AGXT2 Suppresses the Proliferation and Dissemination of Hepatocellular Carcinoma Cells by Modulating Intracellular Lipid Metabolism.

Purpose: Alanine glyoxylate aminotransferase (AGXT) family members are crucial in cancer processes, but their role in hepatocellular carcinoma (HCC) metabolism is unclear. This study investigates AGXT2's function in HCC. Patients and Methods: AGTX2 expression was studied using bioinformatics, real-time reverse transcriptase-polymerase chain reaction (RT-qPCR), Western blot, and Enzyme-linked immunosorbent assay (ELISA). A lentivirus-induced AGTX2 overexpression cell model was analyzed with RNA sequencing (RNA-seq) and liquid chromatography-mass spectrometry (LC-MS). Cholesterol levels were confirmed by Oil Red O staining. AGTX2 effects were evaluated through cell cycle analysis, wound healing, and transwell migration assays.Tumorigenic effects were observed in NOD-SCID IL2Rγnull (NTG) mice in subcutaneous experiments. Protein interaction was examined through co-immunoprecipitation methods. Results: We observed a significant reduction in AGXT2 mRNA and protein levels in both HCC tumor tissues and serum samples from patients with liver cancer, which was associated with a worse prognosis. The activation of AGXT2 has been shown to effectively decrease cholesterol levels in liver cancer cells, serving as an antagonist in the cholesterol metabolism pathway. An increase in low density lipoprotein receptor (LDLR) mRNA was noted in cells overexpressing AGXT2, accompanied by a decrease in LDLR protein and an elevation in proprotein convertase subtilisin/kexin type 9 (PCSK9) mRNA and protein levels. Molecular docking and co-immunoprecipitation experiments further elucidated the interaction between AGXT2 and LDLR proteins. AGXT2 was observed to suppress the migratory and invasive capabilities of HCC cells, inducing cell cycle arrest in the G2/M phase. AGXT2 activation inhibited subcutaneous liver cancer tumor growth in NTG mice. Conclusion: AGXT2 was found to lower cholesterol levels in liver cancer cells, possibly through interactions with the LDLR protein and modulation of PCSK9-mediated LDLR degradation. This mechanism may impede cholesterol transport to liver cancer cells, thereby suppressing their growth and metastasis.

KLRG1-expressing CD8+ T cells are exhausted and polyfunctional in patients with chronic hepatitis B.

Killer cell lectin-like receptor G1 (KLRG1) has traditionally been regarded as an inhibitory receptor of T cell exhaustion in chronic infection and inflammation. However, its exact role in hepatitis B virus (HBV) infection remains elusive. CD8+ T cells from 190 patients with chronic hepatitis B were analyzed ex vivo for checkpoint and apoptosis markers, transcription factors, cytokines and subtypes in 190 patients with chronic hepatitis B. KLRG1+ and KLRG1- CD8+ T cells were sorted for transcriptome analysis. The impact of the KLRG1-E-cadherin pathway on the suppression of HBV replication mediated by virus-specific T cells was validated in vitro. As expected, HBV-specific CD8+ T cells expressed higher levels of KLRG1 and showed an exhausted molecular phenotype and function. However, despite being enriched for the inhibitory molecules, thymocyte selection-associated high mobility group box protein (TOX), eomesodermin (EOMES), and Helios, CD8+ T cells expressing KLRG1 produced significant levels of tumour necrosis factor (TNF)-α, interferon (IFN)-γ, perforin, and granzyme B, demonstrating not exhausted but active function. Consistent with the in vitro phenotypic assay results, RNA sequencing (RNA-seq) data showed that signature effector T cell and exhausted T cell genes were enriched in KLRG1+ CD8+ T cells. Furthermore, in vitro testing confirmed that KLRG1-E-cadherin binding inhibits the antiviral efficacy of HBV-specific CD8+ T cells. Based on these findings, we concluded that KLRG1+ CD8+ T cells are not only a terminally exhausted subgroup but also exhibit functional diversity, despite inhibitory signs in HBV infection.

HOXA-AS2 Epigenetically Inhibits HBV Transcription by Recruiting the MTA1-HDAC1/2 Deacetylase Complex to cccDNA Minichromosome.

