Isthmus progenitor cells contribute to homeostatic cellular turnover and support regeneration following intestinal injury.

The currently accepted intestinal epithelial cell organization model proposes that Lgr5+ crypt-base columnar (CBC) cells represent the sole intestinal stem cell (ISC) compartment. However, previous studies have indicated that Lgr5+ cells are dispensable for intestinal regeneration, leading to two major hypotheses: one favoring the presence of a quiescent reserve ISC and the other calling for differentiated cell plasticity. To investigate these possibilities, we studied crypt epithelial cells in an unbiased fashion via high-resolution single-cell profiling. These studies, combined with in vivo lineage tracing, show that Lgr5 is not a specific ISC marker and that stemness potential exists beyond the crypt base and resides in the isthmus region, where undifferentiated cells participate in intestinal homeostasis and regeneration following irradiation (IR) injury. Our results provide an alternative model of intestinal epithelial cell organization, suggesting that stemness potential is not restricted to CBC cells, and neither de-differentiation nor reserve ISC are drivers of intestinal regeneration.

An optimized protocol for isolation of murine pancreatic single cells with high yield and purity.

Here, we present a protocol for rapidly isolating single cells from the mouse pancreas, minimizing damage caused by digestive enzymes in exocrine cells. We guide you through steps to optimize the dissection sequence, enzyme composition, and operational procedures, resulting in high yields of viable pancreatic single cells. This protocol can be applied across a wide range of research areas, including single-cell sequencing, gene expression profiling, primary cell culture, and even the development of spheroids or organoids. For complete details on the use and execution of this protocol, please refer to Jiang et al. (2023).1.

Identification and Validation of Chromobox Family Members as Potential Prognostic Biomarkers and Therapeutic Targets for Human Esophageal Cancer.

Background: Chromobox family proteins (CBXs) are vital components of epigenetic regulation complexes and transcriptionally inhibit target genes by modifying the chromatin. Accumulating evidence indicates that CBXs are involved in the initiation and progression of multiple malignancies. However, the expression, function, and clinical relevance such as the prognostic and diagnostic values of different CBXs in esophageal carcinoma (ESCA) are still unclear. Methods: We applied Oncomine, TCGA, GEO, GEPIA, UALCAN, Kaplan-Meier plotter, cBioPortal, Metascape, and TIMER to investigate the roles of CBX family members in ESCA. Additionally, quantitative real-time PCR (RT-PCR), western blot, and immunofluorescence were used to verify the expression of CBX family members in ESCA clinical samples. Results: Compared with normal tissues, the mRNA expression levels of CBX1/3/8 were significantly increased in ESCA, whereas CBX7 mRNA expression was reduced in both the TCGA cohort and GEO cohort. In the TCGA cohort, ROC curves suggested that CBX1/2/3/4/8 had great diagnostic value in ESCA, and the AUCs were above 0.9. Furthermore, upregulation of CBX1/3/8 and downregulation of CBX7 were closely related to the clinicopathological parameters in ESCA patients, such as tumor grades, tumor nodal metastasis status, and TP53 mutation status. The survival analysis indicated that higher CBX1/3/8 mRNA expressions and lower CBX7 expression suggested an unfavorable prognosis in ESCA. High genetic change rate (52%) of CBXs was found in ESCA patients. Functions and pathways of mutations in CBXs and their 50 frequently altered neighbor genes in ESCA patients were investigated; the results showed that DNA repair and DNA replication were correlated to CBX alterations. Moreover, we found a significant correlation between the expression level of CBX family members and the infiltration of immune cells in ESCA. Finally, we verified the expression of CBX family members in clinical samples and found the results were consistent with the databases. Conclusion: Our study implied that CBX1/3/7/8 are potential targets of precision therapy for ESCA patients and new biomarkers for the prognosis.

A new murine esophageal organoid culture method and organoid-based model of esophageal squamous cell neoplasia.

