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Autoimmune hepatitis (AIH) is an immune-mediated chronic inflammatory liver disease, and its pathogenesis is not fully understood. Our previous study discovered that receptor interacting protein kinase 3 (RIP3) is correlated with serum transaminase levels in AIH patients. However, its role and underlying mechanism in AIH are poorly understood. Here, we detected the increased expression and activation of RIP3 in livers of patients and animal models with AIH. The inhibition of RIP3 kinase by GSK872 prevented concanavalin A (ConA)-induced immune-mediated hepatitis (IMH) by reduced hepatic proinflammatory cytokines and immune cells including Th17 cells and macrophages. Further experiments revealed that RIP3 inhibition resulted in an increase in CD11b+Gr1+ myeloid-derived suppressor cells (MDSCs) with immunoregulatory properties in the liver, spleen, and peripheral blood. Moreover, the depletion of Gr-1+ MDSCs abrogated the protective effect and immune suppression function of GSK872 in ConA-induced IMH. Altogether, our data demonstrate that RIP3 blockade prevents ConA-induced IMH through promoting MDSCs infiltration. Inhibition of RIP3 kinase may be a novel therapeutic avenue for AIH treatment.

Poly(ADP-ribosyl)ation (PARylation) and SUMO modification (SUMOylation) are novel post-translational modifications (PTMs) mainly induced by PARP1 and SUMO1. Growing evidence has revealed that C/EBPβ plays multiple roles in biological processes and participates in cardiovascular diseases. However, the cross-talk between C/EBPβ PARylation and SUMOylation during cardiovascular diseases is unknown. This study aims to investigate the effects of C/EBPβ PTMs on cardiac hypertrophy and its underlying mechanism. Abdominal aortic constriction (AAC) and phenylephrine (PE) were conducted to induce cardiac hypertrophy. Intramyocardial delivery of recombinant adenovirus (Ad-PARP1) was taken to induce PARP1 overexpression. In this study, we found C/EBPβ participates in PARP1-induced cardiac hypertrophy. C/EBPβ K134 residue could be both PARylated and SUMOylated individually by PARP1 and SUMO1. Moreover, the accumulation of PARylation on C/EBPβ at K134 site exhibits downregulation of C/EBPβ SUMOylation at the same site. Importantly, C/EBPβ K134 site SUMOylation could decrease C/EBPβ protein stability and participates in PARP1-induced cardiac hypertrophy. Taken together, these findings highlight the importance of the cross-talk between C/EBPβ PTMs at K134 site in determining its protein level and function, suggesting that multi-target pharmacological strategies inhibiting PARP1 and activating C/EBPβ SUMOylation would be potential for treating pathological cardiac hypertrophy.

Brain endothelial cells (ECs) are an important component of the blood-brain barrier (BBB) and play key roles in restricting entrance of possible toxic components and pathogens into the brain. However, identifying endothelial genes that regulate BBB homeostasis remains a time-consuming process. Although somatic genome editing has emerged as a powerful tool for discovery of essential genes regulating tissue homeostasis, its application in brain ECs is yet to be demonstrated in vivo. Here, we used an adeno-associated virus targeting brain endothelium (AAV-BR1) combined with the CRISPR/Cas9 system (AAV-BR1-CRISPR) to specifically knock out genes of interest in brain ECs of adult mice. We first generated a mouse model expressing Cas9 in ECs (Tie2Cas9). We selected endothelial β-catenin (Ctnnb1) gene, which is essential for maintaining adult BBB integrity, as the target gene. After intravenous injection of AAV-BR1-sgCtnnb1-tdTomato in 4-week-old Tie2Cas9 transgenic mice resulted in mutation of 36.1% of the Ctnnb1 alleles, thereby leading to a dramatic decrease in the level of CTNNB1 in brain ECs. Consequently, Ctnnb1 gene editing in brain ECs resulted in BBB breakdown. Taken together, these results demonstrate that the AAV-BR1-CRISPR system is a useful tool for rapid identification of endothelial genes that regulate BBB integrity in vivo.

Microtubules, a major target in oral squamous cell carcinoma (OSCC) chemotherapy, contribute to multiple malignant biological behaviors, including proliferation, migration, and epithelial-mesenchymal transition (EMT). Surpassing traditional tubulin inhibitors, ID09 emerges with brilliant solubility, photostability, and drug-sensitivity in multidrug-resistant cells. Its anti-tumor effects have been briefly verified in lung adenocarcinoma and hepatocellular carcinoma. However, whether OSCC is sensitive to ID09 and the potential mechanisms remain ambiguous, which are research purposes this study aimed to achieve. Various approaches were applied, including clone formation assay, flow cytometry, wound healing assay, Transwell assay, cell counting kit-8 assay, Western blot, qRT-PCR, and in vivo experiment. The experimental results revealed that ID09 not only contributed to cell cycle arrest, reduced migration, and reversed EMT, but accelerated mitochondria-initiated apoptosis. Remarkably, Western blot detected diminishment in expression of Mcl-1 due to the deactivation of Ras-Erk pathway, resulting in ID09-induced apoptosis, proliferation and migration suppression, which could be offset by Erk1/2 phosphorylation agonist Ro 67-7476. This study initially explored the essential role Mcl-1 played and the regulatory effect of Ras-Erk pathway in anti-cancer process triggered by tubulin inhibitor, broadening clinical horizon of tubulin inhibitors in oral squamous cell carcinoma chemotherapy application.

Irisin is well-known to contribute to bone homeostasis due to its bidirectional regulation on osteogenesis and osteoclastogenesis. However, the mechanisms of irisin involved in mesenchymal stem/stromal cells (MSCs)-derived osteogenesis are still under investigated. Fibronectin type III domain-containing protein 5 (FNDC5) is the precursor protein of irisin, compare with wild type (WT) littermates, FNDC5-/- mice lost bone mass significantly, collectively evidenced by the decrease of bone mineral density (BMD), impaired bone formation and reduced N-terminal propertied of type I procollagen (P1NP) in sera. Meanwhile, the bone resorbing of FNDC5-/- mice has enhanced accompanied by increased tartrate phosphatase (TRAP) staining cells morphologically and cross-Linked C-telopeptide of type 1 collagen (CTX) level in sera. In vitro study showed that lack of irisin impeded the MSC-derived osteogenesis of FNDC5-/- mice. The addition of irisin promote the osteogenesis of WT and irisin-deficient MSCs, by activating αV integrin-induced ERK/STAT pathway, subsequently enhancing bone morphogenetic protein 2 (BMP2) expression and BMP/SMAD signaling activation. Taken together, these findings further indicate that irisin regulates bone homeostasis. Moreover, irisin promotes MSC-derived osteogenesis by binding to αV integrin and activating BMP/SMAD signaling consequently. Thus, irisin may be a promising therapeutic target for osteoporosis and bone defects.

Pulmonary arterial hypertension (PAH) is characterized by muscularized pulmonary blood vessels, leading to right heart hypertrophy and cardiac failure. However, state-of-the-art therapeutics fail to target the ongoing remodeling process. Here, this study shows that matrix metalloproteinases (MMP)-1 and MMP-10 levels are increased in the medial layer of vessel wall, serum, and M1-polarized macrophages from patients with PAH and the lungs of monocrotaline- and hypoxia-induced PAH rodent models. MMP-10 regulates the malignant phenotype of pulmonary artery smooth muscle cells (PASMCs). The overexpression of active MMP-10 promotes PASMC proliferation and migration via upregulation of cyclin D1 and proliferating cell nuclear antigen, suggesting that MMP-10 produced by infiltrating macrophages contributes to vascular remodeling. Furthermore, inhibition of STAT1 inhibits hypoxia-induced MMP-10 but not MMP-1 expression in M1-polarized macrophages from patients with PAH. In conclusion, circulating MMP-10 could be used as a potential targeted therapy for PAH.