Persistent transcription of HBV covalently closed circular DNA (cccDNA) is critical for chronic HBV infection. Silencing cccDNA transcription through epigenetic mechanisms offers an effective strategy to control HBV. Long non-coding RNAs (lncRNAs), as important epigenetic regulators, have an unclear role in cccDNA transcription regulation. In this study, lncRNA sequencing (lncRNA seq) is conducted on five pairs of HBV-positive and HBV-negative liver tissue. Through analysis, HOXA-AS2 (HOXA cluster antisense RNA 2) is identified as a significantly upregulated lncRNA in HBV-infected livers. Further experiments demonstrate that HBV DNA polymerase (DNA pol) induces HOXA-AS2 after establishing persistent high-level HBV replication. Functional studies reveal that HOXA-AS2 physically binds to cccDNA and significantly inhibits its transcription. Mechanistically, HOXA-AS2 recruits the MTA1-HDAC1/2 deacetylase complex to cccDNA minichromosome by physically interacting with metastasis associated 1 (MTA1) subunit, resulting in reduced acetylation of histone H3 at lysine 9 (H3K9ac) and lysine 27 (H3K27ac) associated with cccDNA and subsequently suppressing cccDNA transcription. Altogether, the study reveals a mechanism to self-limit HBV replication, wherein the upregulation of lncRNA HOXA-AS2, induced by HBV DNA pol, can epigenetically suppress cccDNA transcription.

A novel combined therapeutic strategy of Nano-EN-IR@Lip mediated photothermal therapy and stem cell inhibition for gastric cancer.

Recurrence and metastasis of gastric cancer is a major therapeutic challenge for treatment. The presence of cancer stem cells (CSCs) is a major obstacle to the success of current cancer therapy, often leading to treatment resistance and tumor recurrence and metastasis. Therefore, it is important to develop effective strategies to eradicate CSCs. In this study, we developed a combined therapeutic strategy of photothermal therapy (PTT) and gastric cancer stem cells (GCSCs) inhibition by successfully synthesizing nanoliposomes loaded with IR780 (photosensitizer) and EN4 (c-Myc inhibitor). The nanocomposites are biocompatible and exhibit superior photoacoustic (PA) imaging properties. Under laser irradiation, IR780-mediated PTT effectively and rapidly killed tumor cells, while EN4 synergistically inhibited the self-renewal and stemness of GCSCs by suppressing the expression and activity of the pluripotent transcription factor c-Myc, preventing the tumor progression of gastric cancer. This Nano-EN-IR@Lip is expected to be a novel clinical nanomedicine for the integration of gastric cancer diagnosis, treatment and prevention.

Immunomultiple PCR-based electrochemical and lateral flow strategy for the simultaneous detection of liver cancer tumor markers.

The current use of the single serum biomarker α-fetoprotein (AFP) in clinical practice has limitations in terms of specificity and sensitivity. We propose a strategy that combines antigen capture polymerase chain reaction (AC-PCR), lateral flow assay (LFA), and electrochemical biosensors to detect both AFP and circulating tumor cells (CTCs) in liver cancer serum. First, we used the AC-PCR technique to achieve target separation, purification, signal conversion, and amplification, eliminating target heterogeneity. Then, we achieved rapid results through the LFA and electrochemical biosensor platforms. As a result, the proposed assay has limits of 5 cells/mL for CTCs and 5 µg/L for AFP. The proposed method was applied effectively to simulated blood samples. This method has the potential to play a role in early liver cancer and provide a potential application for the diagnosis and precision treatment of liver cancer.

Identification of cuproptosis-related gene SLC31A1 and upstream LncRNA-miRNA regulatory axis in breast cancer.

Mounting evidence indicate that cuproptosis, a novel form of programmed cell death, contributes to cancer development and progression. However, a comprehensive analysis regarding the expressions, functions, and regulatory network of cuproptosis-related genes is still lacking. In the present work, cuproptosis-related genes, upstream miRNAs and lncRNAs, and clinical data of breast cancer from TCGA database were analyzed by R language including Cox regression analysis, correlation calculation, ROC curve construction, and survival evaluation, and were further verified by public-available databases. Chemosensitivity and immune infiltration were also evaluated by online tools. SLC31A1 was significantly increased in breast cancer samples than those in normal tissues. SLC31A1 was negatively related to a favorable outcome in breast cancer, and the AUC value increased with the prolongation of follow-up time. LINC01614 and miR-204-5p were potential upstream regulators of SLC31A1. Moreover, SLC31A1 was significantly positively correlated with different immune cells infiltration, immune cell biomarkers, and immune checkpoints in breast cancer. SLC31A1 was a potential cuproptosis-related gene in breast cancer, which was significantly upregulated and was able to predict diagnosis, prognosis, chemosensitivity, and immune infiltration. LINC01640/miR-204-5p/SLC31A1 might be a significant and promising axis during cuproptosis in breast cancer.