Organoids mimic the physiologic and pathologic events of organs. However, no consensus on esophageal organoid (EO) culture methods has been reached. Moreover, organoid models reproducing esophageal squamous cell carcinoma (ESCC) initiation have been unavailable. Herein, we sought to develop an esophageal minimum essential organoid culture medium (E-MEOM) for culturing murine EOs and establishing an early ESCC model. We formulated E-MEOM to grow EOs from a single cell with clonal expansion, maintenance, and passage. We found that EOs cultured in E-MEOM were equivalent to the esophageal epithelium by histological analysis and transcriptomic study. Trp53 knockout and Kras G12D expression in EOs induced the development of esophageal squamous neoplasia, an early lesion of ESCC. Here we propose the new formula for EO culture with minimum components and the organoid model recapitulating ESCC initiation, laying the foundation for ESCC research and drug discovery.

Development of Rofecoxib-Based Fluorescent Probes and Investigations on Their Solvatochromism, AIE Activity, Mechanochromism, and COX-2-Targeted Bioimaging.

Cyclooxygenase-2 (COX-2) fluorescent probes are promising tools for early diagnosis of cancer. Traditionally, COX-2 probes were designed by connecting two parts, a fluorophore and a COX-2 binding unit, via a flexible linker. Herein, a new class of COX-2-specific fluorescent probes have been developed via one-step modification from rofecoxib by an integrative approach to combine the binding unit and the fluorophore into one. Among them, several new rofecoxib analogues not only exhibited still potent COX-2 binding ability but also exhibited attractive fluorescence properties, such as tunable blue-red emission, solvatochromism, aggression-induced emission behavior, and mechanochromism. Notably, the emission of 2a16 can be switched between green-yellow in the crystalline state and red-orange in the amorphous state by grinding and fuming treatments. Furthermore, the highly fluorescent compound 2a16 (Φf = 0.94 in powder) displayed a much stronger fluorescence imaging of COX-2 in HeLa cancer cells overexpressing COX-2 than RAW264.7 normal cells with a minimal expression of COX-2. Most importantly, 2a16 can light up human cancer tissues from adjacent normal tissues with a much brighter fluorescence by targeting the COX-2 enzyme. These results demonstrated the potential of 2a16 as a new red fluorescent probe for human cancer imaging in clinical applications.

Resveratrol Improves Liver Steatosis and Insulin Resistance in Non-alcoholic Fatty Liver Disease in Association With the Gut Microbiota.

Resveratrol (RSV) is a potential alternative therapy for non-alcoholic fatty liver disease (NAFLD) that has been evaluated in many clinical trials, but the mechanisms of RSV action have not been fully elucidated. Recent studies suggested that the gut microbiota is an important RSV target; therefore, we speculated that the gut microbiota might mediate the beneficial effects of RSV in NAFLD. To verify this hypothesis, we established a high-fat diet (HFD)-induced NAFLD mouse model, which was subjected to RSV gavage to evaluate the therapeutic effects. We observed that RSV reduced liver steatosis and insulin resistance in NAFLD. RSV significantly changed the diversity and composition of the gut microbiota according to 16S rRNA sequencing. Gut microbiota gene function prediction showed that the enrichment of pathways related to lipid and glucose metabolism decreased after RSV treatment. Furthermore, correlation analysis indicated that the improvements in NAFLD metabolic indicators were closely related to the altered gut microbiota. We further fermented RSV with the gut microbiota in vitro to verify that RSV directly affected the gut microbiota. Our data suggested that the gut microbiota might be an important target through which RSV exerts its anti-NAFLD effect.

TMEM9-v-ATPase Activates Wnt/ß-Catenin Signaling Via APC Lysosomal Degradation for Liver Regeneration and Tumorigenesis.

BACKGROUND AND AIMS: How Wnt signaling is orchestrated in liver regeneration and tumorigenesis remains elusive. Recently, we identified transmembrane protein 9 (TMEM9) as a Wnt signaling amplifier. APPROACH AND RESULTS: TMEM9 facilitates v-ATPase assembly for vesicular acidification and lysosomal protein degradation. TMEM9 is highly expressed in regenerating liver and hepatocellular carcinoma (HCC) cells. TMEM9 expression is enriched in the hepatocytes around the central vein and acutely induced by injury. In mice, Tmem9 knockout impairs hepatic regeneration with aberrantly increased adenomatosis polyposis coli (Apc) and reduced Wnt signaling. Mechanistically, TMEM9 down-regulates APC through lysosomal protein degradation through v-ATPase. In HCC, TMEM9 is overexpressed and necessary to maintain ß-catenin hyperactivation. TMEM9-up-regulated APC binds to and inhibits nuclear translocation of ß-catenin, independent of HCC-associated ß-catenin mutations. Pharmacological blockade of TMEM9-v-ATPase or lysosomal degradation suppresses Wnt/ß-catenin through APC stabilization and ß-catenin cytosolic retention. CONCLUSIONS: Our results reveal that TMEM9 hyperactivates Wnt signaling for liver regeneration and tumorigenesis through lysosomal degradation of APC.

