lncRNA-loc391533通过sFlt-1/VEGF途径在子痫前期发病中的机制研究

Recent studies have shown that long non-coding RNA (lncRNA) plays a key role in various human diseases, especially in tumor, cardiovascular diseases by regulation of gene expression. But the regulation of gene expression of lncRNA in preeclampsia is still unknown. Our previous studies found that lncRNA-loc391533 plays an important role in preeclampsia. lncRNA-loc391533 is a 1,434 bp intragenic lncRNA transcripted from the gene LOC391533 located on Chromosome 3p21.31. This gene is fms-related tyrosine kinase 1 pseudogene 1 (FLT1P1), similar to gene FLT1 which located on Chromosome 13q12. Gene FLT1 encodes a member of the VEGFR family. This protein plays an important role in angiogenesis and vasculogenesis, with different isoforms including a full-length transmembrane receptor isoform and shortened, soluble isoforms. The soluble isoforms are associated with the onset of preeclampsia. Thus we hypothesis that lncRNA-loc391533 via sFlt-1/VEGF involved in the molecular mechanism of preeclampsia. In this study, lncRNA-loc391533 will be introduced into JEG-3/BEWO cell line. The cell proliferation，invasion and cell cycle were detected by CCK8，transwell and FCM. The function of lncRNA on hypertension was observed by lentiviral transfected animal in vivo. The molecular regulation mechanism of lncRNA was studies by siRNA, RNA immunoprecipitation and RNA pulldown assay. These results might reveal insights into the molecular regulation mechanism of lncRNA, lead us to propose that the pathway of lncRNA-loc391533 via sFlt-1/VEGF, may provide a new target for the treatment of preeclampsia.

我们前期研究在子痫前期（PE）的胎盘组织中检测到一个与PE关系密切的lncRNA，将之命名为lncRNA-loc391533。其来源基因loc391533是VEGF 受体1的假基因，我们推测它很可能通过sFlt-1/VEGF途径参与子痫前期的发病。为验证假设，本研究拟通过体外实验，构建表达载体，导入滋养细胞株内，检测细胞增殖、侵袭等能力；通过体内实验，将表达载体注入妊娠小鼠，观察对孕鼠血压、尿蛋白/肌酐比、胎盘病理及胎崽的影响，确认lncRNA-loc391533在子痫前期发病中起作用。随后提取PE患者胎盘中的滋养细胞行原代培养，采用siRNA技术敲除lncRNA-loc391533功能，与未敲除组对比观察其对sFlt-1、Flt-1、VEGF转录和表达的影响，进一步验证它是否通过sFlt-1/VEGF的途径参与子痫前期的发病机制。最后我们将通过RNA pull-down、RNA免疫共沉淀技术了解lncRNA-loc391533在sFlt-1/VEGF作用途径中的上游机制。本研究结果有望揭示其在子痫前期中的作用和机制，也为子痫前期新的治疗靶点提供理论和实践依据。

我们前期研究在子痫前期（PE）的胎盘组织中检测到一个与PE关系密切的lncRNA，将之命名为lncRNA-loc391533。其来源基因loc391533是VEGF 受体1的假基因，我们推测它很可能通过sFlt-1/VEGF途径参与子痫前期的发病。为验证假设，本研究拟通过体外实验，构建表达载体，导入滋养细胞株内，检测细胞增殖、侵袭等能力；采用siRNA技术敲除lncRNA-loc391533功能，与未敲除组对比观察其对sFlt-1、Flt-1、VEGF转录和表达的影响，以验证它是否通过sFlt-1/VEGF的途径参与子痫前期的发病机制。然后我们将通过RNA pull-down、RNA免疫共沉淀技术了解lncRNA-loc391533在sFlt-1/VEGF作用途径中的上游机制。结果显示：在人滋养细胞HTR8，构建过表达质粒lncRNA-loc391533-OE，不影响细胞周期；可增强细胞的增殖、侵袭功能；对相关蛋白（sFlt-1、Flt-1、VEGF）的表达无明显影响；我们利用ChIRP-MS方法研究了FLT1P1相互作用的蛋白质组。我们发现大部分FLT1P1相互作用的蛋白质与翻译过程有关。本研究结果有望揭示其在子痫前期中的作用和机制，也为子痫前期新的治疗靶点提供理论和实践依据。