Background: Circular RNAs (circRNAs), which generally act as microRNA (miRNA) sponges to competitively regulate the downstream target genes of miRNA, play an essential role in cancer biology. However, few studies have been reported on the role of circRNA based competitive endogenous RNA (ceRNA) network in hepatocellular carcinoma (HCC). Herein, we aimed to screen and establish the circRNA/miRNA/mRNA networks related to the prognosis and progression of HCC and further explore the underlying mechanisms of tumorigenesis. Methods: GEO datasets GSE97332, GSE108724, and GSE101728 were utilized to screen the differentially expressed circRNAs (DE-circRNAs), DE-miRNAs, and DEmRNAs between HCC and matched para-carcinoma tissues. After six RNA-RNA predictions and five intersections between DE-RNAs and predicted RNAs, the survival-related RNAs were screened by the ENCORI analysis tool. The ceRNA networks were constructed using Cytoscape software, based on two models of up-regulated circRNA/down-regulated miRNA/up-regulated mRNA and down-regulated circRNA/up-regulated miRNA/down-regulated mRNA. The qRT-PCR assay was utilized for detecting the RNA expression levels in HCC cells and tissues. The apoptosis, Edu, wound healing, and transwell assays were performed to evaluate the effect of miR-106b-5p productions on the proliferation, invasion, and metastasis of HCC cells. In addition, the clone formation, cell cycle, and nude mice xenograft tumor assays were used to investigate the influence of hsa_circ_0001495 (circCCNB1) silencing and overexpression on the proliferation of HCC cells in vitro and in vivo. Furthermore, the mechanism of downstream gene DYNC1I1 and AKT/ERK signaling pathway via the circCCNB1/miR-106b-5p/GPM6A network in regulating the cell cycle was also explored. Results: Twenty DE-circRNAs with a genomic length less than 2000bp, 11 survival-related DE-miRNAs, and 61 survival-related DE-mRNAs were screened out and used to construct five HCC related ceRNA networks. Then, the circCCNB1/miR-106b-5p/GPM6A network was randomly selected for subsequent experimental verification and mechanism exploration at in vitro and in vivo levels. The expression of circCCNB1 and GPM6A were significantly down-regulated in HCC cells and cancer tissues, while miR-106b-5p expression was up-regulated. After transfections, miR-106b-5p mimics notably enhanced the proliferation, invasion, and metastasis of HCC cells, while the opposite was seen with miR-105b-5p inhibitor. In addition, circCCNB1 silencing promoted the clone formation ability, the cell cycle G1-S transition, and the growth of xenograft tumors of HCC cells via GPM6A downregulation. Subsequently, under-expression of GPM6A increased DYNC1I1 expression and activated the phosphorylation of the AKT/ERK pathway to regulate the HCC cell cycle. Conclusions: We demonstrated that circCCNB1 silencing promoted cell proliferation and metastasis of HCC cells by weakening sponging of oncogenic miR-106b-5p to induce GPM6A underexpression. DYNC1I1 gene expression was up-regulated and further led to activation of the AKT/ERK signaling pathway.

Esophageal carcinoma (EC) ranks sixth among cancers in mortality worldwide and effective drugs to reduce EC incidence and mortality are lacking. To explore potential anti-esophageal cancer drugs, we conducted drug screening and discovered that verdinexor, a selective inhibitor of nuclear exportin 1 (XPO1/CRM1), has anti-esophageal cancer effects both in vivo and in vitro. However, the mechanism and role of verdinexor in esophageal cancer remain unknown. In the present study, we observed that verdinexor inhibited the proliferation and migration of EC cells in vitro and suppressed tumor growth in vivo. Additionally, we found that verdinexor induced cleavage of PARP and downregulated XPO1, c-Myc, and FOSL1 expression. RNA-sequence analysis and protein-protein interaction (PPI) analysis revealed that verdinexor regulated the XPO1/c-Myc/FOSL1 axis. The results of immunoprecipitation and proximity ligation assays confirmed that verdinexor disrupted the interaction between XPO1 and c-Myc. Overexpression of c-Myc rescued the inhibition of cell proliferation and cell migration caused by verdinexor. Overexpressed FOSL1 restored the inhibited migration by verdinexor. Taken together, verdinexor inhibited cell proliferation and migration of esophageal cancer via XPO1/c-Myc/FOSL1 axis. Our findings provide a new option for the development of anti-esophageal cancer drugs.

Accumulating evidence indicates a carcinogenic role of environmental arsenic exposure, but mechanisms on how arsenic fosters malignant transformation of the normal cells are not fully established. By applying untargeted global metabolomics approach, we now show that arsenic is highly capable of perturbing the intracellular metabolic programs of the human bronchial epithelial cells, some of which are prominent hallmarks of cancer cell metabolism. To understand the spatiotemporal patterns of arsenic regulation on multiple metabolic pathways, we treated the cells with environmentally relevant concentration of arsenic, 0.25 μM, consecutively for 6 weeks to 24 weeks, and found that arsenic prompted heme metabolism, glycolysis, sphingolipid metabolism, phospholipid catabolism, protein degradation, and cholesterol breakdown constitutively, but inhibited metabolism of uracil-containing pyrimidine, carnitine, serotonin, polyamines, and fatty acid β-oxidation. A strong inhibition of all metabolites in mitochondrial tricarboxylic acid (TCA) cycle was noted in the cells treated with As3+ for 6 to 13 weeks. However, the metabolites in the earlier, but not the later steps of TCA cycle, including citrate, aconitate and isocitrate, were induced at 16 weeks through 24 weeks of arsenic treatment. This comprehensive metabolomics analysis provides new insights into metabolic perturbation by arsenic and may lead to more precise indications of arsenic in molecular carcinogenesis.

Background: Natural killer (NK) cell-based immunotherapy is clinically limited due to insufficient tumor infiltration in solid tumors. We have previously found that the natural product rocaglamide (RocA) can enhance NK cell-mediated killing of non-small cell lung cancer (NSCLC) cells by inhibiting autophagy, and autophagic inhibition has been shown to increase NK cell tumor infiltration in melanoma. Therefore, we hypothesized that RocA could increase NK cell infiltration in NSCLC by autophagy inhibition. Methods: Flow cytometry, RNA-sequencing, real-time PCR, Western blotting analysis, and xenograft tumor model were utilized to assess the infiltration of NK cells and the underlying mechanism. Results: RocA significantly increased the infiltration of NK cells and the expressions of CCL5 and CXCL10 in NSCLC cells, which could not be reversed by the inhibitions of autophagy/ULK1, JNK and NF-κB. However, such up-regulation could be suppressed by the inhibitions of TKB1 and STING. Furthermore, RocA dramatically activated the cGAS (cyclic GMP-AMP synthase)-STING (stimulator of interferon genes) signaling pathway, and the inhibition/depletion of STING ablated the up-regulation of CCL5 and CXCL10, NK cell infiltration, and tumor regression induced by RocA. Besides, RocA damaged mitochondrial DNA (mtDNA) and promoted the cytoplasmic release of mtDNA. The mPTP inhibitor cyclosporin A could reverse RocA-induced cytoplasmic release of mtDNA. Conclusions: RocA could promote NK cell infiltration by activating cGAS-STING signaling via targeting mtDNA, but not by inhibiting autophagy. Taken together, our current findings suggested that RocA was a potent cGAS-STING agonist and had a promising potential in cancer immunotherapy, especially in NK cell-based immunotherapy.

Background: Oral squamous cell carcinoma (OSCC) refers to the malignant tumor of the head and neck with a highest morbidity. It exhibits a poor prognosis and unsatisfactory treatment, which is partially attributed to delayed diagnosis. As indicated from existing reports, the protein histidine phosphatase LHPP acts as a vital factor in tumorigenesis in liver, lung, bladder, breast and pancreatic tumor tissues. Thus far, the expression level of LHPP in OSCC has been rarely studied, and its functional mechanism remains unclear. Methods: DEG analysis, OSCC cell lines and OSCC samples were used to detect the expression of LHPP in adjacent normal and cancerous tissues and its relationship with OSCC differentiation. Immunofluorescence staining was used to detect the over-expressed LHPP in OSCC cell lines. The cell counting kit 8 test, EdU proliferation test, scratch test, invasion test, single clone formation test, mouse xenograft tumor model HE staining and immunohistochemistry were used to estimate the biological characteristics of LHPP in OSCC in vivo and in vitro. GO and KEGG enrichment analysis and LHPP transcription factor analysis were used to further predict the role of LHPP and its related genes. The Western blotting assay, real-time PCR analysis and flow cytometry determined the inhibitory mechanism of LHPP in OSCC cells.Results: LHPP was down-regulated in OSCC tissues and cells than that in normal oral mucosa tissues and cells, LHPP expressing also displays a close association with the degree differentiation of OSCC. LHPP is capable of significantly inhibiting OSCC cells from proliferating, migrating and invading. The increase in LHPP expression facilitated OSCC cell apoptosis through PI3K/AKT signaling pathway.Conclusion: LHPP is a novel tumor suppressor which promotes apoptosis by inhibiting the PI3K/AKT signaling pathway in OSCC, indicating that LHPP is a new diagnostic marker and therapeutic target for OSCC.

Simultaneous P53 loss and activation of the PTEN-restricted PI3K-AKT pathway frequently occur in aggressive breast cancers. P53 loss causes genome instability, while PTEN loss and/or activating mutations of PIK3CA and AKT promote cancer cell proliferation that also increases incidences of genomic aberrations. However, the genomic alterations associated with P53 loss and activated PTEN-PI3K-AKT signaling in breast cancer have not been defined. Spatiotemporally controlled breast cancer models with inactivation of both P53 and Pten in adult mice have not been established for studying genomic alterations. Herein, we deleted both floxed Pten and Tp53 genes in the mammary gland epithelial cells in adult mice using a RCAS virus-mediated Cre-expressing system. These mice developed small tumors in 21 weeks, and poorly differentiated larger tumors in 26 weeks. In these tumors, we identified 360 genes mutated by nonsynonymous point mutations and small insertions and deletions (NSPMs/InDels), 435 genes altered by copy number amplifications (CNAs), and 450 genes inactivated by copy number deletions (CNDs). Importantly, 22.2%, 75.9% and 27.3% of these genes were also altered in human breast tumors with P53 and PTEN losses or P53 loss and activated PI3K-AKT signaling by NSPMs/InDels, CNAs and CNDs, respectively. Therefore, inactivation of P53 and Pten in adult mice causes rapid-growing breast tumors, and these tumors recapitulate a significant number of genetic aberrations in human breast tumors with inactivated P53 and activated PTEN-PI3K-AKT signaling. Further characterization of these commonly altered genes in breast cancer should help to identify novel cancer-driving genes and molecular targets for developing therapeutics.

