Single-cell immune profiling of mouse liver aging reveals Cxcl2+ macrophages recruit neutrophils to aggravate liver injury.

BACKGROUND AND AIMS: Immune cells play a crucial role in liver aging. However, the impact of dynamic changes in the local immune microenvironment on age-related liver injury remains poorly understood. We aimed to characterize intrahepatic immune cells at different ages to investigate key mechanisms associated with liver aging. APPROACH AND RESULTS: We carried out single-cell RNA sequencing on mouse liver tissues at 4 different ages, namely, the newborn, suckling, young, and aged stages. The transcriptomic landscape, cellular classification, and intercellular communication were analyzed. We confirmed the findings by multiplex immunofluorescence staining, flow cytometry, in vitro functional experiments, and chimeric animal models. Nine subsets of 89,542 immune cells with unique properties were identified, of which Cxcl2+ macrophages within the monocyte/macrophage subset were preferentially enriched in the aged liver. Cxcl2+ macrophages presented a senescence-associated secretory phenotype and recruited neutrophils to the aged liver through the CXCL2-CXCR2 axis. Through the secretion of IL-1ß and TNF-α, Cxcl2+ macrophages stimulated neutrophil extracellular traps formation. Targeting the CXCL2-CXCR2 axis limited the neutrophils migration toward the liver and attenuated age-related liver injury. Moreover, the relationship between Cxcl2+ macrophages and neutrophils in age-related liver injury was further validated by human liver transplantation samples. CONCLUSIONS: This in-depth study illustrates that the mechanism of Cxcl2+ macrophage-driven neutrophil activation involves the CXCL2-CXCR2 axis and provides a potential therapeutic strategy for age-related liver injury.

Combined inhibition of surface CD51 and γ-secretase-mediated CD51 cleavage improves therapeutic efficacy in experimental metastatic hepatocellular carcinoma.

BACKGROUND & AIMS: Integrin αv (ITGAV, CD51) is regarded as a key component in multiple stages of tumor progression. However, the clinical failure of cilengitide, a specific inhibitor targeting surface CD51, suggests the importance of yet-unknown mechanisms by which CD51 promotes tumor progression. METHODS: In this study, we used several hepatocellular carcinoma (HCC) cell lines and murine hepatoma cell lines. To investigate the role of CD51 on HCC progression, we used a 3D invasion assay and in vivo bioluminescence imaging. We used periostin-knockout transgenic mice to uncover the role of the tumor microenvironment on CD51 cleavage. Moreover, we used several clinically relevant HCC models, including patient-derived organoids and patient-derived xenografts, to evaluate the therapeutic efficacy of cilengitide in combination with the γ-secretase inhibitor LY3039478. RESULTS: We found that CD51 could undergo transmembrane cleavage by γ-secretase to produce a functional intracellular domain (CD51-ICD). The cleaved CD51-ICD facilitated HCC invasion and metastasis by promoting the transcription of oxidative phosphorylation-related genes. Furthermore, we identified cancer-associated fibroblast-derived periostin as the major driver of CD51 cleavage. Lastly, we showed that cilengitide-based therapy led to a dramatic therapeutic effect when supplemented with LY3039478 in both patient-derived organoid and xenograft models. CONCLUSIONS: In summary, we revealed previously unrecognized mechanisms by which CD51 is involved in HCC progression and uncovered the underlying cause of cilengitide treatment failure, as well as providing evidence supporting the translational prospects of combined CD51-targeted therapy in the clinic. IMPACT AND IMPLICATIONS: Integrin αv (CD51) is a widely recognized pro-tumoral molecule that plays a crucial role in various stages of tumor progression, making it a promising therapeutic target. However, despite early promising results, cilengitide, a specific antagonist of CD51, failed in a phase III clinical trial. This prompted further investigation into the underlying mechanisms of CD51's effects. This study reveals that the γ-secretase complex directly cleaves CD51 to produce an intracellular domain (CD51-ICD), which functions as a pro-tumoral transcriptional regulator and can bypass the inhibitory effects of cilengitide by entering the nucleus. Furthermore, the localization of CD51 in the nucleus is significantly associated with the prognosis of patients with HCC. These findings provide a theoretical basis for re-evaluating cilengitide in clinical settings and highlight the importance of identifying a more precise patient subpopulation for future clinical trials targeting CD51.