Circulating sNinj1 as a novel predictor of prognosis and severity in hepatocellular carcinoma.

BACKGROUND: The occurrence and development of HCC are closely associated with cell death. Recently, researchers found that Ninj1 plays a pivotal role in PMR during different types of cell death. However, the importance of Ninj1 in HCC has not been extensively investigated. METHODS: This study included 102 newly diagnosed HCC patients and 102 sex and age-matched NCs. Circulating sNinj1 was assessed by ELISA. Serum LDH and IL-1ß were detected through a chemiluminescence assay. The correlations of these biomarkers with disease severity and their potential as prognostic predictors for HCC were evaluated. The dynamic changes of sNinj1, LDH, and IL-1ß levels before and after treatment were recorded. RESULTS: Serum levels of sNinj1, IL-1ß, and LDH were significantly higher in HCC patients. Our study found that the sNinj1 level was positively correlated with tumor size, metastasis, and staging. ROC analysis indicated that the AUC of sNinj1 in differentiating HCC from NCs was 0.85. As a result of tumor thrombosis and invasion of the hepatic vein, sNinj1's AUCs were 0.71 and 0.73, respectively. After partial resection and TACE treatment, serum sNinj1 and LDH exhibited similar change trends. A one-year follow-up analysis also demonstrated that HCC patients with high sNinj1 had significantly poorer survival than those with low sNinj1. CONCLUSIONS: The serum sNinj1 is another diagnostic biomarker supporting the HCC diagnosis. More importantly, it has been shown that circulating sNinj1 reveals potential as a novel predictor of HCC severity and prognosis.

Identification of a novel five ferroptosis-related gene signature as a promising prognostic model for breast cancer.

BACKGROUND: Breast cancer (BCa) is a major challenge for women's health worldwide. Ferroptosis is closely related to tumorigenesis and cancer progression. However, the prognostic value of ferroptosis-related genes in BCa remains unclear, and more accurate prognostic models are urgently needed. METHODS: Gene expression profiles and clinical information of BCa patients were collected from public databases. LASSO and multivariate Cox regression analysis were utilized to construct the prognostic gene signature. Kaplan-Meier plotter, receiver operating characteristic (ROC) curves, and nomogram were used to validate the prognostic value of the gene signature. Gene set enrichment analysis was performed to explore the molecular functions and signaling pathways. RESULTS: Differentially expressed ferroptosis-related genes between BCa samples and normal tissues were obtained. A novel five-gene signature including BCL2, SLC40A1, TFF1, APOOL, and PRAME was established for prognosis prediction. Patients stratified into high-risk or low-risk group displayed significantly different survival. Kaplan-Meier and ROC curves showed a good performance for survival prediction in different cohorts. Biological function analysis revealed that the five-gene signature was associated with cancer progression, immune infiltration, immune response, and drug resistance. Nomogram including the five-gene signature was established. CONCLUSION: A novel five ferroptosis-related gene signature and nomogram could be used for prognostic prediction in BCa.

Liver fibrosis-derived exosomal miR-106a-5p facilitates the malignancy by targeting SAMD12 and CADM2 in hepatocellular carcinoma.

The mechanism of hepatocellular carcinoma (HCC) development induced by liver fibrosis is obscure. The objective of this study is to establish miRNAs from exosomes associated with liver fibrosis, and to identify potential biomarkers for the prediction of personalized clinical management effectiveness in HCC. Our research focused on miRNAs from exosomes and mRNA from liver fibrosis, which we found in the gene expression omnibus (GEO) database. Weighted gene co-expression network analysis (WGCNA) evaluated miRNAs from exosomes associated with liver fibrosis, and Wilcoxon analysis assessed differentially expressed mRNAs (DEGs) across liver fibrosis/normal tissues. Following that, DEGs were assessed through gene set enrichment analysis (GSEA), gene ontology (GO), and the Kyoto Encyclopedia of Genes and Genomes (KEGG). In addition, based on the screened targeted genes, including SAMD12 and CADM2, we further elucidated their correlation in HCC patients from the BEST database. The Kaplan-Meier Plotter platform was applied to evaluate the prognostic values of miRNA in HCC. In vitro and vivo experiments validated our findings. Six miRNAs associated with liver fibrosis were evaluated in our investigation. In-depth research presented exosome-derived miR-106a-5p, SAMD12 and CADM2 could exert valuable predictive implications for HCC treatment and illness assessment. Serum miR-106a-5p derived from liver fibrosis was decreased compared with healthy individuals. SAMD12 and CADM2 were diminished in liver cancer cell lines, and their knockdown of them exacerbated the proliferation capacities of liver cells in vitro. Exosome-derived miRNA of liver fibrosis modulated tumorigenesis by targeting SAMD12 and CADM2 in HCC.