HCC has remained one of the challenging cancers to treat, owing to the paucity of drugs targeting the critical survival pathways. Considering the cancer cells are deficient in DNase activity, the increase of an autonomous apoptisis endonuclease should be a reasonable choice for cancer treatment. In this study, we investigated whether DNASE1L3, an endonuclease implicated in apoptosis, could inhibit the progress of HCC. We found DNASE1L3 was down-regulated in HCC tissues, whereas its high expression was positively associated with the favorable prognosis of patients with HCC. Besides, serum DNASE1L3 levels were lower in HCC patients than in healthy individuals. Functionally, we found that DNASE1L3 inhibited the proliferation of tumor cells by inducing G0/G1 cell cycle arrest and cell apoptosis in vitro. Additionally, DNASE1L3 overexpression suppressed tumor growth in vivo. Furthermore, we found that DNASE1L3 overexpression weakened glycolysis in HCC cells and tissues via inactivating the rate-limiting enzymes involved in PTPN2-HK2 and CEBPβ-p53-PFK1 pathways. Finally, we identified the HBx to inhibit DNASE1L3 expression by up-regulating the expression of ZNF384. Collectively, our findings demonstrated that DNASE1L3 could inhibit the HCC progression through inducing cell apoptosis and weakening glycolysis. We believe DNASE1L3 could be considered as a promising prognostic biomarker and therapeutic target for HCC.

Rationale: Myocardial ischemia/reperfusion (I/R) injury is a common clinic scenario that occurs in the context of reperfusion therapy for acute myocardial infarction (AMI). The mitochondrial F1Fo-ATPase inhibitory factor 1 (IF1) blocks the reversal of the F1Fo-ATP synthase to prevent detrimental consumption of cellular ATP and associated demise. In the present study, we study the role and mechanism of IF1 in myocardial I/R injury. Methods: Mice were ligated the left anterior descending coronary artery to build the I/R model in vivo. Rat hearts were isolated and perfused with constant pressure according to Langendorff. Also, neonatal cardiomyocytes hypoxia-reoxygenation (H/R) model was also used. Myocardial infarction area, cardiac function, cellular function, and cell viability was conducted and compared. Results: Our data revealed that IF1 is upregulated in hearts after I/R and cardiomyocytes with hypoxia/re-oxygenation (H/R). IF1 delivered with adenovirus and adeno-associated virus serotype 9 (AAV9) ameliorated cardiac dysfunction and pathological development induced by I/R ex vivo and in vivo. Mechanistically, IF1 stimulates glucose uptake and glycolysis activity and stimulates AMPK activation during in vivo basal and I/R and in vitro OGD/R conditions, and activation of AMPK by IF1 is responsible for its cardioprotective effects against H/R-induced injury. Conclusions: These results suggest that increased IF1 in the I/R heart confer cardioprotective effects via activating AMPK signaling. Therefore, IF1 can be used as a potential therapeutic target for the treatment of pathological ischemic injury and heart failure.

Traumatic brain injury (TBI) is one of the main concerns worldwide as there is still no comprehensive therapeutic intervention. Astrocytic water channel aquaporin-4 (AQP-4) system is closely related to the brain edema, water transport at blood-brain barrier (BBB) and astrocyte function in the central nervous system (CNS). Minocycline, a broad-spectrum semisynthetic tetracycline antibiotic, has shown anti-inflammation, anti-apoptotic, vascular protection and neuroprotective effects on TBI models. Here, we tried to further explore the underlying mechanism of minocycline treatment for TBI, especially the relationship of minocycline and AQP4 during TBI treatment. In present study, we observed that minocycline efficaciously reduces the elevation of AQP4 in TBI mice. Furthermore, minocycline significantly reduced neuronal apoptosis, ameliorated brain edema and BBB disruption after TBI. In addition, the expressions of tight junction protein and astrocyte morphology alteration were optimized by minocycline administration. Similar results were found after treating with TGN-020 (an inhibitor of AQP4) in TBI mice. Moreover, these effects were reversed by cyanamide (CYA) treatment, which notably upregulated AQP4 expression level in vivo. In primary cultured astrocytes, small-interfering RNA (siRNA) AQP4 treatment prevented glutamate-induced astrocyte swelling. To sum up, our study suggests that minocycline improves the functional recovery of TBI through reducing AQP4 level to optimize BBB integrity and astrocyte function, and highlights that the AQP4 may be an important therapeutic target during minocycline treating for TBI.

The limb-bud and heart (LBH) gene was reported to suppress nasopharyngeal carcinoma (NPC) progression in our previous study. Distant metastasis predominantly accounts for the unsatisfactory prognosis of NPC treatment, in which epithelial-mesenchymal transition (EMT) and tumor angiogenesis are of great significance. The roles of exosomes in mediating NPC progression have been highlighted in recent researches, and attempts have been made to explore the clinical application of NPC exosomes. Here we investigated the function of the LBH gene in NPC exosomes, and its potential mechanism. NPC xenografts were constructed, showing that vascular endothelial growth factor A (VEGFA) expression and neovascularity were attenuated by LBH overexpression, together with diminished EMT progression. NPC-derived exosomes were isolated, identified and applied for in vitro/in vivo experiments, and the exosomal distribution of LBH was elevated in exosomes derived from LBH-upregulated cells. Ectopic LBH, αB-crystallin (CRYAB) and VEGFA expression was induced by lentiviral infection or plasmid transfection to explore their functions in modulating EMT and angiogenesis in NPC. The addition of LBH+ NPC exosomes during a Matrigel plug assay in mice suppressed in vivo angiogenesis, and the treatment of human umbilical vein endothelial cells (HUVECs) with LBH+ NPC exosomes inhibited cellular proliferation, migration and tube formation. The interactions among LBH, CRYAB and VEGFA were confirmed by colocalization and fluorescence resonance energy transfer (FRET) assays, and extracellular VEGFA secretion from both HUVECs and NPC cells under the treatment with LBH+ NPC exosomes was diminished according to ELISA results. We concluded that exosomal LBH inhibits EMT progression and angiogenesis in the NPC microenvironment, and that its effects are partially implemented by modulation of VEGFA expression, secretion and related signaling. Thus, LBH could serve as a promising therapeutic target in VEGFA-focused NPC treatment.

Mesenchymal stem cells (MSCs) as a therapeutic promise are often quickly cleared by innate immune cells of the host including natural killer (NK) cells. Efforts have been made to generate immune-escaping human embryonic stem cells (hESCs) where T cell immunity is evaded by defecting β-2-microglobulin (B2M), a common unit for human leukocyte antigen (HLA) class I, and NK cells are inhibited via ectopic expression of HLA-E or -G. However, NK subtypes vary among recipients and even at different pathologic statuses. It is necessary to dissect and optimize the efficacy of the immune-escaping cells against NK subtypes. Here, we first generated B2M knockout hESCs and differentiated them to MSCs (EMSCs) and found that NK resistance occurred with B2M-/- EMSCs expressing HLA-E and -G only when they were transduced via an inducible lentiviral system in a dose-dependent manner but not when they were inserted into a safe harbor. HLA-E and -G expressed at high levels together in transduced EMSCs inhibited three major NK subtypes, including NKG2A+/LILRB1+, NKG2A+/LILRB1-, and NKG2A-/LILRB1+, which was further potentiated by IFN-γ priming. Thus, this study engineers MSCs with resistance to multiple NK subtypes and underscores that dosage matters when a transgene is used to confer a novel effect to host cells, especially for therapeutic cells to evade immune rejection.

X-box binding protein 1(XBP1) is a critical component for unfolded protein response (UPR) in ER stress. According to previous studies performed with different XBP1-deficient mice, the XBP1 gene affects mouse cartilage development and causes other related diseases. However, how the complete transcriptome, including mRNA and ncRNAs, affects the function of cartilage and other tissues when XBP1 is deficient in chondrocytes is unclear. In this study, we aimed to screen the differentially expressed (DE) mRNAs, circRNAs, lncRNAs and miRNAs in XBP1 cartilage-specific knockout (CKO) mice using high throughput sequencing and construct the circRNA-miRNA-mRNA and lncRNA-miRNA-mRNA regulatory networks. DE LncRNAs (DE-LncRNAs), circRNAs (DE-circRNAs), miRNAs (DE-miRNAs), and mRNAs [differentially expressed genes (DEGs)] between the cartilage tissue of XBP1 CKO mice and controls were identified, including 441 DE-LncRNAs, 15 DE-circRNAs, 6 DE-miRNAs, and 477 DEGs. Further, 253,235 lncRNA-miRNA-mRNA networks and 1,822 circRNA-miRNA-mRNA networks were constructed based on the correlation between lncRNAs/circRNAs, miRNAs, mRNAs. The whole transcriptome analysis revealed that XBP1 deficiency in cartilage affects the function of cartilage and other different tissues, as well as associated diseases. Overall, our findings may provide potential biomarkers and mechanisms for the diagnosis and treatment of cartilage and other related diseases.

