The Roles Of Chitinase-3-like Protein 1 In The Progression Of Liver Fibrosis And Glioma

CHI3L1 is a nonnegligible member of the glycosylhydrolase protein 18 family,which is widely expressed in eukaryotes and prokaryotes.CHI3L1 can bind chitin,but lacks off enzymatic activity.As an important innate and acquired immune regulatory molecule,CHI3L1 was secreted by a number of cells,for instance,neutrophils,macrophages,fibroblast-like cells,endothelial cells and cancer cells.CHI3L1 had been proved to be dramatically over-expressed in inflammatory and tumoral disease such as alcoholic liver disease,atherosclerosis,gastric cancer,breast cancer,and colon cancer.CHI3L1 had been verified to play a crucial role in inflammasome activation,apoptosis,macrophage differentiation,Th1/Th2 inflammatory balance,tissue injury and repair.Previous studies had suggested that CHI3L1 was up-regulated in patients with alcoholic disease and hepatic fibrosis,but the definite role of CHI3L1 in the progression of chronic liver fibrosis still remained unclear.Therefore,it is of great clinical significance to explore the role and targeted molecular mechanism of CHI3L1 in inflammatory and neoplastic diseases.Part ?: CHI3L1 inhibites the activation of TGF-β signaling pathway to alleviate hepatic fibrosisBackground and Aims: Hepatic fibrosis is the transitory stage of liver cirrhosis and liver cancer,which results from chronic liver injury,inflammation and repair that was caused by viral hepatitis,alcohol abuse,parasite and autoimmune disease.When the pathogenic factors continue,the fibrotic liver evolves into cancer and liver failure.According to statistics,patients with chronic hepatitis for more than 5 years have a10~20% chance to develop into liver fibrosis,and a 2~5% probability to progress into liver cancer.It had been reported that CHI3L1 was elevated in patients with alcoholic liver disease and cirrhosis,but its role in chronic liver disease and the involved mechanism still needed further studies.This study aims to explore the association between CHI3L1 expression and liver fibrosis progression,and study the role of CHI3L1 and involved mechanisms in liver fibrosis progression,which aimed at providing theoretical basis for clinical diagnosis and treatment of liver fibrosis.Methods: We collected serum samples from patients with clinical diagnosed staged liver fibrosis and healthy volunteers from Center for Digestive Medicine in the Second Affiliated Hospital of Nanjing medical university to compare the serum levels of CHI3L1.Transcriptome profiles and matched clinical data from patients with fibrotic liver were downloaded from GEO and were applied for further analysis to study the association between CHI3L1 expression and liver fibrosis.In vivo,intraperitoneal injection of TAA was applied to induce the mouse liver fibrosis models,and HYP(Hydroxyproline,HYP)assay was performed to detect the content of hydroxyproline in the liver,thus assessing the severity of liver fibrosis between WT and Chil1-/-mice.Collagen and ɑ-SMA expression were measured by Western blot.H&E(Hematoxylin and eosin,H&E),Mason,Sirius Red,Silver staining and IHC(Hmmunohistochemical,IHC)staining were performed on section slides of mice liver to assess the degree of liver fibrosis.Transcriptome analysis were used to compare the genetic changes and altered pathway in the presence of CHI3L1 knockout,which aims at exploring the definite role of CHI3L1 in the progression of liver fibrosis.In vitro,therefor we applied human hepatic stellate cell lines(LX-2 cell)to carry out the mechanism related experiments.We treated LX-2 cells with rh TGF-β1,rh CHI3L1,and rh TGF-β1 combined with rh CHI3L1 with a time gradient up to 2 h,and then detected the phosphorylation of Smad2/3 and expression of Smad7 to study the role of CHI3L1 in TGF-β signaling pathway.Smad7 is known as a negative regulator in TGF-β signaling pathway to inhibit downstream cascade phosphorylation of Smad2/3 when translocate to the membrane to form a complex with TGF-βR?.Therefore,we isolated the membrane protein of LX-2 cells after treated with rh TGF-β1,rh CHI3L1,and rh TGF-β1 combined with rh CHI3L1 to detect the content of Smad7,thus exploring the role of CHI3L1 in Smad7 recruitment.We also performed IF(Immunofluorescence,IF)to observe the Smad7 translocation.In the treatment trial,we performed tail intravenous injection with rm CHI3L1 for WT and Chil1-/-mice with a TAA induced liver fibrosis background,and then detected ɑ-SMA and collagen expression,and the phosphorylation of Smad2/3 in the liver by western blot.H&E,Silver,Mason,Sirius Red staining and IHC staining were operated to compare the level of liver fibrosis in different groups,thus measuring the therapeutic effect of rm CHI3L1 on murine hepatic fibrosis.Results: CHI3L1 was over-expressed in fibrotic liver and associated with the stage of liver fibrosis.In vivo studies