Elevated neutrophil extracellular traps by HBV-mediated S100A9-TLR4/RAGE-ROS cascade facilitate the growth and metastasis of hepatocellular carcinoma.

BACKGROUND: Neutrophil extracellular traps (NETs) are considered significant contributors to cancer progression, especially metastasis. However, it is still unclear whether NETs are involved in hepatitis B virus (HBV)-related hepatocarcinogenesis and have potential clinical significance during evaluation and management for hepatocellular carcinoma (HCC). In this study, we aimed to investigate the functional mechanism of NETs in HBV-related hepatocarcinogenesis and their clinical significance. METHODS: A total of 175 HCC patients with and without HBV infection and 58 healthy controls were enrolled in this study. NETs were measured in tissue specimens, freshly isolated neutrophils and blood serum from these patients, and the correlation of circulating serum NETs levels with malignancy was evaluated. The mechanism by which HBV modulates NETs formation was explored using cell-based studies. In addition, in vitro and in vivo experiments were further performed to clarify the functional mechanism of NETs on the growth and metastasis of HCC. RESULTS: We observed an elevated level of NETs in blood serum and tissue specimens from HCC patients, especially those infected with HBV. NETs facilitated the growth and metastasis of HCC both in vitro and in vivo, which were mainly dominated by increased angiogenesis, epithelial-mesenchymal transition (EMT)-related cell migration, matrix metalloproteinases (MMPs)-induced extracellular matrix (ECM) degradation and NETs-mediated cell trapping. Inhibition of NETs generation by DNase 1 effectively abrogated the NETs-aroused HCC growth and metastasis. In addition, HBV-induced S100A9 accelerated the generation of NETs, which was mediated by activation of toll-like receptor (TLR4)/receptor for advanced glycation end products (RAGE)-reactive oxygen species (ROS) signaling. Further, circulatory NETs were found to correlate with viral load, TNM stage and metastasis status in HBV-related HCC, and the identified NETs could predict extrahepatic metastasis, with an area under the ROC curve (AUC) of 0.83 and 90.3% sensitivity and 62.8% specificity at a cutoff value of 0.32. CONCLUSIONS: Our findings indicated that activation of RAGE/TLR4-ROS signaling by HBV-induced S100A9 resulted in abundant NETs formation, which subsequently facilitated the growth and metastasis of HCC cells. More importantly, the identified circulatory NETs exhibited potential as an alternative biomarker for predicting extrahepatic metastasis in HBV-related HCC.

Association between IL-38 and inflammatory indicators in patients with bacterial pneumonia.

BACKGROUND: IL-38, a recently discovered cytokine of IL-1 family, exerts immunoregulatory activities in multi-type inflammatory diseases. However, its expression level and underlying clinical importance for IL-38 in respiratory bacterial infections remain unknown. METHODS: Thirty-five patients with bacterial pneumonia and twenty age- and gender- matched healthy individuals were enrolled in the study to determine serum IL-38 concentrations by ELISA. Then, the correlation between serum IL-38 levels and clinical features were analyzed and ROC curve was used to evaluate the potential diagnostic value for bacterial infections. In vitro, LPS-stimulated human respiratory epithelial cell model was employed to explore immunomodulatory mechanism of IL-38 in pulmonary infections. RESULTS: Elevated serum levels of IL-38 were determined in patients with bacterial pneumonia when compared with healthy controls. In addition, serum IL-38 levels were negatively correlated with clinical inflammation parameters, including WBC count, CRP, PCT and proinflammatory IL-6 and IL-8. In vitro, we demonstrated that recombinant IL-38 was able to remarkably inhibit expression of proinflammatory IL-6, IL-8, IL-1ß and TNF-α as well as adhesion molecule ICAM-1, which were partially mediated by attenuated activation of STAT3 and NF-κB signal cascades in BEAS-2B cells. Furthermore, we identified the diagnostic efficiency of IL-38 in discriminating patients with bacterial pneumonia from healthy individuals. CONCLUSIONS: Our study indicates higher serum IL-38 levels in patients with bacterial pneumonia are involved in anti-inflammatory activities in respiratory infections revealing a critical role of IL-38 in attenuating excessive pulmonary inflammation against exogenous pathogens. More importantly, IL-38 exhibited a potential novel biomarker for bacterial pneumonia. Thus, our data may provide useful insights for both clinical and basic research for bacterial pneumonia diagnosis.

