。miRNAs在干细胞自我更新和分化过程中发挥重要的调控作用，是细胞"定时"、"定向"分化的"开关"。课题组前期采用miRNAs表达谱芯片检测到miR-424在牙髓干细胞内皮向分化后显著下调，经生物信息学预测和实时荧光定量PCR初步验证其靶基因是VEGF和VEGFR-2。在此基础上，我们拟利用荧光素酶报告基因系统验证miR-424的靶基因结合位点；通过miR-424过表达或抑制表达观察其对牙髓干细胞内皮向分化的作用和相关的下游信号通路；同时结合体内实验观察miR-424对牙髓干细胞介导的促血管生成潜能的影响。本研究将进一步充实人牙髓干细胞定向分化的分子调节机制，以期为组织工程牙髓血管化提供新的切入点。

血管新生在牙髓修复再生中具有重要的作用。近年来研究证实，微小RNAs（miRNAs）参与干细胞的自我更新和分化以及血管新生的过程。牙髓细胞（DPCs）是由不同分化阶段前体细胞组成的异质细胞群，可作为研究牙髓干细胞（DPSCs）的体外模型。本课题组通过建立DPCs内皮向分化模型，采用miRNAs芯片检测内皮向诱导分化过程中miRNA表达谱变化，发现多个miRNAs表达发生变化，生物信息学分析显示差异表达的miRNAs与细胞运动、细胞形态和血管形态/发育，细胞粘附等多种细胞行为相关，可能参与调控血管新生关键信号通路（MAPK和Wnt等）。提示DPCs内皮向分化过程中存在miRNA调控机制，其中，miR-424的表达显著下调，因此，我们推测miR-424可能在牙髓干细胞内皮向分化及促血管新生潜能中发挥作用。据此，我们首先采用实时荧光定量PCR技术检测miR-424在DPCs内皮向分化中的表达；通过慢病毒感染DPCs过表达或抑制miR-424表达，明确其在牙髓细胞内皮向分化中的调控作用，联合生物信息学预测及荧光素酶报告实验预测并验证miR-424的下游靶基因和其结合位点及相关的信号通路等。结果发现miR-424抑制牙髓细胞内皮向分化标记物KDR和vWF的表达和细胞成管能力；生物信息学预测VEGF及其受体KDR是miR-424的靶基因，进一步荧光素酶报告基因结果显示miR-424直接靶向结合VEGF和KDR的3’UTR区域；miR-424过表达或抑制表达则相应降低或促进VEGF和KDR表达，且与ERK/MAPK信号通路有关。上述结果提示，miR-424通过作用于靶基因VEGF和KDR，经由ERK/MAPK信号通路调控DPCs的内皮向分化及促血管生成潜能，这为临床活髓保存和牙髓再生修复提供了新的理论依据。