revealed that the Chil1-/-mice liver underwent more serious hepatic fibrosis by HYP assay and H&E,Mason,Sirius Red,and Silver staining.Western blot analysis uncovered that ɑ-SMA and collagen were higher in the liver in Chil1-/-mice than that of WT mice.GSVA showed the TGF-β pathway enrichment score was higher in Chil1-/-mice compared to WT group based on the transcriptome analysis.Data above suggested that CHI3L1 attenuates liver fibrosis via inhibition of TGF-β signaling pathway.In vitro studies showed that ɑ-SMA and collagen were down-regulated in LX-2cells after treated with rh CHI3L1.Meanwhile,rh CHI3L1 pretreatment also inhibited the phosphorylation of Smad2/3 in LX-2 cells while the expression of Smad7 remained invariable.Furthermore,CHI3L1 was then proved to bind TGF-βR? which interacted with Smad7 through Western blot analysis of membrane proteins.IF also showed that Smad7 was recruited to the membrane after rh CHI3L1 stimulation.In the treatment studies,Western blot illustrated mice administrated with rm CHI3L1 bore less expression of ɑ-SMA and collagen,and less phosphorylation of Smad2/3.Likewise,IHC staining and H&E,Mason,Sirius Red and Silver staining documented that mice were rescued by challenging with rm CHI3L1.Taking together,mice with Chil1+/+ and Chil1-/-background were rescued by tail intravenous injection of rm CHI3L1 both in the CCL4 and TAA induced mouse models.Conclusions: CHI3L1 recriuts Smad7 to the membrane to inhibite the activation of HSC,thus functioning as a negative regulator of hepatic fibrosis,thereby serving as a potential therapeutic target.Part Ⅱ: CHI3L1 promotes glioma progression via the NF-κB signaling pathway and microenvironment reprogrammingBackground and Aims: Glioma,a very heterogeneous group of primary brain tumors,accounts for 80% of the central nervous system tumors.Despite numerous aggressive combination therapy consisting of surgery,along with chemotherapy and radiotherapy,the 5-year overall survival rate for high grade glioma(High grade glioma,HGG)was about 15-35%,while patients with GBM suffered 14.6 months median survival time and 5.8% 5-year survival rate.Hence,further identification of key molecules and specific mechanisms in the occurrence and development of glioma not only contribute to accurate diagnosis,but also provide new therapeutic strategy.Non-enzymatic CHI3L1 has been reported to be highly expressed in a series of tumors,and is associated with lower survival rates.CHI3L1 was proved to promote the growth,proliferation and migration of tumor cells to accelerate the progression of malignancy.What’s more,immune cells in the tumor microenvironment undergo substantial reprogramming and remodeling,thereby acquiring pro-tumor phenotypes that facilitate tumor progression.This study aimed at determining the involved pathway caused by the up-regulated CHI3L1,and its function in the tumor microenvironments(TME)reprogramming.Methods: Gene expression profiles and matched clinical information were obtained from GEO,TCGA and CGGA to determine the m RNA level of CHI3L1 in glioma.CHI3L1 at protein levels were detected by immunohistochemistry(IHC),Western blot,and enzyme linked immunosorbent assay(ELISA).GSEA was used to identify the envolved target pathway,and GSVA was applied to assess the association between CHI3L1 expression and NF-κB signaling pathway enrichment.Chil1-/-BMDMs,U87 MG and A172 cells with CHI3L1 knocking down was treated with recombined TNFɑ to study the relationship between CHI3L1 expression and NF-κB p65 subunit phosphorylation.Single-cell RNA-Seq raw data was used to characterize the habits of tumor cells and landscape delineation of myeloid.TIMER was applied to evaluate the association between CHI3L1 expression and immune cells infiltration.THP-1 cell line was utilized to study the effect of CHI3L1 on M2 macrophages differentiation.Colony formation assay,Ed U cell proliferation assay and wound healing and migration assay were applied to investigate the proliferation and invasion capacity of glioma cell lines by loss of function.Results: CHI3L1 was overexpressed across the disease stage of glioma,which was closely linked with tumor staging,growth and invasion,and survival rates.Nuclear CHI3L1 binds ACTN4 to activate NF-κB signaling pathway by promoting p65 subunit nuclear translocation in glioma cells.Once released into the TME in the presence of F-actin,CHI3L1 interacts with CD44 to activate AKT pathway thus accelerating M2 macrophage polarization.Further,CHI3L1 expression was positively correlated with immune checkpoints,for instance,PD-L1,CTLA-4,LAG3,TIM3 and B7-H3,which reprogrammed the TME to an immunosuppression phenotype.Conclusions: CHI3L1 facilitates NF-κB pathway activation within glioma cells and M2 macrophage polarization via AKT pathway in the TME,which were responsible for the establishment of tumor-promoting and immunosuppression networks in glioma.