CCL25/CCR9 interaction promotes the malignant behavior of salivary adenoid cystic carcinoma via the PI3K/AKT signaling pathway.

Background: CC chemokine receptor 9 (CCR9), an organ-specific chemokine receptor, interacts with its exclusive ligand CCL25 to promote tumor proliferation and metastasis. However, the effect of CCR9 on salivary adenoid cystic carcinoma (SACC) malignant behavior remains unknown. This study aimed to investigate the specific molecular mechanism by which CCR9/CCL25 modulates malignant progression in SACC. Methods: Immunohistochemistry staining and RT-qPCR analyses were performed to detect the correlation of CCR9 expression and tumor progression-associated markers in SACC. In vitro, SACC cell proliferation and apoptosis were evaluated using Cell Counting Kit-8 and colon formation, and cell migration and invasion were detected by wound healing and transwell assays. Vercirnon was used as an inhibitor of CCR9, and LY294002 was used as an inhibitor of the PI3K/AKT pathway in this study. Western blot and RT-qPCR assays were carried out to measure the downstream factors of the interaction of CCL25 and CCR9. The effect of CCL25 on the development of SACC in vivo was examined by a xenograft tumor model in nude mice following CCL25, Vercirnon and LY294002 treatment. Results: CCR9 was highly expressed in SACC compared with adjacent salivary gland tissues, and its level was associated with tumor proliferation and metastases. CCL25 enhanced cell proliferation, migration, and invasion through its interaction with CCR9 and exerted an antiapoptotic effect on SACC cells. Targeting CCR9 via Vercirnon significantly reduced the phosphorylation level of AKT induced by CCL25. CCL25/CCR9 could activate its downstream factors through the PI3K/AKT signaling pathway, such as cyclin D1, BCL2 and SLUG, thus promoting SACC cell proliferation, antiapoptosis, invasion and metastasis. The in vivo data from the xenograft mouse models further proved that CCL25 administration promoted malignant tumor progression by activating the PI3K/AKT pathway. Conclusion: The interaction of CCL25 and CCR9 promotes tumor growth and metastasis in SACC by activating the PI3K/AKT signaling pathway, offering a promising strategy for SACC treatment.

Reduced Circulating Soluble Receptor for Advanced Glycation End-products in Chronic Hepatitis B Are Associated with Hepatic Necroinflammation.

The diagnosis and disease management of chronic hepatitis B (CHB) remain challenging due to the elusive assessment of disease severity. Recently, soluble receptor for advanced glycation end-products (sRAGE) has been implicated in the inflammatory-immune response initiated by liver injury. Nonetheless, its natural behavior and clinical importance in CHB remain elusive. One hundred and twenty CHB patients and forty healthy controls (HCs) were enrolled, and the serum sRAGE as well as RAGE expression in biopsy specimens from these subjects was analyzed, and correlation of sRAGE with clinical features as well as its potential predictive value for monitoring the CHB was also evaluated. Reduced serum sRAGE levels and decreased tissular RAGE expression were observed in CHB patients. sRAGE and RAGE were inversely correlated with gradually increased grades of hepatic necroinflammation as well as the routine indicator ALT. Furthermore, receiver operating characteristic (ROC) analysis showed that combination of ALT and sRAGE exerted better predictive power (area under the ROC curve (AUC) of 0.86) for hepatic necroinflammation than that of ALT (AUC of 0.82), sRAGE (AUC of 0.81), or sRAGE-to-ALT ratio (sRAGE/ALT) (AUC of 0.85) alone. More importantly, circulating sRAGE alone exerted valuable predictive power for hepatic moderate-to-severe necroinflammation in CHB patients but with normal ALT (AUC of 0.81) or minimally elevated ALT (AUC of 0.85). In conclusion, reduced serum sRAGE levels may imply an increased severity for necroinflammation, and it may serve as a potential alternative biomarker for monitoring hepatic necroinflammation in CHB.

DDX17-regulated alternative splicing that produced an oncogenic isoform of PXN-AS1 to promote HCC metastasis.