Chemotherapy combined with radiotherapy can benefit more unresectable HCC patients with portal and/or hepatic vein invasion: a retrospective analysis of the SEER database.

Background: The purpose of this study is to evaluate the effects of chemotherapy and radiotherapy on the prognosis of unresectable HCC patients with portal and/or hepatic vein invasion. Methods: A retrospective analysis of unresectable HCC patients with portal and/or hepatic vein invasion registered in the Surveillance, Epidemiology, End Results (SEER) database was performed. The propensity score-matching (PSM) method was used to balance differences between groups. Overall survival (OS) and cancer-specific survival (CSS) were the interesting endpoints. OS was calculated from the date of diagnosis to the date of death caused by any cause or the last follow-up. CSS was defined as the interval between the date of diagnosis and date of death due only to HCC or last follow-up. OS and CSS were analyzed by using Kaplan-Meier analysis, Cox proportional hazards model, and Fine-Gray competing-risk model. Results: A total of 2,614 patients were included. 50.2% patients received chemotherapy or radiotherapy and 7.5% patients received both chemotherapy and radiotherapy. Compared to the untreated group, chemotherapy or radiotherapy (COR) (HR = 0.538, 95% CI 0.495-0.585, p < 0.001) and chemotherapy and radiotherapy (CAR) (HR = 0.371, 95% CI 0.316-0.436, p < 0.001) showed better OS. In the COR group, Cox analysis results showed AFP, tumor size, N stage and M stage were independent risk factor of OS. Competing-risk analysis results showed AFP, tumor size and M stage were independent risk factor of CSS. In the CAR group, AFP and M stage were independent risk factors of OS. Competing-risk analysis results showed M stage were independent risk factor of CSS. Kaplan Meier analysis showed chemotherapy combined with radiotherapy significantly improves OS (10.0 vs. 5.0 months, p < 0.001) and CSS (10.0 vs. 6.0 months, p = 0.006) than monotherapy. Conclusion: AFP positive and distant metastasis are the main risk factors affecting OS and CSS of unresectable HCC patients with portal and/or hepatic vein invasion. Chemotherapy combined with radiotherapy significantly improves OS and CSS of unresectable HCC patients with portal and/or hepatic vein invasion.

Administration of AG490 decreases the senescence of umbilical cord-mesenchymal stem cells and promotes the cytotherapeutic effect in liver fibrosis.

The therapeutic potential of umbilical cord-mesenchymal stem cell (UC-MSC) transplantation in liver fibrosis has been highlighted. However, the fate of transplanted MSCs in the fibrotic microenvironment remains unclear. In this study, we aim to uncover the fate of transplanted MSCs and develop targeting strategies that could enhance the therapeutic efficacy of MSC therapy in liver fibrosis. We used human UC-MSCs as the study object. For in vitro experiments, we stimulated UC-MSCs with several fibrotic-related factors (Liver fibrotic Factors, LF), including TGFß, TNFα and IFNγ for downstream investigations. We co-cultured LF-treated UC-MSCs with hepatic stellate cell line LX-2 to assess the anti-fibrotic effect. We showed that upon LF stimulation, UC-MSCs exhibited reduced anti-fibrotic activity and underwent rapid senescence. Pathway analysis showed that JAK/STAT3 signaling was highly activated upon LF stimulation, which significantly elevated senescence-associated secretory phenotype (SASP) and senescence in UC-MSCs and could be reversed by a specific JAK inhibitor AG490. Moreover, using both carbon tetrachloride (CCl4) and 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-induce fibrosis models, we demonstrated that AG490 pretreatment promoted UC-MSCs survival within the fibrotic liver microenvironment and exhibited enhance therapeutic efficacy. Overall, we showed that targeting MSC senescence in vivo through AG490 pretreatment could enhance the anti-fibrotic activities of UC-MSCs.

MSC-derived extracellular vesicles as nanotherapeutics for promoting aged liver regeneration.

