Loss of FAM172A gene prompts cell proliferation in liver regeneration.

The present study was designed to explore the function of FAM172A in liver regeneration and HCC. Mice were sacrificed after 70% partial hepatectomy (PH). RNA sequencing was performed on primary hepatocytes of WT and FAM172A-/- mice. We used HepG2 cells to construct cell lines with stably knockdown and overexpression of FAM172A. The expression of FAM172A in liver tissues was investigated by immunohistochemical staining, and we also used public database to perform survival analysis and prognostic model in HCC. Compared with WT mice after PH, normalized liver weight/body weight (LW/BW) ratio and the proliferating cell nuclear antigen (PCNA) protein level of FAM172A-/- mice elevated. The DEGs were mainly enriched in inflammatory response, tumor necrosis factor production, and wound healing. FAM172A knockdown enhanced the NFκB-TNFα and pERK-YAP1-Cyclin D1 axis. FAM172A peptide inhibited proliferation of primary hepatocytes. Moreover, the low expression of FAM172A in human HCC tissues implies a lower likelihood of survival and a valid diagnostic marker for HCC. Loss of FAM172A gene promotes cell proliferation by pERK-YAP1-Cyclin D1 and pNFκB-TNFα pathways during liver regeneration after PH. FAM172A may be a favorable diagnosis marker of HCC.

Development of a prognostic signature based on anoikis-related genes in hepatocellular carcinoma with the utilization of LASSO-cox method.

To develop a signature based on anoikis-related genes (ARGs) for predicting the prognosis of patients with hepatocellular carcinoma (HCC), and to elucidate the molecular mechanisms involved. In this study, bioinformatic algorithms were applied to integrate and analyze 777 HCC RNA-seq samples from the cancer genome atlas and international cancer genome consortium repositories. A prognostic signature was developed via the least absolute shrinkage and selection operator-cox regression method. To evaluate the accuracy of the signature in predicting events, multi-type technical means, such as Kaplan-Meier plots, receiver operating characteristic curve analysis, nomogram construction, and univariate and multivariate Cox regression studies were performed. We investigated the underlying molecular biological mechanisms and immune mechanisms of the signature using gene set enrichment analysis and the CIBERSORT R package, respectively. Meanwhile, immunohistochemical staining acquired from the human protein atlas was used to confirm the differential expression levels of hub genes involved in the prognostic signature. We developed an HCC prognostic signature with a collection of 5 ARGs, and the prognostic value was successfully assessed and verified in both the test and validation cohorts. The risk scores calculated by the prognostic signature were proved to be an independent negative prognostic factor for overall survival. A set of nomograms based on risk scores was established and found to be effective in predicting OS. Further investigation of the underlying molecular biological mechanisms and immune mechanisms indicated that the signature may be relevant to metabolic dysregulation and infiltration of gamma delta T cells in the tumor. The survival prognosis of HCC patients can be predicted by the anoikis-related prognostic signature, and it serves as a valuable reference for individualized HCC therapy.

Upregulation of GLT25D1 in Hepatic Stellate Cells Promotes Liver Fibrosis via the TGF-ß1/SMAD3 Pathway In Vivo and In vitro.