BACKGROUND AND AIMS: The mechanism underlying HCC metastasis remains unclear, many oncogenes are known to regulate this process. However, the role of alternative splicing (AS) in pro-metastatic HCC is poorly understood. APPROACH AND RESULTS: By performing RNA sequencing on nine pairs of primary HCC tissues with extrahepatic metastasis (EHMH) and nine pairs of metastasis-free HCC (MFH) tissues, we depicted the AS landscape in HCC and found a higher frequency of AS events in EHMH compared with MFH. Moreover, 28 differentially expressed splicing regulators were identified in EHMH compared with MFH. Among these, DEAD-box RNA helicase 17 (DDX17) was significantly up-regulated in EHMH and was strongly associated with patient outcome. Functional studies indicated that DDX17 knockout inhibited the degradation of the extracellular matrix, and diminished the invasive ability of HCC cells. A significant reduction in lung metastasis induced by DDX17 deficiency was also demonstrated in a diethylnitrosamine-induced DDX17HKO mouse model. Mechanistically, high DDX17 induced intron 3 retention of PXN-AS1 and produced a transcript (termed PXN-AS1-IR3). The transcript PXN-AS1-IR3 acted as an important promoter of HCC metastasis by inducing MYC transcription activation via recruiting the complex of testis expressed 10 and p300 to the MYC enhancer region, which led to transcriptional activation of several metastasis-associated downstream genes. Finally, the PXN-AS1-IR3 level was significantly higher in serum and HCC tissues with extrahepatic metastasis. CONCLUSIONS: DDX17 and PXN-AS1-IR3 act as important metastatic promoters by modulating MYC signaling, suggesting that DDX17 and PXN-AS1-IR3 may be potential prognostic markers for metastatic HCC.

Classification of the mitochondrial ribosomal protein-associated molecular subtypes and identified a serological diagnostic biomarker in hepatocellular carcinoma.

Purpose: The objective of this study was to sort out innovative molecular subtypes associated with mitochondrial ribosomal proteins (MRPs) to predict clinical therapy response and determine the presence of circulating markers in hepatocellular carcinoma (HCC) patients. Methods: Using an unsupervised clustering method, we categorized the relative molecular subtypes of MRPs in HCC patients. The prognosis, biological properties, immune checkpoint inhibitor and chemotherapy response of the patients were clarified. A signature and nomogram were developed to evaluate the prognosis. Enzyme-linked immunosorbent assay (ELISA) measured serum mitochondrial ribosomal protein L9 (MRPL9) levels in liver disease patients and normal individuals. Receiver operating characteristic (ROC) curves were conducted to calculate the diagnostic effect. The Cell Counting Kit 8 was carried out to examine cell proliferation, and flow cytometry was used to investigate the cell cycle. Transwell assay was applied to investigate the potential of cell migration and invasion. Western blot detected corresponding changes of biological markers. Results: Participants were classified into two subtypes according to MRPs expression levels, which were characterized by different prognoses, biological features, and marked differences in response to chemotherapy and immune checkpoint inhibitors. Serum MRPL9 was significantly higher in HCC patients than in normal individuals and the benign liver disease group. ROC curve analysis showed that MRPL9 was superior to AFP and Ferritin in differentiating HCC from healthy and benign patients, or alone. Overexpressed MRPL9 could enhance aggressiveness and facilitate the G1/S progression in HCC cells. Conclusion: We constructed novel molecular subtypes based on MRPs expression in HCC patients, which provided valuable strategies for the prediction of prognosis and clinical personalized treatment. MRPL9 might act as a reliable circulating diagnostic biomarker and therapeutic target for HCC patients.

Pleural Fluid GSDMD Is a Novel Biomarker for the Early Differential Diagnosis of Pleural Effusion.