One-Step Transformation from Rofecoxib to a COX-2 NIR Probe for Human Cancer Tissue/Organoid Targeted Bioimaging.

COX-2 fluorescent probes are promising tools for cancer diagnosis. Such probes have been conventionally designed by conjugating a fluorophore to COX-2 inhibitors through lengthy synthetic processes. Herein, a type of fluorescent probe for COX-2 imaging has been developed using a single-step process from rofecoxib. In total, six rofecoxib analogues were designed using this unique strategy. Several analogues retained comparative COX-2 targeting activity of rofecoxib and also exhibited attractive fluorescent properties, which were investigated using a combination of experimental and theoretical approaches. The most potent analogue, 2a1, displayed strong fluorescent imaging of COX-2 in HeLa cells overexpressing COX-2 compared to Raw 264.7 cells and celecoxib-treated HeLa cells that expressed low levels of COX-2. Notably, our studies indicate that 2a1 can differentiate human cancer tissue from adjacent tissue with much brighter fluorescence either in histological section or cultured 3D organoids. These results illustrate the potential of 2a1 as a COX-2 near infrared fluorescent probe for human cancer imaging in clinical settings.

HBx promotes the proliferative ability of HL­7702 cells via the COX­2/Wnt/ß­catenin pathway.

Hepatitis B virus X protein (HBx) has been termed a viral oncoprotein, and is involved in the initiation and progression of hepatocellular carcinoma (HCC). Cyclooxygenase­2 (COX­2) and ß­catenin have been attributed to the oncogenic activity of HBx in HBV­associated HCC. The present study aimed to determine whether there is crosstalk between COX­2 and the Wnt/ß­catenin signaling pathway during HL­7702­HBx cell proliferation, and to investigate the associated underlying molecular mechanism.  In the present study, cell proliferation assay, colony formation assay and flow cytometric analysis were used to detect the proliferative ability of cells. Reverse transcription­quantitative polymerase chain reaction and western blotting were performed to examine the mRNA and protein expression of COX­2, ß­catenin, cyclin­D1 and c­myc. The results demonstrated that HL­7702­HBx exhibited increased cell proliferation, higher colony formation efficiency and a shortened G1 period of the cell cycle. In addition, the mRNA and protein expression levels of COX­2 were increased, and this was associated with HL­7702­HBx cell growth. Furthermore, the expression of ß­catenin and its target genes, cyclin­D1 and c­myc proto­oncogene protein, was upregulated by HBx via COX­2. Finally, HBx promoted HL­7702 cell proliferation through the Wnt/ß­catenin signaling pathway. In conclusion, the primary finding of the present study was that HBx may promote HL­7702 cell proliferation via the COX­2/Wnt/ß­catenin pathway. Thus, it may be helpful to further investigate the molecular mechanism of HBV­associated hepatocellular carcinoma.

Hepatitis B Virus X protein elevates Parkin-mediated mitophagy through Lon Peptidase in starvation.

Hepatocellular Carcinoma (HCC) is the fifth most prevalent cancer worldwide. Specially, Hepatitis B viurs X protein (HBx) is a leading factor in the progression of Hepatitis B viurs-related HCC. Nutrient-deprived tumor microenvironment also contributes to tumor development. However, the role of HBx in nutrient-deprived HCC has received little investigation. Here, we show that HBx elevates PINK1-Parkin mediating mitophagy in starvation. HBx not only increases the PINK1/Parkin gene expression but also accelerates Parkin recruitment to partial mitochondria. Further analysis indicates that, HBx either promotes mitochondrial unfolded protein response, with remarkable mitochondrial LONP1 increases, or reduces LONP1 expression in cytosol inducing LONP1-Parkin pathway, both consequently enhancing mitophagy. Moreover, the enhanced mitophagy lowers mitochondrial apoptosis in starved hepatoma cells, and Bax is implied in the machinery. In addition, we define differential centrifuge, 3000 g or 12,000 g to pellet mitochondria, as an effective method to obtain distinct mitochondria. In collect, HBx regulates diverse aspects of LONP1 and Parkin, enhancing mitophagy in starvation. This study may shed new insights into the machinery development of hepatocellular carcinoma.