Background and Aims: Collagen ß(1-O) galactosyltransferase 25 domain 1 (GLT25D1) is associated with collagen production and glycosylation, and its knockout in mice results in embryonic death. However, its role in liver fibrosis remains elusive, particularly in hepatic stellate cells (HSCs), the primary collagen-producing cells associated with liver fibrogenesis. Herein, we aimed to elucidate the role of GLT25D1 in HSCs. Methods: Bile duct ligation (BDL)-induced mouse liver fibrosis models, primary mouse HSCs (mHSCs), and transforming growth factor beta 1 (TGF-ß1)-stimulated LX-2 human hepatic stellate cells were used in in vivo and in vitro studies. Stable LX-2 cell lines with either GLT25D1 overexpression or knockdown were established using lentiviral transfection. RNA-seq was performed to investigate the genomic differences. HPLC-MS/MS were used to identify glycosylation sites. Scanning electronic microscopy (SEM) and second-harmonic generation/two-photon excited fluorescence (SHG/TPEF) were used to image collagen fibril morphology. Results: GLT25D1 expression was upregulated in nonparenchymal cells in human cirrhotic liver tissues. Meanwhile, its knockdown attenuated collagen deposition in BDL-induced mouse liver fibrosis and inhibited mHSC activation. GLT25D1 was overexpressed in activated versus quiescence LX-2 cells and regulated in vitro LX-2 cell activation, including proliferation, contraction, and migration. GLT25D1 also significantly increased liver fibrogenic gene and protein expression. GLT25D1 upregulation promoted HSC activation and enhanced collagen expression through the TGF-ß1/SMAD signaling pathway. Mass spectrometry showed that GLT25D1 regulated the glycosylation of collagen in HSCs, affecting the diameter of collagen fibers. Conclusions: Collectively, the upregulation of GLT25D1 in HSCs promoted the progression of liver fibrosis by affecting HSCs activation and collagen stability.

C6orf120 gene deficiency may be vulnerable to carbon tetrachloride induced acute hepatic injury in rats.

Introduction: The function of the C6orf120 gene, which encodes an N-glycosylated protein, remains unknown. The study was performed to characterize the utility of the C6orf120 gene in carbon tetrachloride-induced acute liver injury and to elucidate the potential underlying mechanisms by establishing a C6orf120 gene-knockout (C6orf120-/-) rat model. Material and methods: C6orf120-/- and wild-type (WT) rats were intraperitoneally administered with CCl4 (1 : 1 v/v in olive oil, 2 µl/g). Rats were sacrificed 24 h after CCl4 administration. Liver tissues were collected for H&E, IHC, qRT-PCR, and Western blot analysis. Results: C6orf120 gene deficiency may be vulnerable to CCl4-induced acute liver injury in rats as indicated by the high levels of alanine aminotransferase (WT: 388.7 ±55.96 vs. C6orf120-/-: 915.9 ±118.8, p < 0.001) and greater degree of pathological damage. Quantitative reverse transcription polymerase chain reaction showed that the mRNA levels of inflammation-associated cytokines, such as interleukin (IL)-1ß, IL-6, and tumor necrosis factor (TNF)-α, in liver tissues were increased in C6orf120-/- rats compared with those in WT rats. Moreover, western blot showed that the protein expression of cytokines nucleotide-binding oligomerization domain leucine rich repeat and pyrin domain containing 3 (NLRP3), caspase-1, IL-1ß, nuclear factor-κB, c-Jun N-terminal kinases, and Bax were increased in C6orf120-/- rats compared with those in WT rats. Conclusions: C6orf120-/- rats were susceptible to CCl4-induced liver injury, which may be related to NLRP3 inflammasome and JNK signaling pathway activation.

Collagen ß(1-O) galactosyltransferase 2 deficiency contributes to lipodystrophy and aggravates NAFLD related to HMW adiponectin in mice.