OBJECTIVE: Gasdermin D (GSDMD), controlling pyroptosis in cells, has multiple physiological functions. The diagnostic role of GSDMD in pleural effusion (PE) remains unknown. METHODS: Sandwich ELISA kits that we developed were applied to measure the level of GSDMD for 335 patients with a definite cause of PE, including transudative PE, tuberculous pleural effusion (TPE), parapneumonic pleural effusion (PPE), and malignant pleural effusion (MPE). The diagnostic accuracy of Light's criteria vs. the new marker GSDMD was performed. Clinical follow-up of 40 cases of PPE was conducted and divided into efficacy and non-efficacy groups according to the therapeutic outcome. Nucleated cells (NCs) in PE were isolated and further infected with bacteria to verify the cell source of GSDMD. RESULTS: The diagnostic accuracy of GSDMD for the diagnosis of PE were 96% (sensitivity) and 94% (specificity). The receiver operating characteristic (ROC) curve indicated that GSDMD can be an efficient biomarker for the differential diagnosis of transudative PE and other groups (all AUC > 0.973). Noteworthily, the highest AUC belonged to tuberculosis diagnosis of 0.990, and the cut-off value was 18.40 ng/mL. Moreover, the same cut-off value of PPE and MPE was 9.35 ng/mL. The combination of GSDMD, adenosine deaminase (ADA), and lactate dehydrogenase (LDH) will further improve the diagnostic efficiency especially between TPE and PPE (AUC = 0.968). The AUC of GSDMD change at day 4, which could predict the therapeutic effect at an early stage, was 0.945 (P < 0.0001). Interestingly, bacterial infection experiments further confirm that the pleural fluid GSDMD was expressed and secreted mainly by the NCs. CONCLUSION: GSDMD and its combination are candidates as a potentially novel biomarker not only to separate PEs early and effectively, but also monitor disease progression.

Combining Calcitonin and Procalcitonin and Rheumatoid Arthritis-Related Biomarkers Improve Diagnostic Outcomes in Early Rheumatoid Arthritis.

OBJECTIVE: To demonstrate whether procalcitonin (PCT) combined with calcitonin (CT) could provide additional diagnostic value to other clinically available rheumatoid arthritis- (RA-) related biomarkers in the early diagnosis of RA. METHOD: The blood samples aseptically collected by venipuncture were centrifuged within 1 hour and frozen at -80°C. PCT and CT levels were measured using electrochemiluminescence immunoassay (ECLIA) in 260 subjects (48 patients with early RA, 34 patients with established RA, 37 patients with systemic lupus erythematosus, 30 with osteoarthritis, 31 with gouty arthritis, and 80 healthy participants). Anti-cyclic citrullinated peptide (Anti-CCP) and anti-RA33 antibodies (Anti-RA33) were analyzed by ELISA. RF was detected by transmission immunoturbidimetry. Mann-Whitney U tests and Kruskal-Wallis tests compared differences among groups. Spearman's rank correlation analysis determined the relationship between biomarkers. Receiver-operator characteristic (ROC) curves were generated, and diagnostic performance was assessed by area under the curve (AUC), as well as specificity, sensitivity, likelihood ratios (LR). RESULTS: Median serum PCT concentrations were significantly higher (p < 0.0001) in patients with early RA (0.065 ng/ml) when compared with healthy controls (0.024 ng/ml), and patients with osteoarthritis (0.025 ng/ml). When compared with gouty arthritis (GA) controls (0.072 ng/ml) and systemic lupus erythematosus (SLE) controls (0.093 ng/ml), median serum PCT concentrations were not significant in patients with early RA (0.065 ng/ml). Median serum CT concentrations were significantly lower (p < 0.0001) in patients with early RA (0.880 pg/ml) compared with healthy controls (3.159 pg/ml), patients with SLE (2.480 pg/ml), and patients with GA (2.550 pg/ml). When compared with osteoarthritis controls (0.586 pg/ml), median serum CT concentrations were not significant in patients with early RA (0.880 pg/ml). ROC curve analysis comparing early RA with healthy controls demonstrated that the AUC of RF, anti-CCP, and anti-RA33 were 0.66, 0.73, and 0.64, respectively; the additions of PCT and CT further improved the diagnostic ability of early RA with the AUC of 0.97, 0.98, and 0.97, respectively (p < 0.01). The sensitivities of RF, anti-CCP, and anti-RA33 for early RA were 33.33%, 44.74%, and 58.33%, respectively, and the additions of PCT and CT showed very high sensitivities of 83.33%, 92.11%, and 87.50%. The high-value groups of PCT moderately correlated with the anti-RA33 levels (r = 0.417, p < 0.05). CT had no significant correlation with disease duration, radiographic progression, or clinical/serological variables, such as ESR levels, CRP levels, RF, anti-CCP, and anti-RA33 levels in early RA. CONCLUSIONS: Serum PCT and CT combined with clinically available RA-related biomarkers could further improve the diagnostic efficiency of early RA.

Exosomal miR-222 from adriamycin-resistant MCF-7 breast cancer cells promote macrophages M2 polarization via PTEN/Akt to induce tumor progression.

