Zhx2 Accelerates Sepsis by Promoting Macrophage Glycolysis via Pfkfb3.

Sepsis is a life-threatening condition with limited therapeutic options, characterized as excessive systemic inflammation and multiple organ failure. Macrophages play critical roles in sepsis pathogenesis. Metabolism orchestrates homeostasis of macrophages. However, the precise mechanism of macrophage metabolism during sepsis remains poorly elucidated. In this study, we identified the key role of zinc fingers and homeoboxes (Zhx2), a ubiquitous transcription factor, in macrophage glycolysis and sepsis by enhancing 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (Pfkfb3) expression. Mice with myeloid Zhx2-specific deletion (abbreviated as MKO) showed more resistance to cecal ligation and puncture and LPS-induced sepsis, exhibiting as prolonged survival, attenuated pulmonary injury, and reduced level of proinflammatory cytokines, such as TNF-α, IL-6, and IL-1ß. Interestingly, Zhx2 deletion conferred macrophage tolerance to LPS-induced glycolysis, accompanied by reduced proinflammatory cytokines and lactate. Consistently, treatment of glycolytic inhibitor 2-deoxyglucose almost completely abrogated the protection of mice from LPS-induced sepsis initiated by Zhx2 deletion in macrophages. RNA sequencing and chromatin immunoprecipitation assays confirmed that Zhx2 enhanced transcription of Pfkfb3, the glycolysis rate-limiting enzyme, via binding with Pfkfb3 promoter. Furthermore, Pfkfb3 overexpression not only rescued the reduction of macrophage glycolysis caused by Zhx2 deficiency, displaying as extracellular acidification rates and lactate production but also destroyed the resistance of mice to LPS-induced sepsis initiated by transfer of bone marrow-derived macrophages from MKO mice. These findings highlight the novel role of transcription factor Zhx2 in sepsis via regulating Pfkfb3 expression and reprogramming macrophage metabolism, which would shed new insights into the potential strategy to intervene sepsis.

ZHX2 inhibits SREBP1c-mediated de novo lipogenesis in hepatocellular carcinoma via miR-24-3p.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death worldwide. Lipogenesis has been considered as a critical player in HCC initiation and progression. However, the underlying mechanism is still not fully understood. Here, we identified zinc fingers and homeoboxes 2 (ZHX2), an HCC-associated tumor suppressor, as an important repressor of de novo lipogenesis. Ectopic expression of ZHX2 significantly inhibited de novo lipogenesis in HCC cells and decreased expression of FASN, ACL, ACC1, and SCD1. In accordance with this, ZHX2 was negatively associated with SREBP1c, the master regulator of de novo lipogenesis, in HCC cell lines and human specimens. Results from silencing and overexpression demonstrated that ZHX2 inhibited de novo lipogenesis and consequent HCC progression via repression of SREBP1c. Furthermore, treatment with the SREBP1c inhibitor fatostatin dampened the spontaneous formation of tumors in liver-specific Zhx2 knockout mice. Mechanistically, ZHX2 increased expression of miR-24-3p transcriptionally, which targeted SREBP1c and led to its degradation. In conclusion, our data suggest a novel mechanism through which ZHX2 suppresses HCC progression, which may provide a new strategy for the treatment of HCC. © 2020 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Tim-3 blockade promotes iNKT cell function to inhibit HBV replication.

Increased expression of T cell immunoglobulin and mucin domain-3 (Tim-3) on invariant natural killer T (iNKT) cells is reported in chronic hepatitis B virus (HBV) infection. However, whether Tim-3 regulates iNKT cells in chronic HBV condition remains unclear. In this study, our results showed that the expression of Tim-3 was up-regulated on hepatic iNKT cells from HBV-transgenic (Tg) mice or iNKT cells stimulated with α-galactosylceramide (α-Galcer). Compared with Tim-3- iNKT cells, Tim-3+ iNKT cells expressed more IFN-γ, IL-4 and CD107a, indicating a strong relationship between Tim-3 and iNKT cell activation. Constantly, treatment of Tim-3 blocking antibodies significantly enhanced the production of IFN-γ, TNF-α, IL-4 and CD107a in iNKT cells both in vivo and in vitro. This Tim-3- mediated suppression of iNKT cells was further confirmed in Tim-3 knockout (KO) mice. Moreover, Tim-3 blockade promoted α-Galcer-triggered inhibition of HBV replication, displaying as the decreased HBV DNA and HBsAg level in serum, and down-regulated pgRNA expression in liver tissues. Collectively, our data, for the first time, demonstrated the potential role of Tim-3 blockade in promoting iNKT cell-mediated HBV inhibition. Therefore, combination of α-Galcer with Tim-3 blockade might be a promising approach in chronic hepatitis B therapy.