Aging is one of the critical factors to impair liver regeneration leading to a high incidence of severe complications after hepatic surgery in the elderly population without any effective treatment for clinical administration. As cell-free nanotherapeutics, mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) have been demonstrated the therapeutic potentials on liver diseases. However, the effects of MSC-EVs on the proliferation of aged hepatocytes are largely unclear. In this study, we found MSCs could reduce the expression of senescence-associated markers in the liver and stimulate its regeneration in aged mice after receiving a two-thirds partial hepatectomy (PHx) through their secreted MSC-EVs. Using RNA-Seq and AAV9 vector, we mechanistically found that these effects of UC-MSC-EVs partially attributed to inducing Atg4B-related mitophagy. This effect repairs the mitochondrial status and functions of aged hepatocytes to promote their proliferation. And protein mass spectrum analysis uncovered that DEAD-Box Helicase 5 (DDX5) enriches in UC-MSC-EVs, which interacts with E2F1 to facilitate its nuclear translocation for activating the expression of Atg4B. Collectively, our data show that MSC-EVs act nanotherapeutic potentials in anti-senescence and promoting regeneration of aged liver by transferring DDX5 to regulate E2F1-Atg4B signaling pathway that induce mitophagy, which highlights the clinical application valuation of MSC-EVs for preventing severe complications in aged population receiving liver surgery.

CircFOXK2 promotes hepatocellular carcinoma progression and leads to a poor clinical prognosis via regulating the Warburg effect.

BACKGROUND: The Warburg effect is well-established to be essential for tumor progression and accounts for the poor clinical outcomes of hepatocellular carcinoma (HCC) patients. An increasing body of literature suggests that circular RNAs (circRNAs) are important regulators for HCC. However, few circRNAs involved in the Warburg effect of HCC have hitherto been investigated. Herein, we aimed to explore the contribution of circFOXK2 to glucose metabolism reprogramming in HCC. METHODS: In the present study, different primers were designed to identify 14 circRNAs originating from the FOXK2 gene, and their differential expression between HCC and adjacent liver tissues was screened. Ultimately, circFOXK2 (hsa_circ_0000817) was selected for further research. Next, the clinical significance of circFOXK2 was evaluated. We then assessed the pro-oncogenic activity of circFOXK2 and its impact on the Warburg effect in both HCC cell lines and animal xenografts. Finally, the molecular mechanisms of how circFOXK2 regulates the Warburg effect of HCC were explored. RESULTS: CircFOXK2 was aberrantly upregulated in HCC tissues and positively correlated with poor clinical outcomes in patients that underwent radical hepatectomy. Silencing of circFOXK2 significantly suppressed HCC progression both in vitro and in vivo. Mechanistically, circFOXK2 upregulated the expression of protein FOXK2-142aa to promote LDHA phosphorylation and led to mitochondrial fission by regulating the miR-484/Fis1 pathway, ultimately activating the Warburg effect in HCC. CONCLUSIONS: CircFOXK2 is a prognostic biomarker of HCC that promotes the Warburg effect by promoting the expression of proteins and miRNA sponges that lead to tumor progression. Overall, circFOXK2 has huge prospects as a potential therapeutic target for patients with HCC.

Extracellular vesicles derived from mesenchymal stromal cells as nanotherapeutics for liver ischaemia-reperfusion injury by transferring mitochondria to modulate the formation of neutrophil extracellular traps.

As nanotherapeutics, mesenchymal stromal cell-derived extracellular vesicles (MSC-EVs) are considered a potent alternative for whole-cell therapy and are gradually entering the clinical field of liver diseases. In this study, neutrophil extracellular traps (NETs) formation in liver tissue was verified as a critical factor for liver ischaemia-reperfusion injury (IRI) in both clinical samples and animal models. Human umbilical cord-derived MSC-EVs (hUC-MSC-EVs) might function to reduce the NETs formation and subsequently improve liver IRI. Mechanistically, we showed that hUC-MSC-EVs contain functional mitochondria that are transferred to intrahepatic neutrophils. This effect triggers mitochondrial fusion and subsequently restores the mitochondrial status and functions in neutrophils to reduce NETs formation. Collectively, our findings suggest that MSC-EVs exert a nanotherapeutic effect on inhibiting local NETs formation by transferring functional mitochondria to intrahepatic neutrophils and repairing their mitochondrial function, which highlights the therapeutic value of hUC-MSC-EVs for liver IRI.

