MicroRNA-34基因簇调控PEG10基因对子痫前期生物学行为的影响

Cytotrophoblast invasion defects are one of the main reasons that lead to preeclampsia. It has been reported recently that some of the abnormal expression of imprinted genes are related to the occurrence and development of preeclampsia. With the use of fiber-optic illumina bead microarray, six pairs of preeclampsia placental tissue and normal placental tissue had been detected preliminarily, as we have reported earlier. More than 900 differentially expressed genes were filtered out, among which 3 imprinted genes, PEG10, ATP10A, and SLC22A2 were further tested. The results confirmed a significant increased expression of PEG10 in placenta with preeclampsia. Immunohistochemical staining results showed that the increased PEG10 was mainly distributed in the trophoblasts of placenta with preeclampsia. Expression levels of PEG10-RF1 protein in the trophoblasts of placenta with preeclampsia were significantly raised. miR-34 gene cluster includes three members: miR-34a, miR-34b, and miR-34c-5p. Firstly, using real-time PCR detection, it was found that expression of miR-34a and miR-34c-5p of the miR-34 family in cytotrophoblasts of placenta with preeclampsia were significantly decreased. After reducing the expression of miR-34 family, it was detected that the reduced expression of miR-34a and miR-34c-5p significantly inhibited the proliferation capacity of these cells. Bioinformatics analysis showed that the binding sites of miR-34a and miR-34c-5p coexisted in PEG10 3'-UTR. By silencing the miR-34a and miR-34c-5p, expression of PEG10-RF1 and MYC were highly raised. Therefore, we inferred that miR-34 cluster members might be involved in the occurrence and development of preeclampsia through the negative regulation of PEG10 expression. Until present, there have been found no reports for the specific mechanism of how miR-34 cluster regulates PEG-10, or for the effects of miR-34 cluster on biological behavior of preeclampsia. This project is planned to prove the direct effects of miR-34a and miR-34c-5p on target gene PEG10 and MYC and to prove their binding sites. Based on this, cytotrophoblasts in which miR-34a and miR-34c-5p are over-expressed or silenced respectively will be established to clarify the effects of miR-34a and miR-34c-5p on the expression of PEG10 and MYC, and on the proliferation, invasion and migration of placental cytotrophoblast. Further study will be carried out about the effects and relevant mechanisms of PEG10 on proliferation, invasion and migration of placental cytotrophoblast through ALK1 and ALK5 signaling pathways. Lastly, the best mode of action to control preeclampsia by the combined application of miR-34a and miR-34c-5p or by applying miR-34a or miR-34c-5p alone will be clarified. The findings of this project can not only reveal the molecular mechanisms of regulation by miR-34 cluster members in biological behavior of placental cytotrophoblast, but also provide a new way to effectively improve the therapy.

我们前期研究发现印迹基因PEG10在子痫前期患者胎盘中的表达显著上调，PEG10主要分布在细胞滋养层细胞，子痫前期患者胎盘细胞滋养细胞中miR-34家族成员的表达水平显著下降，生物信息学软件预测到miR-34家族成员与PEG10的结合位点。为深入研究miR-34基因簇对PEG10的可能调控机制及对子痫前期生物学特性的影响，本项目拟首先验证miR-34家族成员与靶基因PEG10和MYC的直接作用和关键结合位点；进一步分别建立miR-34a和miR-34c-5p过表达和表达沉默的细胞滋养细胞，明确miR-34家族成员对PEG10和MYC基因表达以及对细胞滋养细胞增殖、侵袭和迁移的调节作用；进一步研究PEG10通过ALK1和ALK5信号途径调节细胞滋养细胞生物学行为及其分子机制；最后明确miR-34家族成员单独或联合应用的治疗效果。本项目不仅能深入揭示子痫前期的发病机制，而且可为治疗提供新途径。

我们前期研究发现印迹基因PEG10在子痫前期患者胎盘中的表达与正常胎盘表达相比有显著不同，PEG10主要分布在细胞滋养层细胞。miR-34基因簇包括miR-34a,miR-34b,miR-34c-5p。与正常对照组相比，子痫前期患者胎盘细胞滋养细胞中miR-34家族成员的表达水平有显著差异，生物信息学软件预测到miR-34家族成员与PEG10的结合位点。为深入研究miR-34基因簇对PEG10的可能调控机制及对子痫前期生物学特性的影响，本项目拟首先验证miR-34家族成员与靶基因PEG10和MYC的直接作用和关键结合位点，发现MYC-3’UTR和PEG10-3’UTR分别存在miR-34a和miR-34c-5p的作用靶点，miR-34a和miR-34c-5p能够与MYC-3’UTR和PEG10-3’UTR的关键位点结合，对PEG10和MYC基因的表达起负性转录后调控作用；进一步分别建立miR-34a和miR-34c-5p过表达和表达沉默的细胞滋养细胞，明确miR-34家族成员对PEG10和MYC基因表达以及对细胞滋养细胞增殖、侵袭和迁移的调节作用；进一步研究PEG10通过ALK1和ALK5信号途径调节细胞滋养细胞生物学行为及其分子机制；最后明确miR-34家族在子痫前期疾病治疗中的最佳模式。本项目不仅能深入揭示子痫前期的发病机制，而且可为治疗提供新途径。