HBV suppresses ZHX2 expression to promote proliferation of HCC through miR-155 activation.

Initiation of hepatocellular carcinoma (HCC) by chronic hepatitis B virus (HBV) infection is a complex process that includes both oncogene activation and tumor suppressor inhibition. The HBV X (HBx) protein has an important and complex role in processes leading to HCC. We previously identified the mammalian Zinc fingers and homeoboxes 2 (ZHX2) gene as an HCC-associated tumor suppressor gene. In the present study, we investigated whether the oncogenic properties of HBV and, more specifically, HBx, involved ZHX2 silencing. Our data indicates that ZHX2 expression is significantly decreased in tumor tissues from HBV-positive HCC patients and livers from HBV transgenic mice. In vitro and in vivo studies confirmed that HBV-encoded proteins, particularly HBx, inhibits both the expression and tumor suppression properties of ZHX2. Further analyses identified miR-155, a well-known oncomiR in various cancers, as an important link between HBx and ZHX2 inhibition. Increased miR-155 levels were found in HBV-positive tumors, livers of HBV transgenic mice and HBx-overexpressing hepatoma cell lines. MiR-155 overexpression reduced ZHX2 levels via miR-155 seed sites in the ZHX2 3'UTR, whereas blocking miR-155 levels led to increased ZHX2 levels. Taken together, our data indicate that HCC-promoting properties of HBV may include ZHX2 silencing via a miR-155 dependent pathway and suggests a novel therapy for HBV-related HCC.

Tim-3 fosters HCC development by enhancing TGF-ß-mediated alternative activation of macrophages.

OBJECTIVE: Tumour-associated macrophages (TAMs) and their alternative activation contribute greatly to the development of hepatocellular carcinoma (HCC). Tim-3 is highly expressed on macrophages and regulates macrophage functions in several conditions. However, whether Tim-3 is involved in the activation and the function of TAMs has not been reported. DESIGN: Tim-3 expression in HCC samples was evaluated by flow cytometry, immunohistochemistry and confocal analysis. We analysed the effects of Tim-3 knockdown on macrophages in growth of H22 tumour homografts in BALB/c mice. Tim-3 interference was performed by neutralising antibody, small interfering RNA or short hairpin RNA-expressing lentivirus. Cytokine production was evaluated by reverse transcription PCR, ELISA or Cytometric Bead Array. The effects of Tim-3 interference in macrophages were examined with regard to alternative activation of macrophages and proliferation and migration of Hepa1-6 cells. Cell growth curve, colony formation and transwell assays were involved to estimate cell proliferation and migration. RESULTS: Tim-3 expression was significantly increased in both peripheral blood monocytes and TAMs in patients with HCC. The Tim-3 expression in monocytes/TAMs strongly correlated with higher tumour grades and the poor survival of patients with HCC. Consistently, HCC conditioned medium or transforming growth factor-ß fostered Tim-3 expression and the alternative activation of macrophages. Moreover, Tim-3 interference in macrophages significantly inhibited the alternative activation of macrophages and suppressed HCC cell growth both in vitro and in vivo. Blocking interleukin 6 reversed the Tim-3-mediated effects on HCC cell growth in vitro. CONCLUSIONS: Tim-3 displays critical roles in microenvironment-induced activation and protumoral effects of TAMs in HCC. Interference of Tim-3 might be great potential in HCC therapy.

DAPK1 mediates cognitive dysfunction and neuronal apoptosis in PSD rats through the ERK/CREB/BDNF signaling pathway.