Nestin promotes pulmonary fibrosis via facilitating recycling of TGF-ß receptor I.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disease that is characterised by aberrant proliferation of activated myofibroblasts and pathological remodelling of the extracellular matrix. Previous studies have revealed that the intermediate filament protein nestin plays key roles in tissue regeneration and wound healing in different organs. Whether nestin plays a critical role in the pathogenesis of IPF needs to be clarified. METHODS: Nestin expression in lung tissues from bleomycin-treated mice and IPF patients was determined. Transfection with nestin short hairpin RNA vectors in vitro that regulated transcription growth factor (TGF)-ß/Smad signalling was conducted. Biotinylation assays to observe plasma membrane TßRI, TßRI endocytosis and TßRI recycling after nestin knockdown were performed. Adeno-associated virus serotype (AAV)6-mediated nestin knockdown was assessed in vivo. RESULTS: We found that nestin expression was increased in a murine pulmonary fibrosis model and IPF patients, and that the upregulated protein primarily localised in lung α-smooth muscle actin-positive myofibroblasts. Mechanistically, we determined that nestin knockdown inhibited TGF-ß signalling by suppressing recycling of TßRI to the cell surface and that Rab11 was required for the ability of nestin to promote TßRI recycling. In vivo, we found that intratracheal administration of AAV6-mediated nestin knockdown significantly alleviated pulmonary fibrosis in multiple experimental mice models. CONCLUSION: Our findings reveal a pro-fibrotic function of nestin partially through facilitating Rab11-dependent recycling of TßRI and shed new light on pulmonary fibrosis treatment.

Heat shock preconditioning mesenchymal stem cells attenuate acute lung injury via reducing NLRP3 inflammasome activation in macrophages.

OBJECTIVES: Acute lung injury (ALI) remains a common cause of morbidity and mortality worldwide, and to date, there is no effective treatment for ALI. Previous studies have revealed that topical administration of mesenchymal stem cells (MSCs) can attenuate the pathological changes in experimental acute lung injury. Heat shock (HS) pretreatment has been identified as a method to enhance the survival and function of cells. The present study aimed to assess whether HS-pretreated MSCs could enhance immunomodulation and recovery from ALI. MATERIALS AND METHODS: HS pretreatment was performed at 42 °C for 1 h, and changes in biological characteristics and secretion functions were detected. In an in vivo mouse model of ALI, we intranasally administered pretreated umbilical cord-derived MSCs (UC-MSCs), confirmed their therapeutic effects, and detected the phenotypes of the macrophages in bronchoalveolar lavage fluid (BALF). To elucidate the underlying mechanisms, we cocultured pretreated UC-MSCs with macrophages in vitro, and the expression levels of inflammasome-related proteins in the macrophages were assessed. RESULTS: The data showed that UC-MSCs did not exhibit significant changes in viability or biological characteristics after HS pretreatment. The administration of HS-pretreated UC-MSCs to the ALI model improved the pathological changes and lung damage-related indexes, reduced the proinflammatory cytokine levels, and modulated the M1/M2 macrophage balance. Mechanistically, both the in vivo and in vitro studies demonstrated that HS pretreatment enhanced the protein level of HSP70 in UC-MSCs, which negatively modulated NLR family pyrin domain containing 3 (NLRP3) inflammasome activation in alveolar macrophages. These effects were partially reversed by knocking down HSP70 expression. CONCLUSION: HS pretreatment can enhance the beneficial effects of UC-MSCs in inhibiting NLRP3 inflammasome activation in macrophages during ALI. The mechanism may be related to the upregulated expression of HSP70.

A novel MSC-based immune induction strategy for ABO-incompatible liver transplantation: a phase I/II randomized, open-label, controlled trial.