Lactic acid (LA) metabolism in the tumor microenvironment contributes to the establishment and maintenance of immune tolerance. This pathway is characterized in tumor associated macrophages. However, the role and pathway of LA metabolism at maternal-fetal interface during early pregnancy, especially in decidual macrophage differentiation, are still unclear. Herein, for the first time, we discovered that LA can trigger either M2 or M1 macrophage polarization via oxidative phosphorylation and glycolysis regulation under normoxia or hypoxia, respectively. Also, LA metabolism played a vital role in decidual macrophages-mediated recurrent pregnancy loss (RPL), through HIF-1α/SRC/LDHA pathway. Moreover, blockade of LA intake with AZD3965 (MCT-1 inhibitor) could rescue pregnancy in an abortion-prone mouse model, suggesting a potential therapeutic target in RPL. Collectively, the present study identifies the previously unknown functions of LA metabolism in the differentiation of decidual macrophages in early normal pregnancy and RPL, and provides a potential therapeutic strategy in RPL by manipulating decidual macrophages' functions through LA metabolic pathway.

Myocardial ischemia/reperfusion (I/R) injury is still a lack of effective therapeutic drugs, and its molecular mechanism is urgently needed. Studies have shown that the intestinal flora plays an important regulatory role in cardiovascular injury, but the specific mechanism has not been fully elucidated. In this study, we found that an increase in Ang II in plasma was accompanied by an increase in the levels of myocardial injury during myocardial reperfusion in patients with cardiopulmonary bypass. Furthermore, Ang II treatment enhanced mice myocardial I/R injury, which was reversed by caveolin-1 (CAV-1)-shRNA or strengthened by angiotensin-converting enzyme 2 (ACE2)-shRNA. The results showed that CAV-1 and ACE2 have protein interactions and inhibit each other's expression. In addition, propionate, a bacterial metabolite, inhibited the elevation of Ang II and myocardial injury, while GPR41-shRNA abolished the protective effects of propionate on myocardial I/R injury. Clinically, the propionate content in the patient's preoperative stool was related to Ang II levels and myocardial I/R injury levels during myocardial reperfusion. Taken together, propionate alleviates myocardial I/R injury aggravated by Ang II dependent on CAV-1/ACE2 axis through GPR41, which provides a new direction that diet to regulate the intestinal flora for treatment of myocardial I/R injury.

Chronic Hepatitis B virus (CHB) infection is a global public health problem. Oligodeoxynucleotides (ODNs) containing class C unmethylated cytosine-guanine dinucleotide (CpG-C) motifs may provide potential adjuvants for the immunotherapeutic strategy against CHB, since CpG-C ODNs stimulate both B cell and dendritic cell (DC) activation. However, the efficacy of CpG-C ODN as an anti-HBV vaccine adjuvant remains unclear. In this study, we demonstrated that CpG M362 (CpG-C ODN) as an adjuvant in anti-HBV vaccine (cHBV-vaccine) successfully and safely eliminated the virus in HBV-carrier mice. The cHBV-vaccine enhanced DC maturation both in vivo and in vitro, overcame immune tolerance, and recovered exhausted T cells in HBV-carrier mice. Furthermore, the cHBV-vaccine elicited robust hepatic HBV-specific CD8+ and CD4+ T cell responses, with increased cellular proliferation and IFN-γ secretion. Additionally, the cHBV-vaccine invoked a long-lasting follicular CXCR5+ CD8+ T cell response following HBV re-challenge. Taken together, CpG M362 in combination with rHBVvac cleared persistent HBV and achieved long-term virological control, making it a promising candidate for treating CHB.

Diabetic keratopathy (DK) is an important diabetic complication at the ocular surface. Chronic low-grade inflammation mediated by the NLRP3 inflammasome promotes pathogenesis of diabetes and its complications. However, the effect of the NLRP3 inflammasome on DK pathogenesis remains elusive. Wild-type (WT) and Nlrp3 knockout (KO) C57 mice were used to establish a type I diabetes model by intraperitoneal injection of streptozotocin. The effect of the NLRP3 inflammasome on diabetic corneal wound healing and never regeneration was examined by a corneal epithelial abrasion model. Western blot, immunofluorescence staining, enzyme-linked immunosorbent assay (ELISA) and pharmacological treatment were performed to investigate the regulatory mechanism of advanced glycation end products (AGEs) on NLRP3 inflammasome activation and corneal wound healing in vivo. The cultured mouse corneal epithelial cells (TKE2) were used to evaluate the effect and mechanism of AGEs on NLRP3 inflammasome activation in vitro. We revealed that NLRP3 inflammasome-mediated inflammation and pyroptosis contributed to DK pathogenesis. Under physiological conditions, the NLRP3 inflammasome was required for corneal wound healing and nerve regeneration. However, under a diabetic scenario, sustained activation of the NLRP3 inflammasome resulted in postponed corneal wound healing and impaired nerve regeneration. Mechanistically, the accumulated AGEs promoted hyperactivation of the NLRP3 inflammasome through ROS production. Moreover, genetically and pharmacologically blocking the AGEs/ROS/NLRP3 inflammasome axis significantly expedited diabetic corneal epithelial wound closure and nerve regeneration. Our results revealed that AGEs-induced hyperactivation of the NLRP3 inflammasome resulted in delayed diabetic corneal wound healing and impaired nerve regeneration, which further highlighted the NLRP3 inflammasome as a promising target for DK treatment.

Background: G-protein-coupled receptor 43 (GPR43) is a posttranscriptional regulator involved in cholesterol metabolism. This study aimed to investigate the possible roles of GPR43 activation in podocyte lipotoxicity in diabetic nephropathy (DN) and explore the potential mechanisms. Methods: The experiments were conducted by using diabetic GPR43-knockout mice and a podocyte cell culture model. Lipid deposition and free cholesterol levels in kidney tissues were measured by BODIPY staining and quantitative cholesterol assays, respectively. The protein expression of GPR43, LC3II, p62, beclin1, low-density lipoprotein receptor (LDLR) and early growth response protein 1 (EGR1) in kidney tissues and podocytes was measured by real-time PCR, immunofluorescent staining and Western blotting. Results: There were increased LDL cholesterol levels in plasma and cholesterol accumulation in the kidneys of diabetic mice. However, GPR43 gene knockout inhibited these changes. An in vitro study further demonstrated that acetate treatment induced cholesterol accumulation in high glucose-stimulated podocytes, which was correlated with increased cholesterol uptake mediated by LDLR and reduced cholesterol autophagic degradation, as characterized by the inhibition of LC3 maturation, p62 degradation and autophagosome formation. Gene knockdown or pharmacological inhibition of GPR43 prevented these effects on podocytes. Furthermore, GPR43 activation increased extracellular regulated protein kinases 1/2 (ERK1/2) activity and EGR1 expression in podocytes, which resulted in an increase in cholesterol influx and autophagy inhibition. In contrast, after GPR43 deletion, these changes in podocytes were improved, as shown by the in vivo and in vitro results. Conclusion: GPR43 activation-mediated lipotoxicity contributes to podocyte injury in DN by modulating the ERK/EGR1 pathway.

The use of large molecules for immunotherapy has led to exciting developments in cancer treatment, such as the development of PD-1/PD-L1 antibodies. However, small molecule targeted therapies still lack effective immune-functional classes. Ideal anticancer drugs should simultaneously generate immune memory when killing cancer cells to prevent tumor relapse and metastasis. To this end, we carried out a rationally designed strategy to develop novel classes of small molecule compounds with bifunctional targeting and immunostimulatory abilities by conjugating targeting compounds with TLR7 agonists, generating immune-targeting conjugates (ImmunTacs). GY161, as a representative ImmunTac, was synthesized via chemical conjugation of ibrutinib with a TLR7 agonist. In vitro, GY161 stimulated the production of cytokines by mouse spleen lymphocytes, promoted the maturation of dendritic cells (DCs), and inhibited the growth and induced the apoptosis of B16 melanoma cells by regulating the c-Met/β-catenin pathway. In vivo, GY161 enhanced the frequency of CD8+ T cells in spleens and tumors, suppressed the growth of B16 melanoma cell-derived tumors and prolonged the survival time of mice. In summary, GY161 could prevent melanoma progression through direct tumor killing and by triggering specific immunity. These results strongly suggest that ImmunTacs are a reliable and promising strategy for developing small molecule immunogenic anticancer drugs.

RNA-binding motif protein 10 (RBM10), one of the members of the RNA-binding protein (RBP) family, has a tumor suppressor role in multiple cancers. However, the functional role of RBM10 in lung adenocarcinoma (LUAD) and the underlying molecular mechanism remains unclear. In this study, we observed that RBM10 is significantly downregulated in LUAD tissues compared with normal tissues. Low RBM10 expression is significantly associated with poor outcome of LUAD patients. In vitro and in vivo experiments show that RBM10 inhibits cell proliferation, metastasis and EMT progression in LUAD. Mechanistically, we demonstrate that RBM10 interacts with β-catenin interacting protein 1 (CTNNBIP1) and positively regulates its expression, disrupting the binding of β-catenin to the transcription factor TCF/LEF, thereby inactivating the Wnt/β-catenin pathway. In conclusion, this is the first study reporting the role of RBM10 in suppressing LUAD progression at least via partly inactivating the Wnt/β-catenin pathway, which provides new insights into the tumorigenesis and metastasis of LUAD. Thus, RBM10 may be a promising new therapeutic target or clinical biomarker for LUAD therapy in the future.