Post-stroke depression (PSD) is one of the most common mental sequelae after a stroke and can damage the brain. Although PSD has garnered increasing attention in recent years, the precise mechanism remains unclear. Studies have indicated that the expression of DAPK1 is elevated in various neurodegenerative conditions, including depression, ischemic stroke, and Alzheimer's disease. However, the specific molecular mechanism of DAPK1-mediated cognitive dysfunction and neuronal apoptosis in PSD rats is unclear. In this study, we established a rat model of PSD, and then assessed depression-like behaviors and cognitive dysfunction in rats using behavioral tests. In addition, we detected neuronal apoptosis and analyzed the expression of DAPK1 protein and proteins related to the ERK/CREB/BDNF signaling pathway. The findings revealed that MCAO combined with CUMS can induce more severe depression-like behaviors and cognitive dysfunction in rats, while overexpression of DAPK1 may hinder the downstream ERK/CREB/BDNF signaling pathways, resulting in neuronal loss and exacerbation of brain tissue damage. In this study, we will focus on DAPK1 and explore its role in PSD.

UBASH3B-mediated MRPL12 Y60 dephosphorylation inhibits LUAD development by driving mitochondrial metabolism reprogramming.

BACKGROUND: Metabolic reprogramming plays a pivotal role in tumorigenesis and development of lung adenocarcinoma (LUAD). However, the precise mechanisms and potential targets for metabolic reprogramming in LUAD remain elusive. Our prior investigations revealed that the mitochondrial ribosomal protein MRPL12, identified as a novel mitochondrial transcriptional regulatory gene, exerts a critical influence on mitochondrial metabolism. Despite this, the role and regulatory mechanisms underlying MRPL12's transcriptional activity in cancers remain unexplored. METHODS: Human LUAD tissues, Tp53fl/fl;KrasG12D-driven LUAD mouse models, LUAD patient-derived organoids (PDO), and LUAD cell lines were used to explored the expression and function of MRPL12. The posttranslational modification of MRPL12 was analyzed by mass spectrometry, and the oncogenic role of key phosphorylation sites of MRPL12 in LUAD development was verified in vivo and in vitro. RESULTS: MRPL12 was upregulated in human LUAD tissues, Tp53fl/fl;KrasG12D-driven LUAD tissues in mice, LUAD PDO, and LUAD cell lines, correlating with poor patient survival. Overexpression of MRPL12 significantly promoted LUAD tumorigenesis, metastasis, and PDO formation, while MRPL12 knockdown elicited the opposite phenotype. Additionally, MRPL12 deletion in a Tp53fl/fl;KrasG12D-driven mouse LUAD model conferred a notable survival advantage, delaying tumor onset and reducing malignant progression. Mechanistically, we discovered that MRPL12 promotes tumor progression by upregulating mitochondrial oxidative phosphorylation. Furthermore, we identified UBASH3B as a specific binder of MRPL12, dephosphorylating tyrosine 60 in MRPL12 (MRPL12 Y60) and inhibiting its oncogenic functions. The decrease in MRPL12 Y60 phosphorylation impeded the binding of MRPL12 to POLRMT, downregulating mitochondrial metabolism in LUAD cells. In-depth in vivo, in vitro, and organoid models validated the inhibitory effect of MRPL12 Y60 mutation on LUAD. CONCLUSION: This study establishes MRPL12 as a novel oncogene in LUAD, contributing to LUAD pathogenesis by orchestrating mitochondrial metabolism reprogramming towards oxidative phosphorylation (OXPHOS). Furthermore, it confirms Y60 as a specific phosphorylation modification site regulating MRPL12's oncogenic functions, offering insights for the development of LUAD-specific targeted drugs and clinical interventions.

Acute Macular Neuroretinopathy Associated with COVID-19 Pandemic: A Real-world Observation Study.

PURPOSE: To evaluate the clinical and retinal imaging features of Chinese patients with acute macular neuroretinopathy (AMN) associated with COVID-19. DESIGN: A prospective observational study. METHODS: Retinal imaging, including color fundus photography, near-infrared imaging (NIR), swept-source optical coherence tomography (SS-OCT), optical coherence tomography angiography (OCTA), and Humphrey perimetry, were conducted for each case. RESULTS: All cases were included within the first three months following the pandemic outbreak. A total of 12 male patients (36.36 %) and 21 female patients (63.64 %) were prospectively recruited, and 29 cases (87.88 %) were bilaterally affected. The median interval between the onset of fever and the appearance of ocular symptoms was two days (range, 0.5-5.0 days). Apart from the outer retinal changes typical of AMN, changes in the inner retinal layers were observed, including intraretinal hemorrhage (8.06 %), cotton wool spots (9.68 %), and paracentral acute middle maculopathy (PAMM) (8.06 %). Smaller retinal inner nuclear layer hyperreflective speckles (RIHS) (41.94 %) were identified as a distinguishing feature from typical PAMM. Voids of vessel signals were found in the superficial (11.54 %), intermediate (82.69 %), and deep capillary plexus (98.08 %), and in the choriocapillaris (19.23 %) on OCTA. Humphrey perimetry illustrated central, paracentral, and peripheral scotomas. The occult lesions associated with AMN, PAMM, and some of the RIHS illustrated by OCT were visualized topographically and further confirmed by OCTA as perfusion defects. CONCLUSION: An increase in AMN cases correlated with the SARS-CoV-2 outbreak. Additional features, including widespread inner retinal perfusion deficits, were observed and may serve as potential biomarkers for systemic microcirculation dysregulation in COVID-19.