BACKGROUND: ABO-incompatible liver transplantation (ABO-i LT) has become a rescue therapeutic option for patients with severe hepatic failure. Although the use of rituximab greatly reduces the morbidity of antibody-mediated rejection (AMR), severe adverse effects, such as infection and biliary complications, still seriously threaten the survival of transplant recipients. The aim of this study was to evaluate the safety and feasibility of using mesenchymal stem cells (MSCs) to replace rituximab in ABO-i LT. METHODS: Twenty-two patients with severe hepatic failure undergoing ABO-i LT were enrolled and randomly divided into two groups: the MSC group and the rituximab group. The safety of the application of MSCs and the incidence of allograft rejection, including antibody-mediated rejection (AMR) and acute cellular rejection (ACR), were evaluated in both groups at the 2-year follow-up period as primary endpoints. Recipients and graft survival and other postoperative complications were compared as secondary endpoints. RESULTS: No severe MSC-related adverse events were observed during the trial. MSC treatment yielded comparable, if not better, results than rituximab at decreasing the incidence of acute rejection (9.1% vs 27.3%). Inspiringly, compared to those in the rituximab group, the rates of biliary complications (0% vs 45.5%) and infection (9.1% vs 81.8%) were significantly decreased in the MSC group. In addition, there were no significant differences in 2-year graft and recipient survival between the two groups (81.8% vs 72.7%). CONCLUSIONS: Our data show that MSC transfusion is comparable to rituximab treatment for AMR prophylaxis following ABO-i LT. Additionally, the results indicate that MSCs are more beneficial to the prevention of infection and biliary complications and may be introduced as a novel immunosuppressive approach for ABO-i LT. TRIAL REGISTRATION: Trial registration: chictr.org.cn , ChiCTR2000037732. Registered 31 August 2020- Retrospectively registered, http://www.chictr.org.cn/showproj.aspx?proj=57074 .

Inhibition of TGFß improves hematopoietic stem cell niche and ameliorates cancer-related anemia.

BACKGROUND: Cancer cachexia is a wasting syndrome that is quite common in terminal-stage cancer patients. Cancer-related anemia is one of the main features of cancer cachexia and mostly results in a poor prognosis. The disadvantages of the current therapies are obvious, but few new treatments have been developed because the pathological mechanism remains unclear. METHODS: C57BL/6 mice were subcutaneously injected with Lewis lung carcinoma cells to generate a cancer-related anemia model. The treated group received daily intraperitoneal injections of SB505124. Blood parameters were determined with a routine blood counting analyzer. Erythroid cells and hematopoietic stem/progenitor cells were analyzed by flow cytometry. The microarchitecture changes of the femurs were determined by micro-computed tomography scans. Smad2/3 phosphorylation was analyzed by immunofluorescence and Western blotting. The changes in the hematopoietic stem cell niche were revealed by qPCR analysis of both fibrosis-related genes and hematopoietic genes, fibroblastic colony-forming unit assays, and lineage differentiation of mesenchymal stromal cells. RESULTS: The mouse model exhibited hematopoietic suppression, marked by a decrease of erythrocytes in the peripheral blood, as well as an increase of immature erythroblasts and reduced differentiation of multipotent progenitors in the bone marrow. The ratio of bone volume/total volume, trabecular number, and cortical wall thickness all appeared to decrease, and the increased osteoclast number has led to the release of latent TGFß and TGFß signaling over-activation. Excessive TGFß deteriorated the hematopoietic stem cell niche, inducing fibrosis of the bone marrow as well as the transition of mesenchymal stromal cells. Treatment with SB505124, a small-molecule inhibitor of TGFß signaling, significantly attenuated the symptoms of cancer-related anemia in this model, as evidenced by the increase of erythrocytes in the peripheral blood and the normalized proportion of erythroblast cell clusters. Meanwhile, hindered hematopoiesis and deteriorated hematopoietic stem cell niche were also shown to be restored with SB505124 treatment. CONCLUSION: This study investigated the role of TGFß released by bone remodeling in the progression of cancer-related anemia and revealed a potential therapeutic approach for relieving defects in hematopoiesis.

Targeting Nestin+ hepatic stellate cells ameliorates liver fibrosis by facilitating TßRI degradation.