Hepatitis B virus X protein sensitizes HL-7702 cells to oxidative stress-induced apoptosis through modulation of the mitochondrial permeability transition pore.

Chronic hepatitis B virus (HBV) infection is a leading cause of liver cirrhosis and cancer. Among the pathogenic factors of HBV, HBV X protein (HBx) is attracting increased attention. Although it is documented that HBx is a multifunctional regulator that modulates cell inflammation and apoptosis, the exact mechanism remains controversial. In the present study, we explored the effect of HBx on oxidative stress-induced apoptosis in normal liver cell line, HL-7702. Our results showed that the existence of HBx affected mitochon-drial biogenesis by modulating the opening of the mitochondrial permeability transition pore (MPTP). Notably, this phenomenon was associated with a pronounced translocation of Bax from the cytosol to the mitochon-dria during the period of exposure to oxidative stress with a release of cytochrome c and activation of cleaved caspase-3 and PARP. Moreover, MPTP blockage with cyclosporin A prevented the translocation of Bax, and inhibited oxidative stress-induced apoptotic killing in the HBx-expressing HL-7702 cells. Our findings suggest that HBx exhibits pro-apoptotic effects upon normal liver cells following exposure to oxidative stress by modulating the MPTP gateway.

HBx co-localizes with COXIII in HL-7702 cells to upregulate mitochondrial function and ROS generation.

Hepatocellular carcinoma (HCC) is one of the most common malignant diseases, and HBx leads to the development of HBV-associated HCC. Mitochondria are key organelles that regulate apoptosis, cellular energetics and signal transduction pathways, and are the source of HBx-induced reactive oxygen species (ROS). Recent findings have shown that HBx interacts with the inner mitochondrial membrane protein, COXIII, via the yeast two-hybrid system, mating experiment and coimmunoprecipitation. The aim of the present study was to examine the co-localizaiton of HBx and COXIII in HL-7702 cells and to investigate ensuing alterations of mitochondrial function. An HL-7702 cell line stably expressing the HBx gene by lentivirus vectors was constructed. Confocal microscopy was utilized to assess the interaction between HBx protein and COXIII. Expression of COXIII, activities of cytochrome c oxidase (COX) and the mitochondrial membrane potential, which were functionally relevant to the HBx protein-COXIII interaction, were investigated in cell cultures. Moreover, the intracellular ROS levels were detected by flow cytometry. The results demonstrated that HBx co-localized with the inner mitochondrial protein, COXIII, in HL-7702 cells, causing the upregulation of COXIII protein expression as well as COX activity. However, HBx did not alter the mitochondrial membrane potential and mitochondria exhibited only slight swelling in HL-7702-HBx cells. Moreover, HBx elevated the generation of mitochondrial ROS in HL-7702-HBx cells. The main finding of the present study was that the co-localization of HBx and COXIII leads to upregulation of the mitochondrial function and ROS generation, which are associated with the oncogenesis of HBV-associated HCC.

The co-localization of HBx and COXIII upregulates COX-2 promoting HepG2 cell growth.

HBx is a multifunctional regulator that interacts with host factors to contribute to the development of hepatocellular carcinoma. In this study, to explore the co-localization of HBx and COXIII in HepG2 cells and to investigate the molecular mechanism of HBx in HepG2 cell growth promotion, we first constructed a HepG2 cell line stably expressing the HBx gene in vitro by lentivirus vectors. In addition, we found that HBx co-localized with the inner mitochondrial protein, COXIII, in HepG2 cells by confocal laser scanning microscopy. It led to changes of mitochondrial biogenesis and morphology, including upregulation of COXIII protein expression, increased cytochrome c oxidase activity and higher mitochondrial membrane potential. The upregulation of COX-2 caused by HBx through generation of mitochondrial reactive oxygen species promoted cell growth. Thus, we conclude that co-localization of HBx and COXIII leads to upregulation of COX-2 that promotes HepG2 cell growth. Such a mechanism provides deeper insights into the molecular mechanism of HBV-associated hepatocellular carcinoma.