miR-139-5p and miR-451a as a Diagnostic Biomarker in LUSC.

Background: Lung squamous cell carcinoma (LUSC) is a type of lung cancer that originates from segmental or subsegmental bronchial mucosa. There is evidence that miRNA plays an important role in the occurrence and progression of tumors. Methods: In this study, plasma samples of patients with early LUSC and healthy volunteers were subjected to miRNA sequencing, and the levels of differentially expressed miRNAs (DEMs) in LUSC tissues were analyzed using R language. Cox regression and Kaplan-Meier (K-M) survival curve analyses were performed to determine the relationship between DEMs and prognosis in LUSC, and PCR method was verified for the plasma expression level of DEMs in patients with LUSC. The levels of CYFRA21-1 and SCC-Ag in plasma were measured, and area under curve (AUC) was used to evaluate the diagnostic value of the DEMs. Results: A total of 21 DEMs were screened out by sequencing. The expression levels of DEMs in tissue samples in the TCGA database were analyzed, and four DEMs with consistent expression levels were further screened from plasma and tissue samples. Regression analysis and K-M curve were performed to select two DEMs (miR-139-5p, miR-451a) that were correlated with the prognosis. PCR verification results showed that the levels of miR-451a and miR-139-5p were low in patients, and the level of miR-139-5p in late stages III & IV with the patients of LUSC was higher than that in stages I & II. The AUC values of the four indicators (SCC-Ag, CYFRA21-1, miR-451a and miR-139-5p) in the diagnosis of LUSC, early and late cases were 0.884, 0.935 and 0.778, respectively. Conclusion: The detection of miR-139-5p and miR-451a levels in plasma has a certain potential in the non-invasive diagnosis, especially in patients with early stages of LUSC.

ZHX2 emerges as a negative regulator of mitochondrial oxidative phosphorylation during acute liver injury.

Mitochondria dysfunction contributes to acute liver injuries, and mitochondrial regulators, such as PGC-1α and MCJ, affect liver regeneration. Therefore, identification of mitochondrial modulators may pave the way for developing therapeutic strategies. Here, ZHX2 is identified as a mitochondrial regulator during acute liver injury. ZHX2 both transcriptionally inhibits expression of several mitochondrial electron transport chain genes and decreases PGC-1α stability, leading to reduction of mitochondrial mass and OXPHOS. Loss of Zhx2 promotes liver recovery by increasing mitochondrial OXPHOS in mice with partial hepatectomy or CCl4-induced liver injury, and inhibition of PGC-1α or electron transport chain abolishes these effects. Notably, ZHX2 expression is higher in liver tissues from patients with drug-induced liver injury and is negatively correlated with mitochondrial mass marker TOM20. Delivery of shRNA targeting Zhx2 effectively protects mice from CCl4-induced liver injury. Together, our data clarify ZHX2 as a negative regulator of mitochondrial OXPHOS and a potential target for developing strategies for improving liver recovery after acute injuries.

CD169-positive macrophages enhance abscopal effect of radiofrequency ablation therapy in liver cancer.