BACKGROUND & AIMS: Liver fibrosis is a wound healing response that arises from various aetiologies. The intermediate filament protein Nestin has been reported to participate in maintaining tissue homeostasis during wound healing responses. However, little is known about the role Nestin plays in liver fibrosis. This study investigated the function and precise regulatory network of Nestin during liver fibrosis. METHODS: Nestin expression was assessed via immunostaining and quantitative real-time PCR (qPCR) in fibrotic/cirrhotic samples. The induction of Nestin expression by transforming growth factor beta (TGFß)-Smad2/3 signalling was investigated through luciferase reporter assays. The functional role of Nestin in hepatic stellate cells (HSCs) was investigated by examining the pathway activity of profibrogenic TGFß-Smad2/3 signalling and degradation of TGFß receptor I (TßRI) after interfering with Nestin. The in vivo effects of knocking down Nestin were examined with an adeno-associated virus vector (serotype 6, AAV6) carrying short-hairpin RNA targeting Nestin in fibrotic mouse models. RESULTS: Nestin was mainly expressed in activated HSCs and increased with the progression of liver fibrosis. The profibrogenic pathway TGFß-Smad2/3 induced Nestin expression directly. Knocking down Nestin promoted caveolin 1-mediated TßRI degradation, resulting in TGFß-Smad2/3 pathway impairment and reduced fibrosis marker expression in HSCs. In AAV6-treated murine fibrotic models, knocking down Nestin resulted in decreased levels of inflammatory infiltration, hepatocellular damage, and a reduced degree of fibrosis. CONCLUSION: The expression of Nestin in HSCs was induced by TGFß and positively correlated with the degree of liver fibrosis. Knockdown of Nestin decreased activation of the TGFß pathway and alleviated liver fibrosis both in vitro and in vivo. Our data demonstrate a novel role of Nestin in controlling HSC activation in liver fibrosis. LAY SUMMARY: Liver fibrosis has various aetiologies but represents a common process in chronic liver diseases that is associated with high morbidity and mortality. Herein, we demonstrate that the intermediate filament protein Nestin plays an essential profibrogenic role in liver fibrosis by forming a positive feedback loop with the TGFß-Smad2/3 pathway, providing a potential therapeutic target for the treatment of liver fibrosis.

Transplantation of hPSC-derived pericyte-like cells promotes functional recovery in ischemic stroke mice.

Pericytes play essential roles in blood-brain barrier (BBB) integrity and dysfunction or degeneration of pericytes is implicated in a set of neurological disorders although the underlying mechanism remains largely unknown. However, the scarcity of material sources hinders the application of BBB models in vitro for pathophysiological studies. Additionally, whether pericytes can be used to treat neurological disorders remains to be elucidated. Here, we generate pericyte-like cells (PCs) from human pluripotent stem cells (hPSCs) through the intermediate stage of the cranial neural crest (CNC) and reveal that the cranial neural crest-derived pericyte-like cells (hPSC-CNC PCs) express typical pericyte markers including PDGFRß, CD146, NG2, CD13, Caldesmon, and Vimentin, and display distinct contractile properties, vasculogenic potential and endothelial barrier function. More importantly, when transplanted into a murine model of transient middle cerebral artery occlusion (tMCAO) with BBB disruption, hPSC-CNC PCs efficiently promote neurological functional recovery in tMCAO mice by reconstructing the BBB integrity and preventing of neuronal apoptosis. Our results indicate that hPSC-CNC PCs may represent an ideal cell source for the treatment of BBB dysfunction-related disorders and help to model the human BBB in vitro for the study of the pathogenesis of such neurological diseases.

A novel protein encoded by circFNDC3B inhibits tumor progression and EMT through regulating Snail in colon cancer.

BACKGROUND: Colon cancer (CC) is a common malignant cancer. Recently, circFNDC3B was found to exert biological function in multiple cancers. However, it was unclear whether the potential protein encoded by circFNDC3B is involved in carcinogenesis of CC. METHODS: We used Sanger sequence and RNase R digestion assay to confirm the existence of circFNDC3B, and quantitative real-time PCR was used to evaluate the circRNA's expression. Then fluorescence in situ hybridization (FISH) was performed to study location of circFNDC3B. The identification of protein encoded by circFNDC3B was performed using LC-MS/MS. The function of circFNDC3B-218aa on proliferation, invasion and migration were assessed by CCK8 assays, colony formation assays, transwell assays, wound-healing assays and animal experiments. RNA-sequencing and western blot were used to identify the gene regulated by circFNDC3B-218aa. Finally, glucose metabolism-related assays were performed to further investigate function of circFNDC3B-218aa. RESULTS: CircFNDC3B was localized mostly in the cytoplasm, and was decreased in CC cell lines and tissues. The patients with low circFNDC3B expression had a shorter OS (P = 0.0014) than patients with high expression. Moreover, circFNDC3B inhibited the proliferation, invasion and migration of CC cells. Next, we identified that circFNDC3B could encode a novel protein circFNDC3B-218aa. Furthermore, circFNDC3B-218aa, not circFNDC3B, inhibited the proliferation, invasion and migration of CC. Additionally, the in vivo experiments implied that up-regulated circFNDC3B-218aa exhibited an inhibitory effect on CC progression. By RNA-sequencing, western blot and glucose metabolism-related assays, we found that circFNDC3B-218aa inhibited the expression of Snail, and subsequently promoted the tumor-suppressive effect of FBP1 in CC. CONCLUSIONS: The novel circFNDC3B-218aa may serve as a tumor suppressive factor and potential biomarker which may supply the potential therapeutic target for CC.