AIM: Our previous results showed that Colgalt1 knock-out resulted in fetal death on day E11.5, and collagen secretion was retarded. This study aimed to elucidate the role of Collagen ß(1-O) galactosyltransferase 2 (Colgalt2) in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). METHODS: Colgalt2-/- mice were fed a high-fat diet (HFD) or methionine-and choline-deficient diet (MCD). Nanopore long-read RNA-Seq analysis of liver tissues was used to profile genomic variation. In vitro, hepatocyte steatosis and differentiation of primary pre-adipocytes were induced. RESULTS: Colgalt2-/- mice exhibited lipodystrophy, increased body weight, and hepatic lipid accumulation at 6 weeks of age. Colgalt2 deficiency aggravated hepatic steatosis in mice fed an HFD or a standard laboratory chow diet. Colgalt2 deficiency promotes steatohepatitis in MCD-fed mice. In HFD mice, Colgalt2 deficiency caused lipodystrophy and decreased plasma HMW, total adiponectin, and leptin levels. Colgalt2 deficiency also reduced circulating HMW/Total adiponectin in mice fed a HFD diet without differences of adiponectin mRNA and protein level in WT and Colgalt2-/- mice. The nanopore long-read RNA-Seq analysis results revealed transcriptional changes in the adiponectin receptor downstream signaling pathway and lipogenic genes, including the AMPK signaling pathway, adipocytokine signaling pathway, and lipid metabolism (Cidea, Cidec, CD36, and PPARγ). Colgalt2 deficiency did not promote lipid accumulation in OA-induced HepG2 cells or primary hepatocytes. However, Colgalt2 deficiency inhibited adipogenesis and reduced PPARγ, adipogenesis-related transcription factors, and expression during adipocyte differentiation. CONCLUSIONS: In mice, Colgalt2 deficiency contributes to lipodystrophy and promotes NAFLD related to HMW adiponectin. These results suggest that Colgalt2 could be a novel and promising therapeutic strategy for the treatment of NAFLD.

FAM172A Deletion May Enhance Hepatic Steatosis by Promoting ER Stress.

BACKGROUND: Endoplasmic reticulum (ER) stress is one of the major causes to the pathogenesis of nonalcoholic fatty liver disease (NAFLD). Our previous study showed that maintains the homeostasis of ER could effectively alleviate NAFLD. In this study, we found that the loss of FAM172A increased ER stress. AIMS: The aims of this study were to explore whether FAM172A could improve NAFLD by inhibiting ER stress. METHODS: The expression levels of FAM172A and ER stress were detected by western blot. The method of immunofluorescence was used to determine FAM172A location. The interacted proteins of FAM172A were identified by immunocoprecipitation. The methods of MTS and caspase-3/7 activity were taken to confirm the effect of FAM172A on cell viability and proliferation. The expression levels of inflammation were detected by qPCR. RESULTS: We confirmed that FAM172A might alleviate NAFLD through inhibiting ER stress. Loss of FAM172A increased the expressions of ATF6, peIF2α, but decreased the expression of IRE1α. Then, it was shown that FAM172A located in ER and FAM172A directly interacted with ATF6 and peIF2α and IRE1α. More importantly, the binding of FAM172A and eIF2a in tunicamycin-treated group increased significantly compared with the control group. However, the binding of FAM172A and ATF6 or IRE1α did not change. Next, we found that the lack of FAM172A could produce more apoptosis and inflammation. CONCLUSIONS: Our results suggest that FAM172A improve steatosis by alleviating ER stress.

EOGT Correlated With Immune Infiltration: A Candidate Prognostic Biomarker for Hepatocellular Carcinoma.

Background: A preliminary study by our group revealed that the deficiency of EGF domain-specific O-linked N-acetylglucosamine transferase (EOGT) impaired regulatory T-cell differentiation in autoimmune hepatitis. Nevertheless, the prognostic value of EOGT in advanced hepatocellular carcinoma (HCC) and its relationship with immune infiltration remain obscured. Methods: Initially, EOGT expression was evaluated by Oncomine, TIMER, GEO, and UALCAN databases. Besides, the prognostic potential of EOGT expression was analyzed using GEPIA, Kaplan-Meier plotter, CPTAC, Cox regression, and nomogram in HCC samples. Furthermore, we investigated the association between EOGT expression and tumor mutation burden, DNA methylation, and immune infiltration in addition to its possible mechanism via cBioPortal, TIMER, GEPIA, ESTIMATE, CIBERSORT, GSEA, STRING, and Cytoscape. Results: The expression of EOGT in HCC was significantly higher than that in normal tissues. Additionally, elevated EOGT expression was correlated with advanced tumor staging and linked to poor overall survival and relapse-free survival, serving as a significant unfavorable prognostic indicator in HCC patients. Remarkably, our results revealed that high-EOGT expression subgroups with elevated TP53 or low CTNNB1 mutations have worse clinical outcomes than the others. Regarding immune infiltration, immunofluorescent staining showed that immune cells in HCC were positive for EOGT. Besides, elevated EOGT expression was linked to exhausted T cells and immune suppressor cells in HCC samples. More importantly, the proportion of CD8+ T cells was reduced in HCC samples with a high level of EOGT expression, but EOGT did not exhibit prognostic potential in HCC samples with increased CD8+ T cells. Conclusions: EOGT may hold great potential as a novel biomarker to distinguish prognosis and immune profiles of HCC patients.