Osteosarcoma (OS) is a malignant bone tumor among adolescents and young adults. IRF7 belongs to the transcription factor family of interferon regulatory factors (IRFs) and has previously been described to function as a tumor suppressor in multiple cancer types. However, the biological functions and cellular mechanism of IRF7 in OS remain elusive. In this study, by quantitative real-time PCR (qRT-PCR) and western blotting, we found that IRF7 was downregulated in OS, and the higher expression of IRF7 was correlated with a better survival prognosis. Moreover, loss-of-function and gain-of-function studies have proved the critical functions of IRF7 in suppressing aerobic glycolysis of osteosarcoma cells as evidenced by glucose uptake, lactate production, extracellular acidification rate, and oxygen consumption rate. Mechanistically, IRF7 inhibited the expression of key glycolytic gene PKM2 via direct transcriptional regulation. Moreover, the in vitro and in vivo tumor-suppressive roles of IRF7 were uncovered in OS and these effects were largely glycolysis-dependent. Therefore, our study unveils a previous unprecedented role of IRF7 in glucose metabolism reprogram and suggests that IRF7 might serve as a potential therapeutic target for patients with OS.

Chronic obstructive pulmonary disease (COPD) affects the health of more than 300 million people worldwide; at present, there is no effective drug to treat COPD. Smoking is the most important risk factor, but the molecular mechanism by which smoking causes the disease is unclear. The senescence of lung epithelial cells is related to development of COPD. Regulation of miRNAs is the main epigenetic mechanism related to aging. β-Galactose staining showed that the lung tissues of smokers have a higher degree of cellular senescence, and the expression of miR-125a-5p is high. This effect is obvious for smokers with COPD/emphysema, and there is a negative correlation between miR-125a-5p levels and values for forced expiratory volume in one second (FEV1)/forced vital capacity (FVC). After Balb/c mice were chronically exposed to various concentrations of cigarette smoke (CS), plethysmography showed that lung function was impaired, lung tissue senescence was increased, and the senescence-associated secretory phenotype (SASP) in bronchoalveolar lavage fluid was increased. For mouse lung epithelial (MLE)-12 cells treated with cigarette smoke extract (CSE), Sp1 and SIRT1 levels were low, HIF-1α acetylation levels were high, and cell senescence and secretion of SASP factors were elevated. Down-regulation of miR-125a-5p or up-regulation of Sp1 reversed these effects. In addition, compared with mice exposed to CS, knockdown of miR-125a-5p reduced lung epithelial cell senescence and COPD/emphysema. Therefore, in smoking-induced COPD, elevated miR-125a-5p participates in the senescence of lung epithelial cells through Sp1/SIRT1/HIF-1α. These findings provide evidence related to the pathogenesis of COPD/emphysema caused by chronic smoking.

Resistance to radiotherapy is frequently observed in the clinic and leads to poor prognosis of non-small cell lung cancer (NSCLC). How to overcome resistance to radiotherapy is a challenge in the treatment of NSCLC. In this study, PPDPF was found to be upregulated in NSCLC tissues and cell lines, and its expression negatively correlated with the overall survival of patients with NSCLC. PPDPF promoted the growth, colony formation and invasion of lung cancer cells. Moreover, knockout of PPDPF inhibited tumorigenesis in the KL (KrasG12D; LKB1f/f) mouse model of lung cancer. Additionally, overexpression of PPDPF led to radioresistance in lung cancer cells, and knockdown of PPDPF sensitized lung cancer cells to radiotherapy. Mechanistically, PPDPF interacted with BABAM2 (an antiapoptotic protein) and blocked its ubiquitination by MDM2, thus stabilizing BABAM2 and promoting the radioresistance of lung cancer cells. Our present study suggested PPDPF as a therapeutic target in NSCLC.

Hepatitis B virus (HBV) infection is a major risk factor for hepatocellular carcinoma (HCC), which required developing novel therapies targeting the inhibition of HBV transcription and replication due to current limited treatment options. We explored novel target for the development of novel therapies targeting the inhibition of HBV replication and transcription. The expression of Id1 and E2F4 in HCC cells and tissues was detected by qRT-PCR and western blot. We investigated the Id1 and E2F4-mediated transcription of HBV infection by using HepG2.2.15, HepAD38, HepG2-NTCP cell lines and AAV/HBV-infected mice. Interactions between the two host proteins and viral covalently closed circular DNA (cccDNA) were assessed using subcellular localization, protein-protein interaction, chromatin immunoprecipitation, and luciferase assays. Ectopic Id1 significantly reduced HBV transcription and replication in both HBV-expressing cells and AAV/HBV-infected mice. Id1 and E2F4 could form a heterodimer to prevent E2F4 from promoting HBV transcription and replication. E2F4 could directly bind to cccDNA and activate the HBV core promoter in cell lines. Furthermore, in vitro binding experiments confirmed that the sequence 1758'-TTAAAGGTC-1766', which is highly conserved among HBV genotypes, is the target site of the E2F4 homodimer. The findings suggest that E2F4 function as novel cccDNA-binding protein to directly activate HBV transcription by binding to Cp promoter region. Our results highlight the ability that E2F4 represent a pan-potential therapeutic target against HBV transcription and provide more clues to better understand the life cycle of HBV.

Diabetic cardiomyopathy (DCM) is associated with oxidative stress and augmented inflammation in the heart. Neuraminidases (NEU) 1 has initially been described as a lysosomal protein which plays a role in the catabolism of glycosylated proteins. We investigated the role of NEU1 in the myocardium in diabetic heart. Streptozotocin (STZ) was injected intraperitoneally to induce diabetes in mice. Neonatal rat ventricular myocytes (NRVMs) were used to verify the effect of shNEU1 in vitro. NEU1 is up-regulated in cardiomyocytes under diabetic conditions. NEU1 inhibition alleviated oxidative stress, inflammation and apoptosis, and improved cardiac function in STZ-induced diabetic mice. Furthermore, NEU1 inhibition also attenuated the high glucose-induced increased reactive oxygen species generation, inflammation and, cell death in vitro. ShNEU1 activated Sirtuin 3 (SIRT3) signaling pathway, and SIRT3 deficiency blocked shNEU1-mediated cardioprotective effects in vitro. More importantly, we found AMPKα was responsible for the elevation of SIRT3 expression via AMPKα-deficiency studies in vitro and in vivo. Knockdown of LKB1 reversed the effect elicited by shNEU1 in vitro. In conclusion, NEU1 inhibition activates AMPKα via LKB1, and subsequently activates sirt3, thereby regulating fibrosis, inflammation, apoptosis and oxidative stress in diabetic myocardial tissue.

Respiratory syncytial virus (RSV) infection is the main cause of bronchiolitis in children. Excessive mucus secretion is one of the primary symbols in RSV related lower respiratory tract infections (RSV-related LRTI), which is closely associated with the occurrence and development of asthma in later life. Integrin β4 (ITGB4) is down-regulated in the airway epithelial cells (AECs) of asthma patients which plays a critical role in the pathogenesis of asthma. However, whether ITGB4 is involved in the pathological processes of RSV infection remains unclear. In this study, we found that decreased expression of ITGB4 was negatively correlated with the level of MUC5AC in childhood AECs following RSV infection. Moreover, ITGB4 deficiency led to mucus hypersecretion and MUC5AC overexpression in the small airway of RSV-infected mice. MUC5AC expression was upregulated by ITGB4 in HBE cells through EGFR, ERK and c-Jun pathways. EGFR inhibitors treatment inhibited mucus hypersecretion and MUC5AC overexpression in ITGB4-deficient mice after RSV infection. Together, these results demonstrated that epithelial ITGB4 deficiency induces mucus hypersecretion by upregulating the expression of MUC5AC through EGFR/ERK/c-Jun pathway, which further associated with RSV-related LRTI.

Anti-cancer chemo-drugs can cause a rapid elevation of intracellular reactive oxygen species (ROS) levels. An imbalance in ROS production and elimination systems leads to cancer cell resistance to chemotherapy. This study aimed to evaluate the mechanism and effect of ROS on multidrug resistance in various human chemoresistant cancer cells by detecting the changes in the amount of ROS, the expression of ROS-related and glycolysis-related genes, and cell death. We found that ROS was decreased while oxidative phosphorylation was increased in chemoresistant cells. We verified that the chemoresistance of cancer cells was achieved in two ways. First, chemoresistant cells preferred oxidative phosphorylation instead of anaerobic glycolysis for energy generation, which increased ATPase activity and produced much more ATP to provide energy. Second, ROS-scavenging systems were enhanced in chemoresistant cancer cells, which in turn decreased ROS amount and thus inhibited chemo-induced cell death. Our in vitro and in vivo photodynamic therapy further demonstrated that elevated ROS production efficiently inhibited chemo-drug resistance and promoted chemoresistant cell death. Taken together, targeting ROS systems has a great potential to treat cancer patients with chemoresistance.