MSCs ameliorate hepatocellular apoptosis mediated by PINK1-dependent mitophagy in liver ischemia/reperfusion injury through AMPKα activation.

Hepatocyte apoptosis is the main pathophysiological process underlying liver ischemia/reperfusion (I/R) injury. Mitochondrial abnormalities have a vital role in hepatocellular damage. The hepatoprotective effects of mesenchymal stem cells (MSCs) have been previously demonstrated. In this study, we aim to investigate the effect and potential mechanism of MSCs against liver I/R injury. Effects of MSCs were studied in mice liver I/R injury model and in a hypoxia/reoxygenation (H/R) model of L02 hepatocytes. The potential mechanisms of MSCs on these in vivo and in vitro I/R-induced hepatocellular apoptosis models were studies. Accompanied by the improvement of hepatic damage, MSCs exhibited capabilities of controlling mitochondrial quality, shown by reduced mitochondrial reactive oxygen species (mtROS) overproduction, decreased the accumulation of mitochondrial fragmentation, restored ATP generation and upregulated mitophagy. Furthermore, we descripted a potential mechanism of MSCs on upregulating mitophagy and found that the reduced Parkin and PINK1 expression and inactivated AMPKα pathway were observed in the liver tissue in I/R model. These effects were reversed by MSCs treatment. In vitro study showed that MSC-conditioned medium (MSC-CM) suppressed hepatocellular apoptosis and inhibited mtROS accumulation in the H/R environment. And these effects of MSC-CM were partially blocked after the cells were transfected with PINK1 siRNA or added with dorsomorphin. Collectively, our findings provide a novel pharmacological mechanism that MSCs exert hepatoprotective effect in liver I/R injury via upregulating PINK1-dependent mitophagy. In addition, this effect might be attributed to the modulation of AMPKα activation.

Prognostic Value of Albumin-to-Alkaline Phosphatase Ratio in Hepatocellular Carcinoma Patients Treated with Liver Transplantation.

Background: The albumin-to-alkaline phosphatase ratio (AAPR) is a newly developed index which was used to predict prognosis of HCC patients. However, its prognostic role in HCC patients undergoing liver transplantation (LT) remains unclear. This study aimed to investigate the correlation between AAPR and prognosis of these patients. Methods: A total of 210 patients who underwent LT from January 2003 to January 2014 were retrospectively analyzed (149 for discovery and 61 for validation). Univariate and multivariate analyses were performed to determine the discriminative ability of the AAPR in predicting long-term survival. The area under the receiver operating characteristic (AUC) was calculated to compare the accuracy of different factors. Results: Patients with high AAPR level were associated with less ascites rate (30.6% versus 53.2%, P=0.033) as well as more frequencies of Child-Pugh class A (73.6% versus 35.1%, P=0.001). Univariate and multivariate analyses suggested the AAPR was independent prognostic factor in predicting overall survival (HR: 0.585, 95% CI: 0.363-0.941, P=0.027). Validation cohort confirmed prognostic value of AAPR. Subgroup analysis demonstrated that reduced AAPR level was associated with worse prognosis in HCC patients categorized in Child-Pugh class A (P=0.029). The AUCs of the AAPR were 0.710 and 0.744 in predicting 3-year and 5-year survival outcomes, respectively. Conclusions: The study showed in two independent cohorts of HCC patients treated by LT that elevated AAPR was associated with better OS. As a low-cost routine laboratory test, it should be considered as biomarker in the clinical management of HCC.

An Integrated Nomogram Combining Clinical Factors and Microtubule-Associated Protein 1 Light Chain 3B Expression to Predict Postoperative Prognosis in Patients with Intrahepatic Cholangiocarcinoma.