Exosome-mediated intercellular communication is considered to be an effective mode for malignant cells to transform biological behaviors in stromal cells. However, the mechanisms by which exosomes modulate macrophages within tumor microenvironment remain largely unclear. In this study, we found that both adriamycin-resistant breast cancer (BCa) cells and the corresponding exosomes (A/exo) were capable of inducing macrophages M2 polarization, which promoted the mobility, proliferation, migration and invasion of BCa cells. Since exosomes deliver microRNAs to affect cellular functions in recipient cells, we confirmed that miR-222 was significantly enriched in A/exo and could be successfully transferred to macrophages. Increased miR-222 level was also detected in exosomes derived from plasma and tissues of chemoresistant patients. Moreover, exosomal miR-222 from A/exo polarized M2 macrophages by targeting PTEN and activating Akt signaling pathway, which promoted BCa cells progression in a feed back loop. Co-culture of adriamycin-resistant BCa cells with macrophages in which miR-222 was upregulated or treated with A/exo facilitated tumor growth in vivo. Collectively, our data demonstrated that chemoresistant BCa cells could remodel macrophages within tumor microenvironment by secreting exosomal miR-222, which directly targeted PTEN and caused Akt cascade activation and macrophages M2 polarization. Our findings may provide a foundation for a promising strategy of BCa treatment by targeting exosomes or exosomal miR-222.

Breast metastasis from rectal carcinoma: A case report and review of the literature.

BACKGROUND: Metastasis from extramammary primary tumor to breast is extremely rare. CASE SUMMARY: A 59-year-old woman with 1-year history of rectal cancer presented with asymptomatic breast mass. At 16 months after the diagnosis of rectal mucinous adenocarcinoma, a breast mass was confirmed by ultrasonography and identified by pathology and immunohistochemistry as a metastasis from the rectal cancer. Treatments included chemotherapy (6 cycles: 300 mg irinotecan on day 1, 4.5 mg raltitrexed on day 2, 450 mg bevacizumab on day 3), radiotherapy, and surgical resection. Two years of follow-up examinations (6-months intervals) showed no evidence of recurrence or novel distant metastasis. CONCLUSION: Breast metastasis from rectal carcinoma is a rare secondary malignancy. Final diagnosis can be established by histopathology and immunohistochemistry.

Dicoumarol, an NQO1 inhibitor, blocks cccDNA transcription by promoting degradation of HBx.

BACKGROUND & AIMS: Current antiviral therapies help keep HBV under control, but they are not curative, as they are unable to eliminate the intracellular viral replication intermediate termed covalently closed circular DNA (cccDNA). Therefore, there remains an urgent need to develop strategies to cure CHB. Functional silencing of cccDNA is a crucial curative strategy that may be achieved by targeting the viral protein HBx. METHODS: We screened 2,000 small-molecule compounds for their ability to inhibit HiBiT-tagged HBx (HiBiT-HBx) expression by using a HiBiT lytic detection system. The antiviral activity of a candidate compound and underlying mechanism of its effect on cccDNA transcription were evaluated in HBV-infected cells and a humanised liver mouse model. RESULTS: Dicoumarol, an inhibitor of NAD(P)H:quinone oxidoreductase 1 (NQO1), significantly reduced HBx expression. Moreover, dicoumarol showed potent antiviral activity against HBV RNAs, HBV DNA, HBsAg and HBc protein in HBV-infected cells and a humanised liver mouse model. Mechanistic studies demonstrated that endogenous NQO1 binds to and protects HBx protein from 20S proteasome-mediated degradation. NQO1 knockdown or dicoumarol treatment significantly reduced the recruitment of HBx to cccDNA and inhibited the transcriptional activity of cccDNA, which was associated with the establishment of a repressive chromatin state. The absence of HBx markedly blocked the antiviral effect induced by NQO1 knockdown or dicoumarol treatment in HBV-infected cells. CONCLUSIONS: Herein, we report on a novel small molecule that targets HBx to combat chronic HBV infection; we also reveal that NQO1 has a role in HBV replication through the regulation of HBx protein stability. LAY SUMMARY: Current antiviral therapies for hepatitis B are not curative because of their inability to eliminate covalently closed circular DNA (cccDNA), which persists in the nuclei of infected cells. HBV X (HBx) protein has an important role in regulating cccDNA transcription. Thus, targeting HBx to silence cccDNA transcription could be an important curative strategy. We identified that the small molecule dicoumarol could block cccDNA transcription by promoting HBx degradation; this is a promising therapeutic strategy for the treatment of chronic hepatitis B.