Radiofrequency ablation (RFA) is a widely used and effective treatment for primary or metastatic liver cancer with small-size lesions. However, the therapeutic effectiveness of RFA in controlling metastatic lesion or recurrence is still limited. As the major cell population in tumor microenvironment (TME), macrophages have been reported to be recruited to RFA-treated lesion, but their roles are still unclear. Herein, we successfully established the mouse model mimicking RFA-induced abscopal effect, in which RFA eliminated the local orthotopic liver tumor but failed to control growth of distant tumor. Correspondently, RFA suppressed protumoral activation of local tumor-associated macrophages (TAMs), but failed to reprogram TAMs in distance. Importantly, although RFA led to reduced proportion of hepatic CD169+ macrophages in local and decreased expression of immune inhibitory molecules Tim-3 and PD-L1, these alterations were not observed for CD169+ macrophages in distant TME. Further RNA-seq and flow cytometry analysis showed that hepatic CD169+ macrophages contributed to reprograming TME through recruiting CD8+ T/NK cells and suppressing accumulation of MDSCs/Tregs. Consistently, depletion of CD169+ macrophages in CD169-DTR mouse greatly promoted liver tumor progression and largely dampened RFA-induced tumor suppression. Notably, transfer of CD169+ macrophages synergistically enhanced RFA-induced inhibition of distant tumor. To our knowledge, this is the first study which demonstrates hepatic CD169+ macrophages as a key factor responsible for RFA-induced abscopal effect. Our data suggest RFA with transfer of CD169+ macrophages as a promising combination therapy to lessen metastasis or recurrence of liver cancer in patients.

LINC01431 Promotes Histone H4R3 Methylation to Impede HBV Covalently Closed Circular DNA Transcription by Stabilizing PRMT1.

Covalently closed circular DNA (cccDNA) is the transcriptional template of hepatitis B virus (HBV), which interacts with both host and viral proteins to form minichromosome in the nucleus and is resistant to antiviral agents. Identification of host factors involved in cccDNA transcriptional regulation is expected to prove a new venue for HBV therapy. Recent evidence suggests the involvement of long noncoding RNAs (lncRNAs) in mediating the interaction of host factors with various viruses, however, lncRNAs that HBV targets and represses cccDNA transcription have not been fully elucidated. Here, the authors identified LINC01431 as a novel host restriction factor for HBV transcription. Mechanically, LINC01431 competitively bound with type I protein arginine methyltransferase (PRMT1) to block the HBx-mediated PRMT1 ubiquitination and degradation. Consequently, LINC01431 increased the occupancy of PRMT1 on cccDNA, leading to enhanced H4R3me2a modification and reduced acetylation of cccDNA-bound histones, thereby repressing cccDNA transcription. In turn, to facilitate viral replication, HBV transcriptionally repressed LINC01431 expression by HBx-mediated repression of transcription factor Zinc fingers and homeoboxes 2 (ZHX2). Collectively, the study demonstrates LINC01431 as a novel epigenetic regulator of cccDNA minichromosome and highlights a feedback loop of HBx-LINC01431-PRMT1 in HBV replication, which provides potential therapeutic targets for HBV treatment.

Adipose-derived stem cell sheets combined with ß-tricalcium phosphate/collagen-I fiber scaffold improve cell osteogenesis.

Transplantation of cell-based material is a promising approach for the treatment of critical bone defects. However, it is still limited by the lack of suitable scaffold material or abundant seeding cell sources. The present study aimed to establish a novel composite of an adipose-derived stem cell (ADSC) sheet and a synthetic porous ß-tricalcium phosphate/collagen-I fiber (ß-TCP/COL-I) scaffold to enhance osteogenic activity. ADSCs were isolated from 3-week-old female Sprague Dawley rats and the ADSC sheets were prepared in an osteoinductive medium. The study groups included the ADSC sheets/scaffold, scattered ADSCs/scaffold, ADSC sheet alone and scaffold alone. Scanning electron microscopy and energy-dispersive spectrometry were used to observe cell-scaffold interactions and analyze the relative calcium content on the composites' surface. Alizarin red S staining was used to examine the calcium deposition. ELISA and reverse transcription-quantitative PCR were used to detect the expression levels of alkaline phosphatase (ALP), osteocalcin (OCN) and osteopontin (OPN). The results revealed that ADSCs were able to tightly adhere to the ß-TCP/COL-I scaffold with no cytotoxicity. The calcifying nodules reaction was positive on ADSC sheets and gradually increased after osteogenic induction. In addition, the ß-TCP/COL-I scaffold combined with ADSC sheets was able to significantly enhance the expression levels of ALP, OCN and OPN and increase the superficial relative calcium content compared to scattered ADSCs/scaffold or the ADSC sheet alone (P<0.05). The results indicated that ADSCs possess a strong osteogenic potential, particularly in the cell-sheet form and when compounded with the ß-TCP/COL-I scaffold, compared to scattered ADSCs with a ß-TCP/COL-I scaffold or an ADSC sheet alone. This novel composite may be a promising candidate for bone engineering.

