Hu Yu-Jie, Zhou Qian, Li Zhu-Yin, Feng Dan, Sun Lei, Shen Yun-Lin, Huang Wen-Yan
Department of Nephrology and Rheumatology, Shanghai Children's Hospital, Shanghai Jiao Tong UniversityShanghai 200062, China.
Am J Transl Res. 2018 Mar 15;10(3):847-856. eCollection 2018.
This study aimed to investigate the exact function of RGC-32 in kidney diseases and explore the potential mechanism of RGC-32 in regulating cell cycle. RGC-32 knockout (RGC-32) mice were generated from C57BL/6 embryonic stem cells. Differentially expressed proteins in the kidney were investigated with the isobaric tags for relative and absolute quantification (iTRAQ) technique. Gene ontology analyses (GO), Kyoto encyclopedia of genes and genomes (KEGG) pathway mapping analysis and functional network analysis were also performed. The expressions of Smc3, Smad 2-3, DNA-PK were further confirmed by qPCR. Results showed that 4690 proteins were quantified on the basis of 25165 unique peptides. Comparative proteomic analysis revealed 361 differentially expressed proteins in RGC-32 mice (knockout/wild ratio >+/- 1.2 and <0.05). GO and KEGG pathway mapping analyses showed differentially expressed proteins were involved in spliceosome, fluid shear stress and atherosclerosis protein processing in endoplasmic reticulum, pathways in cancer, viral carcinogenesis, epithelial cell signaling in infection, HTLV-I infection, PI3K-Akt signaling pathway, ubiquitin mediated proteolysis, Parkinson's disease, MAPK signaling pathway, carbon metabolism, Alzheimer's disease, NOD-like receptor signaling pathway, tight junction, Proteoglycans in cancer, phagosome, ribosome, mTOR signaling pathway, and AMPK signaling pathway. Differentially expressed proteins Smc3 (0.821), DNA-PK (0.761), Smad 2-3 (0.631) were involved in cell cycle regulation. mRNA expression of Smad2-3, DNA-PK, and Smc3 was consistent with that from iTRAQ. It is concluded that RGC-32 may affect the expression of many proteins (76 up-regulated and 285 down-regulated) in the kidney, and may regulate the expression of Smc3, DNA-PK and Smad 2-3 to affect the cell cycle.
本研究旨在探究RGC - 32在肾脏疾病中的具体功能，并探索RGC - 32调节细胞周期的潜在机制。从C57BL / 6胚胎干细胞培育出RGC - 32基因敲除（RGC - 32−/−）小鼠。采用相对和绝对定量等压标签（iTRAQ）技术研究肾脏中差异表达的蛋白质。还进行了基因本体分析（GO）、京都基因与基因组百科全书（KEGG）通路映射分析和功能网络分析。通过qPCR进一步证实了Smc3、Smad 2 - 3、DNA - PK的表达。结果显示，基于25165个独特肽段对4690种蛋白质进行了定量。比较蛋白质组学分析揭示，RGC - 32−/−小鼠中有361种差异表达蛋白质（敲除/野生型比率> + / - 1.2且<0.05）。GO和KEGG通路映射分析表明，差异表达蛋白质涉及剪接体、流体剪切力与动脉粥样硬化、内质网中的蛋白质加工、癌症通路、病毒致癌作用、感染中的上皮细胞信号传导、HTLV - I感染、PI3K - Akt信号通路、泛素介导的蛋白水解、帕金森病、MAPK信号通路、碳代谢、阿尔茨海默病、NOD样受体信号通路、紧密连接、癌症中的蛋白聚糖、吞噬体、核糖体、mTOR信号通路和AMPK信号通路。差异表达蛋白质Smc3（0.821）、DNA - PK（0.761）、Smad ２ - 3（0.631）参与细胞周期调控。Smad2 - 3、DNA - PK和Smc3的mRNA表达与iTRAQ结果一致。结论是，RGC - 32可能影响肾脏中许多蛋白质的表达（76种上调和285种下调），并可能通过调节Smc3、DNA - PK和Smad 2 - 3的表达来影响细胞周期。
