Identification Molecular Targets For Advanced Liver Disease Through Proteomics Combined NTR-Ⅱ Imaging

Liver disease,especially advanced liver diseases,such as liver cirrhosis and hepatocellular carcinoma（HCC）were with high morbidity and mortality.In order to improve the prognosis of patients,proteomics and the NIR-II imaging were integrated to gain quantitative sights into new molecular targets in a non-invasive manner for advanced liver diseases.The strategy of proteomics combined NIR-II imaging was developed,and the potential therapeutic targets MCM2 and CTSS with high specificity for HCC and liver cirrhosis were identified through this strategy,respectively.My thesis included two parts:1.Poteomics analysis and NIR-II imaging of MCM2 with high specificity for HCC.（1）MCM2 was significantly up-regulated in HCC tumor tissues through an iTRAQ quantitative proteomic approach in paired tumor and adjacent HCC tissues,and was identified as one of the most significantly altered proteins in HCC tumor tissues.（2）The over-espression of MCM2 protein in HCC tumor tissues was further confirmed through tissue microarray and immunostaining with 100 paired HCC tumor and adjacent tissues.Over 90%cases exhibited positive staining while liver tissues adjacent to HCC demonstrated negative or weak staining.（3）MCM2-targeted of HCC tumors through NIR-II Imaging in vivo.The first small-molecule based MCM2-targeted NIR-II probe CH1055-MCM2 was concisely generated and subsequently evaluated in mice bearing HepG2 xenograft.The excellent imaging properties such as good tumor uptake,high tumor contrast and specificity were achieved in the small-animal models.（4）The mRNA expression of MCM2 was gradually increased along with the development of HCC and significantly decreased the survival rate of HCC cases according to the TCGA database.（5）MCM2 impacted the proliferation and cell cycle of HepG2 cells.The S stage was block and the proliferation was reduced after MCM2-siRNA transfection.siRNAs-mediated repression of MCM2 expression resulted in the significant suppression of HepG2 cell cycle and proliferation through CDK 2/7 pathway.2.Dyregulation of ECM remodeling was one of key pathological features in cirrhotic tissues.In order to explore the mechanism and identify potential therapeutic targets for liver cirrhosis,ECM associated proteins were analyzed through proteomics analysis.（1）CTSS was up-regulated by proteomics analysis.CTSS was upregulated in cirrhotic tissues compared with normal liver tissues through an iTRAQ quantitative proteomics analysis.According to the tissue microarray staining with CTSS antibodies,most cirrhotic tissues showed positive staining,especially cirrhotic tissues at advanced stages.（2）The RNAseq data from NCBI database indicated that mRNA expression of CTSS was correlated positively with the clinical stage of liver cirrhosis.（3）The over-expression of CTSS in liver cirrhosis was examined through CCl₄-induced WT mice by NIR-II imaging.（4）The function of CTSS in the development of liver cirrhosis was investgated through CCl₄-induced WT and CTSS KO mice by HE staining and Masson staining.（5）A series of peptides derived from collagens such as Canstatin and Endosatin were identified.Furthermore,Endostatin might be the degradation products of CTSS through Western blot among CCl₄-induced WT and CTSS KO mice.（6）CTSS might involve in the development of liver cirrhosis through inhibiting the differentiation of LSEC by Endostatin.CCl₄ induced LSEC dedifferentiation in WT mice,while open fenestraes of LSEC were maintained in CTSS KO mice.In addition,open fenestraes of LSEC were decreased after induced by the recombinant Endostatin（Endostar）in vitro.This indicated that CTSS might improve the development of liver cirrhosis through promoting LSEC dedifferentiation by Endostatin.