Hepatitis B virus evades immune recognition via RNA adenosine deaminase ADAR1-mediated viral RNA editing in hepatocytes.

HBV is considered as a "stealth" virus that does not invoke interferon (IFN) responses; however, the mechanisms by which HBV bypasses innate immune recognition are poorly understood. In this study, we identified adenosine deaminases acting on RNA 1 (ADAR1), which is a key factor in HBV evasion from IFN responses in hepatocytes. Mechanically, ADAR1 interacted with HBV RNAs and deaminated adenosine (A) to generate inosine (I), which disrupted host immune recognition and thus promoted HBV replication. Loss of ADAR1 or its deficient deaminase activity promoted IFN responses and inhibited HBV replication in hepatocytes, and blocking the IFN signaling pathways released the inhibition of HBV replication caused by ADAR1 deficiency. Notably, the HBV X protein (HBx) transcriptionally promoted ADAR1 expression to increase the threshold required to trigger intrinsic immune activation, which in turn enhanced HBV escape from immune recognition, leading to persistent infection. Supplementation with 8-azaadenosine, an ADAR1 inhibitor, efficiently enhanced liver immune activation to promote HBV clearance in vivo and in vitro. Taken together, our results delineate a molecular mechanism by which HBx promotes ADAR1-derived HBV immune escape and suggest a targeted therapeutic intervention for HBV infection.

Tim-3 Hampers Tumor Surveillance of Liver-Resident and Conventional NK Cells by Disrupting PI3K Signaling.

Natural killer (NK) cells are enriched within the liver. Apart from conventional NK (cNK) cells, recent studies identified a liver-resident NK (LrNK) subset, which constitutes about half of hepatic NK cells and exhibits distinct developmental, phenotypic, and functional features. However, it remains unclear whether and how LrNK cells, as well as cNK cells, participate in the development of hepatocellular carcinoma (HCC) individually. Here, we report that both LrNK and cNK cells are significantly decreased in HCC. The T-cell immunoglobulin and mucin domain-containing protein 3 (Tim-3) was significantly upregulated in both tumor-infiltrating LrNK and cNK cells and suppressed their cytokine secretion and cytotoxic activity. Mechanistically, phosphatidylserine (PtdSer) engagement promoted phosphorylation of Tim-3, which then competed with PI3K p110 to bind p85, inhibiting downstream Akt/mTORC1 signaling and resulting in malfunctioning of both NK-cell subsets. Tim-3 blockade retarded HCC growth in a NK-cell-dependent manner. These studies for the first time report the presence and dysfunction of LrNK cells in HCC and show that Tim-3-mediated PI3K/mTORC1 interference is responsible for the dysfunction of both tumor-infiltrating cNK and LrNK cells, providing a new strategy for immune checkpoint-based targeting. SIGNIFICANCE: Tim-3 enhances hepatocellular carcinoma growth by blocking natural killer cell function.

Tumor suppressor ZHX2 inhibits NAFLD-HCC progression via blocking LPL-mediated lipid uptake.

Non-alcoholic fatty liver disease (NAFLD) leads to hepatocellular carcinoma (HCC). However, the underlying mechanism remains largely unclear. Here, we investigated the role of the tumor suppressor Zinc fingers and homeoboxes 2 (ZHX2) in the progression of NAFLD to HCC. ZHX2 expression was significantly decreased in fatty liver tissues, especially in the liver with NAFLD-HCC. ZHX2 overexpression disturbed lipid homeostasis of cultured HCC cells, and inhibited lipid deposition in hepatocytes both in vitro and in vivo. Moreover, ZHX2 inhibited uptake of exogenous lipids through transcriptional suppression of lipid lipase (LPL), leading to retarded proliferation of HCC cells. Importantly, LPL overexpression significantly reversed ZHX2-mediated inhibition of HCC cell proliferation, xenograft tumor growth, lipid deposition, and spontaneous liver tumor formation. Consistently, IHC staining demonstrated a negative correlation of ZHX2 with LPL in an HCC cohort. Collectively, ZHX2 protects hepatocytes from abnormal lipid deposition in NAFLD through transcriptional repression of LPL, which subsequently retards cell growth and NAFLD-HCC progression. These findings illustrate a novel mechanism of NAFLD progression into HCC.