Bone marrow-derived mesenchymal stem cells (BM-MSCs) are multipotent stromal cells that have a critical role in the maintenance of skeletal tissues such as bone, cartilage, and the fat in bone marrow. In addition to providing microenvironmental support for hematopoietic processes, BM-MSCs can differentiate into various mesodermal lineages including osteoblast/osteocyte, chondrocyte, and adipocyte that are crucial for bone metabolism. While BM-MSCs have high cell-to-cell heterogeneity in gene expression, the cell subtypes that contribute to this heterogeneity in vivo in humans have not been characterized. To investigate the transcriptional diversity of BM-MSCs, we applied single-cell RNA sequencing (scRNA-seq) on freshly isolated CD271+ BM-derived mononuclear cells (BM-MNCs) from two human subjects. We successfully identified LEPRhiCD45low BM-MSCs within the CD271+ BM-MNC population, and further codified the BM-MSCs into distinct subpopulations corresponding to the osteogenic, chondrogenic, and adipogenic differentiation trajectories, as well as terminal-stage quiescent cells. Biological functional annotations of the transcriptomes suggest that osteoblast precursors induce angiogenesis coupled with osteogenesis, and chondrocyte precursors have the potential to differentiate into myocytes. We also discovered transcripts for several clusters of differentiation (CD) markers that were either highly expressed (e.g., CD167b, CD91, CD130 and CD118) or absent (e.g., CD74, CD217, CD148 and CD68) in BM-MSCs, representing potential novel markers for human BM-MSC purification. This study is the first systematic in vivo dissection of human BM-MSCs cell subtypes at the single-cell resolution, revealing an insight into the extent of their cellular heterogeneity and roles in maintaining bone homeostasis.

Objective: Angiogenesis is involved in multiple biological processes, including atherosclerosis (AS) and cancer. Dickkopf1 (DKK1) plays many roles in both tumors and AS and has emerged as a potential biomarker of cancer progression and prognosis. Targeting DKK1 is a good choice for oncological treatments. Many anticancer therapies are associated with specific cardiovascular toxicity. However, the effects of DKK1 neutralizing therapy on AS are unclear. We focused on how DKK1 affected angiogenesis in AS and ox-LDL-induced human umbilical vein endothelial cells (HUVECs). Methods: ApoE-/- mice were fed a high-fat diet and then injected with DKK1i or DKK1 lentivirus to study the effects of DKK1. In vitro, promoter assays, protein analysis, database mining, dual-luciferase reporter assay (DLR), electrophoretic mobility shift assay (EMSA), chromatin immunoprecipitation (ChIP), and coimmunoprecipitation (co-IP) were used to study the mechanism of DKK1 biogenesis. Cell migration and angiogenesis assays were performed to investigate the function and regulatory mechanisms of DKK1. Results: DKK1 participated in angiogenesis both in the plaques of ApoE-/- mice by knockdown or overexpression of DKK1 and ox-LDL-induced HUVECs. DKK1 induced angiogenesis (increasing migration and capillary formation, inducing expression of VEGFR-2/VEGF-A/MMP) via the CKAP4/PI3K pathway, independent of Wnt/β-catenin. ox-LDL increased the expression and nuclear transfer of Ets-1 and c-jun, and induced the transcriptional activity of DKK1 in HUVECs. Ets-1, along with c-jun and CBP, could bind to the promoter of DKK1 and enhance DKK1 transcription. MiR33a-5p was downregulated in ox-LDL induced HUVECs and aortic artery of high-fat diet ApoE-/- mice. Ets-1 was a direct target of miR33a-5p. MiR33a-5p/Ets-1/ DKK1 axis contributed to angiogenesis. Conclusions: MiR33a-5p/Ets-1/DKK1 signaling participated in ox-LDL-induced angiogenesis of HUVECs via the CKAP4/PI3K pathway. These new findings provide a rationale and notable method for tumor therapy and cardiovascular protection.

Rationale: Nonalcoholic steatohepatitis (NASH), as one of the key stages in the development of nonalcoholic fatty liver disease (NAFLD), can directly progress to HCC, but the underlying mechanism is not fully understood. Methods: Differentially expressed genes (DEGs) in each stage of disease development were studied through a GEO dataset deriving from a Stelic Animal Model (STAM), which can simulate the evolution of NAFLD/NASH to HCC in humans. GSVA analysis was performed to analyze the differentially expressed oncogenic signatures in each stage. A human NAFLD-related dataset from GEO database was utilized for gene expression verification and further validated in the protein level in STAM mice. Small molecule inhibitors were applied to STAM mice for investigating whether inhibition of the LPL/FABP4/CPT1 axis could prevent the occurrence of NASH-related HCC in vivo. Microsphere formation and clonal formation assays in vitro were applied to study if inhibition of the LPL/FABP4/CPT1 axis can reduce the viability of liver cancer stem cells (LCSCs). Results: We found that upregulation of the LPL/FABP4/CPT1 molecular axis, as a fatty acid metabolic reprogramming process, occurred specifically during the NASH phase. GSVA analysis showed widespread activation of a large number of oncogenic signals, which may contribute to malignant transformation during NASH. Furthermore, inhibition of the LPL/FABP4/CPT1 axis could effectively delay the tumor growth in STAM mice. Cell assays revealed inhibitors targeting this axis can significantly reduce the sphere-forming, proliferation, and clonality of LCSCs. Conclusion: These results suggest that activation of the LPL/FABP4/CPT1 axis is essential for LCSCs maintenance, which acts synergistically with a variety of up-regulated oncogenic signals that drive the hepatocyte-LCSCs transdifferentiation during NASH to HCC progression. Thus, targeting the LPL/FABP4/CPT1 axis may provide a potential direction for NASH-related HCC prevention.

Bone remodeling is a dynamic process between bone formation mediated by osteoblasts and bone resorption mediated by osteoclasts. Disrupted bone remodeling is a key factor in postmenopausal osteoporosis, a metabolic disorder characterized by deteriorated bone microarchitecture and increased risk of fracture. Recent studies have shown that piwi-binding RNA (piRNA) is involved in the pathogenesis of certain diseases at the post-transcriptional level. Here, we analyzed piRNA-63049 (piR-63049), which may play an essential role in bone remodeling. The expression of piR-63049 significantly increased in both bone tissues and plasma of osteoporotic rats and postmenopausal osteoporotic patients. Overexpressing piR-63049 could inhibit the osteoblastogenesis of bone marrow stromal cells (BMSCs) while knocking down piR-63049 could promote the osteoblastogenesis of BMSCs through the Wnt2b/β-catenin signaling pathway. Moreover, knocking-down piR-63049 (piR-63049-antagonist) in vivo could attenuate the bone loss in ovariectomized rats by promoting bone formation. Taken together, the current study shows that piR-63049 inhibits bone formation through the Wnt2b/β-catenin signaling pathway. This novel piRNA may be a potential target to increase bone formation in bone loss disorders such as postmenopausal osteoporosis.

BET bromodomain BRD4 and RAC1 oncogenes are considered important therapeutic targets for cancer and play key roles in tumorigenesis, survival and metastasis. However, combined inhibition of BRD4-RAC1 signaling pathways in different molecular subtypes of breast cancer including luminal-A, HER-2 positive and triple-negative breast (TNBC) largely remains unknown. Here, we demonstrated a new co-targeting strategy by combined inhibition of BRD4-RAC1 oncogenic signaling in different molecular subtypes of breast cancer in a context-dependent manner. We show that combined treatment of JQ1 (inhibitor of BRD4) and NSC23766 (inhibitor of RAC1) suppresses cell growth, clonogenic potential, cell migration and mammary stem cells expansion and induces autophagy and cellular senescence in molecular subtypes of breast cancer cells. Mechanistically, JQ1/NSC23766 combined treatment disrupts MYC/G9a axis and subsequently enhances FTH1 to exert antitumor effects. Furthermore, combined treatment targets HDAC1/Ac-H3K9 axis, thus suggesting a role of this combination in histone modification and chromatin modeling. C-MYC depletion and co-treatment with vitamin-C sensitizes different molecular subtypes of breast cancer cells to JQ1/NSC23766 combination and further reduces cell growth, cell migration and mammosphere formation. Importantly, co-targeting RAC1-BRD4 suppresses breast tumor growth in vivo using xenograft mouse model. Clinically, RAC1 and BRD4 expression positively correlates in breast cancer patient's samples and show high expression patterns across different molecular subtypes of breast cancer. Both RAC1 and BRD4 proteins predict poor survival in breast cancer patients. Taken together, our results suggest that combined inhibition of BRD4-RAC1 pathways represents a novel and potential therapeutic approach in different molecular subtypes of breast cancer and highlights the importance of co-targeting RAC1-BRD4 signaling in breast tumorigenesis via disruption of C-MYC/G9a/FTH1 axis and down regulation of HDAC1.

Introduction and Aims: Elevated plasma levels of C-reactive protein (CRP) are closely associated with progressive renal injury in patients with chronic kidney disease (CKD). Here, we tested a hypothesis that CRP may promote renal fibrosis and inflammation via a TGF-β/Smad3-dependent mechanism. Methods: Role and mechanisms of TGF-β/Smad3 in CRP-induced renal fibrosis and inflammation were examined in a mouse model of unilateral ureteral obstruction (UUO) induced in CRP Tg/Smad3 KO mice and in a rat tubular epithelial cell line in which Smad3 gene is stably knocked down (S3KD-NRK52E). Results: We found that mice overexpressing the human CRP gene were largely promoted renal inflammation and fibrosis as evidenced by increasing IL-1β, TNF-α, MCP-1 expression, F4/80+ macrophages infiltration, and marked accumulation of α-smooth muscle actin (α-SMA), collagen I and fibronectin in the UUO kidney, which were blunted when Smad3 gene was deleted in CRPtg-Smad3KO. Mechanistically, we found that the protection of renal inflammation and fibrosis in the UUO kidney of CRPtg-Smad3KO mice was associated with the inactivation of CD32-NF-κB and TGF-β/Smad3 signaling. Conclusion: In conclusion, Smad3 deficiency protects against CRP-mediated renal inflammation and fibrosis in the UUO kidney by inactivating CD32-NF-κB and TGF-β/Smad3 signaling.