Deficiency of O-linked-glycosylation regulates activation of T cells and aggravates Concanavalin A-induced liver injury.

OBJECTIVE: Protein glycosylation is involved in immunological recognition and immune cell activation. The role of O-glycosylation in Concanavalin A (Con A)-induced autoimmune hepatitis (AIH) was elucidated in the present study. METHODS: Mice were intravenously injected with Con A (10 mg/kg) to establish an AIH mouse model. Here, 24 h prior to administration of Con A, experimental mice were intragastrically administrated with O-glycosylation inhibitor (benzyl-α-GalNAc) at doses of 1 and 5 mg/kg, respectively, while control mice were administrated with the same volume of saline. Before and after administration of Con A for 6 and 12 h, mice were sacrificed and their plasma and livers were collected to score liver injury. Peripheral blood, spleen, and thymus were collected for flow cytometry analysis. The expression levels of neutrophilic alkaline phosphatase-3 (NALP3) and NALP6 in liver were evaluated as well. RESULTS: Pre-treatment with benzyl-α-GalNAc increased the serum transaminase levels and induced more infiltration and necrosis in livers of Con A administrated mice. The levels of some pro-inflammation cytokines also increased in administrated mice. In addition, pretreatment with benzyl-α-GalNAc up-regulated the expression levels of NALP3 and NALP6. And benzyl-α-GalNAc inhibited the levels of apoptosis of thymus cells and influenced activation of T cells in peripheral blood and spleen of Con A administrated mice, especially that accelerated the physiological progression of CD4+CD25-CD69+ subset. CONCLUSION: The present research demonstrated that benzyl-α-GalNAc aggravated Con A-induced AIH, and the role of the O-glycosylation inhibitor as the aggravation may be related to regulation of the levels of cytokines, as well as influencing proliferation of T cells.

Down-regulation of GLT25D1 inhibited collagen secretion and involved in liver fibrogenesis.

Collagen ß (1-O) galactosyltransferase 1 (GLT25D1) has been reported to transfer galactose to hydroxylysine residues via ß (1-O) linkages in collagen. However, the role of Glt25d1 in liver fibrogenesis is still unknow. Recently, we generated a Glt25d1 knockout mouse to elucidate the role of Glt25d1 in vivo. However, we found that complete deletion of the Glt25d1 gene resulted in embryonic lethality at E11.5. Histopathological analysis revealed that dysplasia in Glt25d1-/- labyrinth with defects of the vascular network. Immunohistochemical showed that the decrease in proliferation of Glt25d1-/- liver and the developing central nervous system (CNS). The role of Glt25d1 in liver fibrogenesis was explored by Glt25d1+/- mice. Glt25d1+/- mice and wild-type (WT) mice were injected intraperitoneally with the same dose of CCl4. The higher level of serum alanine aminotransferase was observed in Glt25d1+/- mice. Reverse transcription-quantitative polymerase chainreaction demonstrated that the mRNA expression levels of the inflammatory cytokines such as, Tnf-α, Cxcl-1 and Mcp-1, showed a significantly increase in CCl4-treated Glt25d1+/- mice. Collagen-I, collagen-III and α-SMA transcripts accumulation was markedly increased in the Glt25d1+/- mice. However, Masson's trichrome staining revealed a trend to decrease in the ECM proteins deposition of Glt25d1+/- liver. Immunohistochemistry and Western blots revealed that the protein expression of Collagen-III was reduced and a trend to a decrease in collagen-I was observed in the Glt25d1+/- liver compared with those of WT mice. Our results demonstrate that Glt25d1 knockout results in embryonic lethality and down-regulation of Glt25d1 may inhibit collagen secretion during liver fibrogenesis.