ZHX2 restricts hepatocellular carcinoma by suppressing stem cell-like traits through KDM2A-mediated H3K36 demethylation.

BACKGROUND: Liver cancer stem cells (CSCs) are critical determinants of HCC relapse and therapeutic resistance, but the mechanisms underlying the maintenance of CSCs are poorly understood. We aimed to explore the role of tumor repressor Zinc-fingers and homeoboxes 2 (ZHX2) in liver CSCs. METHODS: CD133+ or EPCAM+ stem-like liver cancer cells were sorted from tumor tissues of HCC patients and HCC cell lines by flow cytometry. In addition, sorafenib-resistant cells, tumor-sphere forming cells and side population (SP) cells were respectively cultured and isolated as hepatic CSCs. The tumor-initiating and chemoresistance properties of ZHX2-overexpressing and ZHX2-knockdown cells were analyzed in vivo and in vitro. Microarray, luciferase reporter assay, chromatin immunoprecipitation (ChIP) and ChIP-on-chip analyses were performed to explore ZHX2 target genes. The expression of ZHX2 and its target gene were determined by quantitative RT-PCR, western blot, immunofluorescence and immunohistochemical staining in hepatoma cells and tumor and adjacent tissues from HCC patients. RESULTS: ZHX2 expression was significantly reduced in liver CSCs from different origins. ZHX2 deficiency led to enhanced liver tumor progression and expansion of CSC populations in vitro and in vivo. Re-expression of ZHX2 restricted capabilities of hepatic CSCs in supporting tumor initiation, self-renewal and sorafenib-resistance. Mechanically, ZHX2 suppressed liver CSCs via inhibiting KDM2A-mediated demethylation of histone H3 lysine 36 (H3K36) at the promoter regions of stemness-associated transcription factors, such as NANOG, SOX4 and OCT4. Moreover, patients with lower expression of ZHX2 and higher expression of KDM2A in tumor tissues showed significantly poorer survival. CONCLUSION: ZHX2 counteracts stem cell traits through transcriptionally repressing KDM2A in HCC. Our data will aid in a better understanding of molecular mechanisms underlying HCC relapse and drug resistance.

Tim-3 aggravates podocyte injury in diabetic nephropathy by promoting macrophage activation via the NF-κB/TNF-α pathway.

OBJECTIVE: Macrophage-mediated inflammation plays a significant role in the development and progression of diabetic nephropathy (DN). However, the underlying mechanisms remain unclear. Studies suggest that T cell immunoglobulin domain and mucin domain-3 (Tim-3) has complicated roles in regulating macrophage activation, but its roles in the progression of DN are still completely unknown. METHODS: We downregulated Tim-3 expression in kidney (intrarenal injection of Tim-3 shRNA expressing lentivirus or global Tim-3 knockout mice) and induced DN by streptozotocin (STZ). We analyzed the degree of renal injury, especially the podocyte injury induced by activated macrophages in vitro and in vivo. Then, we transferred different bone marrow derived macrophages (BMs) into STZ-induced Tim-3 knockdown mice to examine the effects of Tim-3 on macrophages in DN. RESULTS: First, we found that Tim-3 expression on renal macrophages was increased in patients with DN and in two diabetic mouse models, i.e. STZ-induced diabetic mice and db/db mice, and positively correlated with renal dysfunction of DN patients. Tim-3 deficiency ameliorated renal damage in STZ-induced diabetes with concurrent increase in protein levels of Nephrin and WT-1. Similar effects were observed in mice with Tim-3 knockdown diabetic mice. Second, adoptive transfer of Tim-3-expressing macrophages, but not Tim-3 knockout macrophages, accelerated diabetic renal injury in DN mice, suggesting a key role for Tim-3 on macrophages in the development of DN. Furthermore, we found NF-κB activation and TNF-α excretion were upregulated by Tim-3 in diabetic kidneys, and podocyte injury was associated with the Tim-3-mediated activation of the NF-κB/TNF-α signaling pathway in DN macrophages both in vivo and in vitro. CONCLUSIONS: These results suggest that Tim-3 functions as a key regulator in renal inflammatory processes and serves as a potential therapeutic target for renal injury in DN.