内质网膜复合亚基3调控内质网应激影响人畸胎瘤细胞的存活

内质网膜蛋白复合物(endoplasmic reticulum membrane complex, EMC)在跨膜蛋白质的生物发生和膜整合中发挥重要作用。内质网膜复合亚基3(endoplasmic reticulum membrane complex 3,EMC3)是EMC的重要组成部分,但其在生殖细胞中发挥的作用未见报道。本研究通过实时荧光定量PCR法检测18周龄小鼠睾丸、肺、脾、下丘脑组织中的EMC3 mRNA表达水平差异。结果显示,小鼠睾丸中EMC3 mRNA表达水平较高。体外培养人畸胎癌细胞NCCIT,通过不同浓度衣霉素诱导细胞产生内质网应激(endoplasmic reticulum stress, ERS),应用实时荧光定量PCR法、蛋白质印迹法检测其中EMC3、葡萄糖调节蛋白78(glucose regulatory protein, GRP78)、CCAAT-增强子结合蛋白同源蛋白(CCAAT-enhancer-binding protein homologous protein, CHOP)的mRNA及其蛋白质表达水平。结果显示,相对于对照组,EMC3、GRP78、CHOP的mRNA与蛋白质水平表达极显著升高(P<0.01),表明衣霉素成功诱导了NCCIT细胞产生内质网应激,EMC3在衣霉素诱导的内质网应激的精原细胞中,mRNA与蛋白质水平表达升高。以NCCIT细胞cDNA为模板,利用PCR法扩增EMC3基因片段,并将其与pRK5-myc载体连接,构建pRK5-myc-EMC3重组质粒,经双酶切鉴定及DNA测序表明:pRK5-myc-EMC3重组质粒构建成功。将重组质粒和空载体分别转染至NCCIT细胞中进行表达,应用实时荧光定量PCR法与CCK8法检测细胞中GRP78、CHOP的mRNA转录水平以及细胞活力。结果显示,EMC3转染组的GRP78、CHOP的mRNA水平表达显著升高(P<0.05),细胞活性极显著降低(P<0.01),表明EMC3可以在NCCIT细胞中调控内质网应激并抑制细胞存活的发生。综上表明,过表达EMC3能够在精原细胞中调控内质网应激抑制细胞存活,EMC3可能在精原细胞的内质网应激中发挥重要作用。

The ER membrane protein complex subunit Emc3 controls angiogenesis via the FZD4/WNT signaling axis

The endoplasmic reticulum(ER) membrane protein complex(EMC) regulates the synthesis and quality control of membrane proteins with multiple transmembrane domains. One of the membrane spanning subunits, EMC3, is a core member of the EMC complex that provides essential hydrophilic vestibule for substrate insertion. Here, we show that the EMC subunit Emc3 plays critical roles in the retinal vascular angiogenesis by regulating Norrin/Wnt signaling. Postnatal endothelial cell(EC)-specific deletion of Emc3 led to retarded retinal vascular development with a hyperpruned vascular network, the appearance of bluntended, aneurysm-like tip endothelial cells(ECs) with reduced numbers of filopodia and leakage of erythrocytes at the vascular front. Diminished tube formation and cell proliferation were also observed in EMC3 depleted human retinal endothelial cells(HRECs). We then discovered a critical role for EMC3 in expression of FZD4 receptor of β-catenin signaling using RNA sequencing, real-time quantitative PCR(RT-q PCR) and luciferase reporter assay. Moreover, augmentation of Wnt activity via lithium chloride(Li Cl) treatment remarkably enhanced β-catenin signaling and cell proliferation of HRECs. Additionally, Li Cl partially reversed the angiogenesis defects in Emc3-c KO mice. Our data reveal that Emc3 plays essential roles in angiogenesis through direct control of FZD4 expression and Norrin/β-catenin signaling.

内质膜蛋白复合体亚基6对海拉细胞自噬的调控作用

目的 探讨内质膜蛋白复合体亚基6（EMC6）基因在海拉（HeLa）细胞中的自噬调解活动。 方法 （1）构建3种质粒：EMC6真核表达质粒、特异性干扰EMC6质粒、空白质粒。分别将3种质粒转染HeLa细胞，筛选3组中的稳定表达细胞株。（2）用实时定量聚合酶链反应（Real-time PCR）、Western blot检测3组质粒转染后Hela细胞中EMC6 mRNA表达结果。（3）电镜下观察HeLa细胞内自噬体；用自噬抑制剂Bafilomycin A1和3-甲基腺嘌呤（3-MA）分别作用于转染EMC6质粒的处理组、空白质粒组和HeLa组，在共焦显微镜下观察比较细胞株中绿色荧光蛋白（GFP）-微管相关蛋白1轻链3（LC3）的变化。 结果 （1）Real-time PCR法检测转染不同质粒进入HeLa细胞株后，处理组、干扰组及空白质粒组各5例，2－ΔΔCt的平均值分别为3.29±0.62、0.38±0.04、1.11±0.20，EMC6 mRNA拷贝数量在3组之间差异有统计学意义（P＝0.020）。（2）在转染了EMC6质粒的HeLa细胞组中可观察到自噬体形成，并观察到Bafilomycin A1作用于3组细胞后，可见GFP-LC3斑点聚集，而EMC6过表达组的LC3斑点明显较空白质粒组及HeLa组丰富。 结论 过表达EMC6蛋白可使HeLa细胞内自噬体数目增加，可能使宫颈癌细胞内自噬活动增强；EMC6过表达细胞株中，磷酸肌醇3激酶（PI3K）抑制剂抑制了EMC6所诱发的自噬过程。