Rationale: In intracranial arterial dolichoectasia (IADE) development, the feedback loop between inflammatory cytokines and macrophages involves TNF-α and NF-κB signaling pathways and leads to subsequent MMP-9 activation and extracellular matrix (ECM) degeneration. In this proof-of-concept study, melittin-loaded L-arginine-coated iron oxide nanoparticle (MeLioN) was proposed as the protective measure of IADE formation for this macrophage-mediated inflammation and ECM degeneration. Methods: IADE was created in 8-week-old C57BL/6J male mice by inducing hypertension and elastase injection into a basal cistern. Melittin was loaded on the surface of ION as a core-shell structure (hydrodynamic size, 202.4 nm; polydispersity index, 0.158). Treatment of MeLioN (2.5 mg/kg, five doses) started after the IADE induction, and the brain was harvested in the third week. In the healthy control, disease control, and MeLioN-treated group, the morphologic changes of the cerebral arterial wall were measured by diameter, thickness, and ECM composition. The expression level of MMP-9, CD68, MCP-1, TNF-α, and NF-κB was assessed from immunohistochemistry, polymerase chain reaction, and Western blot assay. Results: MeLioN prevented morphologic changes of cerebral arterial wall related to IADE formation by restoring ECM alterations and suppressing MMP-9 expression. MeLioN inhibited MCP-1 expression and reduced CD68-positive macrophage recruitments into cerebral arterial walls. MeLioN blocked TNF-α activation and NF-κB signaling pathway. In the Sylvian cistern, co-localization was found between the CD68-positive macrophage infiltrations and the MeLioN distributions detected on Prussian Blue and T2* gradient-echo MRI, suggesting the role of macrophage harboring MeLioN. Conclusions: The macrophage infiltration into the arterial wall plays a critical role in the MMP-9 secretion. MeLioN, designed for ION-mediated melittin delivery, effectively prevents IADE formation by suppressing macrophage-mediated inflammations and MMP activity. MeLioN can be a promising strategy preventing IADE development in high-risk populations.

Background: SARS-CoV-2, the cause of the worldwide COVID-19 pandemic, utilizes the mechanism of binding to ACE2 (a crucial component of the renin-angiotensin system [RAS]), subsequently mediating a secondary imbalance of the RAS family and leading to severe injury to the host. However, very few studies have been conducted to reveal the mechanism behind the effect of SARS-CoV-2 on tumors. Methods: Demographic data extracted from 33 cancer types and over 10,000 samples were employed to determine the comprehensive landscape of the RAS. Expression distribution, pretranscriptional and posttranscriptional regulation and posttranslational modifications (PTMs) as well as genomic alterations, DNA methylation and m6A modification were analyzed in both tissue and cell lines. The clinical phenotype, prognostic value and significance of the RAS during immune infiltration were identified. Results: Low expression of AGTR1 was common in tumors compared to normal tissues, while very low expression of AGTR2 and MAS1 was detected in both tissues and cell lines. Differential expression patterns of ACE in ovarian serous cystadenocarcinoma (OV) and kidney renal clear cell carcinoma (KIRC) were correlated with ubiquitin modification involving E3 ligases. Genomic alterations of the RAS family were infrequent across TCGA pan-cancer program, and ACE had the highest alteration frequency compared with other members. Low expression of AGTR1 may result from hypermethylation in the promoter. Downregulation of RAS family was linked to higher clinical stage and worse survival (as measured by disease-specific survival [DSS], overall survival [OS] or progression-free interval [PFI]), especially for ACE2 and AGTR1 in KIRC. ACE-AGTR1, a classical axis of the RAS family related to immune infiltration, was positively correlated with M2-type macrophages, cancer-associated fibroblasts (CAFs) and immune checkpoint genes in most cancers. Conclusion: ACE, ACE2, AGT and AGTR1 were differentially expressed in 33 types of cancers. PTM of RAS family was found to rely on ubiquitination. ACE2 and AGTR1 might serve as independent prognostic factors for LGG and KIRC. SARS-CoV-2 might modify the tumor microenvironment by regulating the RAS family, thus affecting the biological processes of cancer.

Background: Nonalcoholic fatty liver disease (NAFLD) is a serious threat to human health worldwide, with a high genetic susceptibility. Rs2302685, a functional germline variant of LRP6, has been recently found to associate with NAFLD risk. This study was aimed to clarify the underlying mechanism associated with rs2302685 risk and its impact on pharmacotherapy in treatment of NAFLD. Methods: Venous blood samples were collected from NAFLD and non-NAFLD patients for SNP genotyping by using mass spectrometry. The Lrp6-floxdel mouse (Lrp6 (+/-)) was generated to model the partial function associated with human rs2302685. The liver injury and therapeutic effects of silibinin were compared between Lrp6 (+/-) and Lrp6 (+/+) mice received a methionine-choline deficient (MCD) diet or normal diet. The effect of Lrp6 functional alteration on Wnt/β-catenin-Cyp2e1 signaling activities was evaluated by a series of cellular and molecular assays. Results: The T allele of LRP6 rs2302685 was confirmed to associate with a higher risk of NAFLD in human subjects. The carriers of rs2302685 had reduced level of AST and ALT as compared with the noncarriers. The Lrp6 (+/-) mice exhibited a less severe liver injury induced by MCD but a reduced response to the treatment of silibinin in comparison to the Lrp6 (+/+) mice, suggesting Lrp6 as a target of silibinin. Wnt/β-catenin-Cyp2e1 signaling together with ROS generation could be exacerbated by the overexpression of Lrp6, while decreased in response to Lrp6 siRNA or silibinin treatment under NAFLD modeling. Conclusions: The Lrp6 function affects individual susceptibility to NAFLD and the therapeutic effect of silibinin through the Wnt/β-catenin-Cyp2e1 signaling pathway. The present work has provided an underlying mechanism for human individual susceptibility to NAFLD associated with Lrp6 polymorphisms as well as a rationale for the effective use of silibinin in NAFLD patients.

Ferroptosis is a newly recognized mechanism of regulated cell death. It was reported to be highly associated with immune therapy and chemotherapy. However, its mechanism of regulation in the tumor microenvironment (TME) and influence on oral squamous cell carcinoma (OSCC) therapy are unknown. We identified a ferroptosis-specific gene-expression signature, an FPscore, developed by a principal component analysis (PCA) algorithm to evaluate the ferroptosis regulation patterns of individual tumor. Multi-omics analysis of ferroptosis regulation patterns was conducted. Three distinct ferroptosis regulation subtypes, which linked to outcomes and the clinical relevance of each patient, were established. A high FPscore of patients with OSCC was associated with a favorable prognosis, a ferroptosis-related immune-activation phenotype, potential sensitivities to the chemotherapy and immunotherapy. Importantly, a high FPscore correlated with a low gene copy number burden and high immune checkpoint expressions. We validated the prognostic value of the FPscore using independent immunotherapy and pan-cancer cohorts. Comprehensive evaluation of individual tumors with distinct ferroptosis regulation patterns provides new mechanistic insights, which may be clinically relevant for the application of combination therapies in OSCC.

Glioma is the most frequent and aggressive adult brain tumor with maximum mortality. However, the gene alteration and mechanism underlying malignant transformation of glioma remain largely unknown. We aimed to find key factors regulating tumor progression and malignant transformation of glioma. Here we compared the gene expression profiles of 693 glioma patients by HGG vs. LGG model, and identified a key factor CCNB2 for malignant transformation in glioma. CCNB2 induced a senescence-associated secretory phenotype (SASP) of glioma cells, and the malignant progression, such as invasion and excessive proliferation was mediated by secreting SASP cytokines, Cathepsin B and PGE2. These findings demonstrated a previously undiscovered link between senescence, CCNB2/SASP/Cathepsin B & PGE2 axis and malignant transformation in glioma. This might provide novel insights on developing new therapeutic regimens for abrogating aggressiveness of glioma.

Rutin, the main component of Potentilla discolor Bunge, was proven to exhibit anti-tumor properties. Sorafenib (SO) is conventionally used in chemotherapy against hepatocellular carcinoma (HCC), but acquired resistance developed during long-term therapy limits its benefits. This study aimed to explore the molecular mechanism of rutin in SO-induced autophagy and chemoresistance in HCC. Sixty-eight paired HCC patients who received the same chemotherapy treatment were obtained. We also established two SO resistance cell lines and then utilized high-throughput RNA sequencing to explore their long non-coding RNA (lncRNA) expression profiles. The target microRNA (miRNA) and downstream mRNA were also explored. Our results indicated that rutin treatment attenuates autophagy and BANCR expression in SO resistance cells. Transmission electron microscopy clearly showed a significantly decreased number of autophagosomes after rutin-treated HepG2/SO and HCCLM3/SO cells. BANCR knockdown promotes the sensitivity of SO resistance cells to SO. Further study found that BANCR acts as a molecular sponge of miR-590-5P to sequester miR-590-5P away from oxidized low-density lipoprotein receptor 1 (OLR1) in HCC cells. Furthermore, in vivo study demonstrated that rutin could inhibit autophagy through the BANCR/miRNA-590-5P/OLR1 axis. Our findings suggest that rutin could regulate autophagy by regulating BANCR/miRNA-590-5P/OLR1 axis.