PURPOSE: Microtubule-associated protein 1 light chain 3B (LC3B) serves as a key component of autophagy, which is associated with the progression of carcinoma. Yet, it is still unclear whether LC3B is also an independent risk factor for intrahepatic cholangiocarcinoma (ICC). We aim to explore the predictive value of LC3B on prognosis of ICC, and to establish a novel and available nomogram to predict relapse-free survival (RFS) and overall survival (OS) for these patients after curative-intent hepatectomy. MATERIALS AND METHODS: From August 2004 to March 2017, 105 ICC patients were eligibly enrolled in the Third Affiliated Hospital of Sun Yat-sen University. Preoperative clinical information of enrolled patients was collected. Expression LC3B in the ICC specimen was detected by immunohistochemistry. RESULTS: The 5-year RFS and OS in this cohort were 15.7% and 29.6%, respectively. On multivariate Cox regression analysis, independent risk factors for 5-year OS were cancer antigen 125, microvascular invasion, LC3B expression and lymph node metastasis. Except for the above 4 factors, neutrophil/lymphocyte ratio and tumor differentiation were independent factors for 5-year RFS. The area under the curve of nomograms for OS and RFS were 0.820 and 0.747, respectively. CONCLUSION: The nomograms based on LC3B can be considered as effective models to predict postoperative survival for ICC patients.

Nestin regulates cellular redox homeostasis in lung cancer through the Keap1-Nrf2 feedback loop.

Abnormal cancer antioxidant capacity is considered as a potential mechanism of tumor malignancy. Modulation of oxidative stress status is emerging as an anti-cancer treatment. Our previous studies have found that Nestin-knockdown cells were more sensitive to oxidative stress in non-small cell lung cancer (NSCLC). However, the molecular mechanism by which Nestin protects cells from oxidative damage remains unclear. Here, we identify a feedback loop between Nestin and Nrf2 maintaining the redox homeostasis. Mechanistically, the ESGE motif of Nestin interacts with the Kelch domain of Keap1 and competes with Nrf2 for Keap1 binding, leading to Nrf2 escaping from Keap1-mediated degradation, subsequently promoting antioxidant enzyme generation. Interestingly, we also map that the antioxidant response elements (AREs) in the Nestin promoter are responsible for its induction via Nrf2. Taken together, our results indicate that the Nestin-Keap1-Nrf2 axis regulates cellular redox homeostasis and confers oxidative stress resistance in NSCLC.

Preconditioning of umbilical cord-derived mesenchymal stem cells by rapamycin increases cell migration and ameliorates liver ischaemia/reperfusion injury in mice via the CXCR4/CXCL12 axis.

OBJECTIVES: Transfusion of umbilical cord-derived mesenchymal stem cells (UC-MSCs) is a novel strategy for treatment of various liver diseases. However, the therapeutic effect of UC-MSCs is limited because only a few UC-MSCs migrate towards the damaged regions. In this study, we observed the effects of autophagy on the migration of UC-MSCs in vitro and in a model of liver ischaemia/reperfusion (I/R) injury. MATERIALS AND METHODS: We investigated the effects of autophagy on the status of the cell, release of anti-inflammatory factors and migration of UC-MSCs in vitro. The therapeutic effects and in vivo migration of rapamycin-preconditioned UC-MSCs were observed in a C57/B6 mouse model of liver I/R injury. RESULTS: Induction of autophagy by rapamycin enhanced the ability of UC-MSCs to migrate and release anti-inflammatory cytokines as well as increased expression of CXCR4 without affecting cell viability. Inhibition of CXCR4 activation markedly decreased migration of these cells. In a mouse model of liver I/R injury, we found significantly upregulated expression of CXCR12 in the damaged liver. More rapamycin-preconditioned UC-MSCs migrated towards the ischaemic regions than 3-methyladenine-preconditioned or non-preconditioned UC-MSCs, leading to improvement in hepatic performance, pathological changes and levels of inflammatory cytokines. These effects were abolished by AMD3100. CONCLUSIONS: Preconditioning of UC-MSCs by rapamycin afforded increased protection against liver I/R injury by enhancing immunosuppression and strengthening the homing and migratory capacity of these cells via the CXCR4/CXCL12 axis.