Diammonium Glycyrrhizinate Mitigates Liver Injury Via Inhibiting Proliferation Of NKT Cells And Promoting Proliferation Of Tregs.

PURPOSE: Diammonium glycyrrhizinate (DG) is a replacement for glycyrrhizic acid, which is used as a hepatic protector in clinical practice for most liver diseases. The potential role of immune response during autoimmune hepatitis-induced by concanavalin A (Con A)-remains to be elucidated. METHODS: C57BL/6J mice were treated with two different doses of DG (75 and 200 mg/kg) 2 hrs before administering Con A. The mice were sacrificed after administering Con A for 0, 6, and 24 hrs. Liver damage grade and serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and total bilirubin levels were evaluated. The expression level of cleaved-caspase 3 in liver was detected by Western blotting. Inflammatory cytokines such as tumor necrosis factor α (TNF-α), interleukin-6 (IL-6), and interferon γ (IFN-γ) in liver were detected by RT-PCR. Thymus, peripheral blood, spleen, and liver tissues were collected to analyze the percentages of NKT cells, subsets of CD4+CD25-CD69+ and CD8+CD69+ T cells, and subsets of regulatory T cells (Tregs). RESULTS: Our results revealed that DG pre-treatment significantly decreased the serum ALT and AST levels and improved the histological damage in Con A-induced autoimmune liver injury. Pre-treatment with DG down-regulated the inflammatory cytokines upon challenge with Con A. The DG pre-treatment inhibited the apoptosis of T lymphocytes in the thymus. Further, it effectively suppressed the proliferation of CD4+CD25-CD69+ and CD8+CD69+ subsets in the peripheral blood and spleen. In addition, the DG pretreatment significantly downregulated the frequency of NKT cells, while upregulating the frequency of Tregs in the liver. CONCLUSION: We believe that the potential protective effect of DG against Con A-induced hepatitis may be partially attributed to its inhibitory activities on inflammatory cytokines in the livers, lymphocyte apoptosis in the thymus, NKT cells proliferation, and activation of CD8+T cells; further, there may also be a possibility of DC promoting Tregs proliferation.

Deficient O-GlcNAc Glycosylation Impairs Regulatory T Cell Differentiation and Notch Signaling in Autoimmune Hepatitis.

Post-translational modifications such as glycosylation play an important role in the functions of homeostatic proteins, and are critical driving factors of several diseases; however, the role of glycosylation in autoimmune hepatitis is poorly understood. Here, we established an O-GlcNAc glycosylation-deficient rat model by knocking out the Eogt gene by TALEN-mediated gene targeting. O-GlcNAc glycosylation deficiency overtly aggravated liver injury in concanavalin-A induced autoimmune hepatitis, and delayed self-recovery of the liver. Furthermore, flow cytometry analysis revealed increased CD4+ T cell infiltration in the liver of rats with O-GlcNAc glycosylation deficiency, and normal differentiation of regulatory T cells (Tregs) in the liver to inhibit T cell infiltration could not be activated. Moreover, in vitro experiments showed that O-GlcNAc glycosylation deficiency impaired Treg differentiation to inhibit the Notch signaling pathway in CD4+ T cells. These finding indicate that O-GlcNAc glycosylation plays a critical role in the activation of Notch signaling, which could promote Treg differentiation in the liver to inhibit T cell infiltration and control disease development in autoimmune hepatitis. Therefore, this study reveals a regulatory role for glycosylation in the pathogenesis of autoimmune hepatitis, and highlights glycosylation as a potential treatment target.