Esophageal squamous cell carcinoma (ESCC) is one of the most common gastrointestinal tumors, accounting for almost half a million deaths per year. Cancer-associated fibroblasts (CAFs) are the major constituent of the tumor microenvironment (TME) and dramatically impact ESCC progression. Recent evidence suggests that exosomes derived from CAFs are able to transmit regulating signals and promote ESCC development. In this study, we compared different the component ratios of miRNAs in exosomes secreted by CAFs in tumors and with those from normal fibroblasts (NFs) in precancerous tissues. The mRNA level of hsa-miR-3656 was significantly upregulated in the former exosomes. Subsequently, by comparing tumor cell development in vitro and in vivo, we found that the proliferation, migration and invasion capabilities of ESCC cells were significantly improved when miR-3656 was present. Further target gene analysis confirmed ACAP2 was a target gene regulated by miR-3656 and exhibited a negative regulatory effect on tumor proliferation. Additionally, the downregulation of ACAP2 triggered by exosomal-derived miR-3656 further promotes the activation of the PI3K/AKT and β-catenin signaling pathways and ultimately improves the growth of ESCC cells both in vitro and in xenograft models. These results may represent a potential therapeutic target for ESCC and provide a new basis for clinical treatment plans.

Repeated cycles of first-line chemotherapy drugs such as doxorubicin (DOX) and cisplatin (CIS) trigger frequent chemoresistance in recurrent urothelial bladder cancer (UBC). Nitroxoline (NTX), an antibiotic to treat urinary tract infections, has been recently repurposed for cancer treatment. Here we aimed to investigate whether NTX suppresses drug-resistant UBC and its molecular mechanism. The drug-resistant cell lines T24/DOX and T24/CIS were established by continual exposure of parental cell line T24 to DOX and CIS, respectively. T24/DOX and T24/CIS cells were resistant to DOX and CIS, respectively, but they were sensitive to NTX time- and dose-dependently. Overexpressions of STAT3 and P-glycoprotein (P-gp) were identified in T24/DOX and T24/CIS, which could be reversed by NTX. Western blot revealed that NTX downregulated p-STAT3, c-Myc, Cyclin D1, CDK4, CDK6, Bcl-xL, Mcl-1, and Survivin, which were further confirmed by Stattic, a selective STAT3 inhibitor. In vivo, NTX exhibited the significant anti-tumor effect in T24/DOX and T24/CIS tumor-bearing mice. These results suggested that NTX-induced P-gp reversal, G0/G1 arrest, and apoptosis in drug-resistant UBC were mediated by inhibition of STAT3 signaling. Our findings repurpose NTX as a novel STAT3 inhibitor to induce P-gp reversal, G0/G1 arrest, and apoptosis in drug-resistant UBC.

Hepatitis C virus (HCV) infection involves a variety of viral and host factors, which leads to the dysregulation of number of relevant genes including long noncoding RNAs (LncRNAs). LncRNA urothelial carcinoma-associated 1 (UCA1) has been reported to be upregulated in HCV-infected individuals. In a bid to elucidate on the contribution of UCA1 on HCV replication, we infected Huh7.5 cells with cell culture-derived HCV and found that UCA1 expression was elevated in time- and dose-dependent manners. Functionally, UCA1 knockdown by siRNA upregulated interferon (IFN) responses, thereby increasing the expression of interferon-stimulating genes (ISGs), and subsequently suppressing HCV replication. Bioinformatics analysis and experimental results indicated that, functioning as competitive endogenous RNA, UCA1 could sponge microRNA (miR)-145-5p, which targeted suppressor of cytokine signaling 7 (SOCS7) mRNA and subsequently mediated SOCS7 silencing. Moreover, SOCS7 protein exerted an inhibitory effect on IFN responses, thereby facilitating HCV replication. Taken together, at first, our findings demonstrate that UCA1 can counteract the expression of miR-145-5p, thereby upregulating the level of SOCS7, and in turn leading to the suppression of antiviral response in Huh7.5 cells.

Background: Chronic diabetes accelerates vascular dysfunction often resulting in cardiomyopathy but underlying mechanisms remains unclear. Recent studies have shown that the deregulated unfolded protein response (UPR) dependent on highly conserved IRE1α-spliced X-box- binding protein (XBP1s) and the resulting endoplasmic reticulum stress (ER-Stress plays a crucial role in the occurrence and development of diabetic cardiomyopathy (DCM). In the present study, we determined whether targeting MAPK/ERK pathway using MEK inhibitor U0126 could ameliorate DCM by regulating IRE1α-XBP1s pathway.Method: Three groups of 8-week-old C57/BL6 mice were studied: one group received saline injection as control (n=8) and two groups were made diabetic by streptozotocin (STZ) (n=10 each). 18 weeks after STZ injection and stable hyperglycemia, one group had saline treatment while the second group was treated with U0126 (5mg/kg/day), 8 weeks later, all groups were sacrificed. Cardiac function/histopathological changes were determined by echocardiogram examination, Millar catheter system, hematoxylin-eosin staining and western blot analysis. H9c2 cardiomyocytes were employed for in vitro studies.Results: Echocardiographic, hemodynamic and histological data showed overt myocardial hypertrophy and worsened cardiac function in diabetic mice. Chronic diabetic milieu enhanced SUMOylation and impaired nuclear translocation of XBP1s. Intriguingly, U0126 treatment significantly ameliorated progression of DCM, and this protective effect was achieved through enriching XBP1s’ nuclear accumulation. Mechanistically, U0126 inhibited XBP1s’ phosphorylation on S348 and SUMOylation on K276 promoting XBP1s’ nuclear translocation. Collectively, these results identify that MEK inhibition restores XBP1s-dependent UPR and protects against diabetes-induced cardiac remodeling. Conclusion: The current study identifies previously unknown function of MEK/ERK pathway in regulation of ER-stress in DCM. U0126 could be a therapeutic target for the treatment of DCM.

Mammalian target of rapamycin (mTOR) is one of the most commonly activated pathways in human cancers, including lung cancer. Targeting mTOR with molecule inhibitors is considered as a useful therapeutic strategy. However, the results obtained from the clinical trials with the inhibitors so far have not met the original expectations, largely because of the drug resistance. Thus, combined or multiple drug therapy can bring about more favorable clinical outcomes. Here, we found that activation of ERK pathway was responsible for rapamycin drug resistance in non-small-cell lung cancer (NSCLC) cells. Accordingly, rapamycin-resistant NSCLC cells were more sensitive to ERK inhibitor (ERKi), trametinib, and in turn, trametinib-resistant NSCLC cells were also susceptible to rapamycin. Combining rapamycin with trametinib led to a potent synergistic antitumor efficacy, which induced G1-phase cycle arrest and apoptosis. In addition, rapamycin synergized with another ERKi, MEK162, and in turn, trametinib synergized with other mTORi, Torin1 and OSI-027. Mechanistically, rapamycin in combination with trametinib resulted in a greater decrease of phosphorylation of AKT, ERK, mTOR and 4EBP1. In xenograft mouse model, co-administration of rapamycin and trametinib caused a substantial suppression in tumor growth without obvious drug toxicity. Overall, our study identifies a reasonable combined strategy for treatment of NSCLC.

Background: Esophageal cancer is the sixth-most common fatal malignant tumor worldwide. Little is known regarding the genetic drivers that influence targeted therapy outcomes in patients with esophageal cancer. Exploring the pathogenesis of this lethal tumor could provide clues for developing appropriate therapeutic drugs. Ubiquitin-protein ligase E3A (UBE3A) reportedly promotes or suppresses various types of malignant tumors. However, the cancer-related role of UBE3A in esophageal cancer remains unclear. Methods: The relationship of UBE3A with the clinicopathological features of pancreatic tumors was bioinformatically investigated in the TCGA dataset. The protein levels of UBE3A and ZNF185 were assessed by Western blot and immunohistochemistry. The role of UBE3A and ZNF185 in esophageal cancer growth was assessed by MTS assays, colony formation assays, and experiments in mouse xenograft models. The interaction between UBE3A and ZNF185 was investigated by co-immunoprecipitation. The relationship between UBE3A, ZNF185, and NOTCH signaling pathway was explored by Western blot and quantitative real-time PCR. Results: We found that UBE3A was upregulated in patients with esophageal cancer and enhanced the cellular progression of esophageal cancer. Moreover, we found that UBE3A degraded ZNF185 in esophageal cancer. Additionally, ZNF185 suppressed the progression of esophageal cancer by inactivating the NOTCH pathway. Conclusions: These data demonstrated that aberrant expression of UBE3A led to enhanced progression of esophageal cancer through the ZNF185/NOTCH signaling axis. Therefore, UBE3A might be an ideal therapeutic candidate for esophageal cancer.