Tumor-suppressive miR-4732-3p is sorted into fucosylated exosome by hnRNPK to avoid the inhibition of lung cancer progression.

BACKGROUND: Aberrant fucosylation observed in cancer cells contributes to an augmented release of fucosylated exosomes into the bloodstream, where miRNAs including miR-4732-3p hold promise as potential tumor biomarkers in our pilot study. However, the mechanisms underlying the sorting of miR-4732-3p into fucosylated exosomes during lung cancer progression remain poorly understood. METHODS: A fucose-captured strategy based on lentil lectin-magnetic beads was utilized to isolate fucosylated exosomes and evaluate the efficiency for capturing tumor-derived exosomes using nanoparticle tracking analysis (NTA). Fluorescence in situ hybridization (FISH) and qRT-PCR were performed to determine the levels of miR-4732-3p in non-small cell lung cancer (NSCLC) tissue samples. A co-culture system was established to assess the release of miRNA via exosomes from NSCLC cells. RNA immunoprecipitation (RIP) and miRNA pull-down were applied to validate the interaction between miR-4732-3p and heterogeneous nuclear ribonucleoprotein K (hnRNPK) protein. Cell functional assays, cell derived xenograft, dual-luciferase reporter experiments, and western blot were applied to examine the effects of miR-4732-3p on MFSD12 and its downstream signaling pathways, and the impact of hnRNPK in NSCLC. RESULTS: We enriched exosomes derived from NSCLC cells using the fucose-captured strategy and detected a significant upregulation of miR-4732-3p in fucosylated exosomes present in the serum, while its expression declined in NSCLC tissues. miR-4732-3p functioned as a tumor suppressor in NSCLC by targeting 3'UTR of MFSD12, thereby inhibiting AKT/p21 signaling pathway to induce cell cycle arrest in G2/M phase. NSCLC cells preferentially released miR-4732-3p via exosomes instead of retaining them intracellularly, which was facilitated by the interaction of miR-4732-3p with hnRNPK protein for selective sorting into fucosylated exosomes. Moreover, knockdown of hnRNPK suppressed NSCLC cell proliferation, with the elevated levels of miR-4732-3p in NSCLC tissues but the decreased expression in serum fucosylated exosomes. CONCLUSIONS: NSCLC cells escape suppressive effects of miR-4732-3p through hnRNPK-mediated sorting of them into fucosylated exosomes, thus supporting cell malignant properties and promoting NSCLC progression. Our study provides a promising biomarker for NSCLC and opens a novel avenue for NSCLC therapy by targeting hnRNPK to prevent the "exosome escape" of tumor-suppressive miR-4732-3p from NSCLC cells.

Artesunate-loaded solid lipid nanoparticles resist esophageal squamous cell carcinoma by inducing Ferroptosis through inhibiting the AKT/mTOR signaling.

Esophageal squamous cell carcinoma (ESCC) is a severe malignancy with high incidence and mortality rate in China, while the application of standard chemotherapeutic drugs for ESCC meets the barriers of high toxicity and multiple drug resistance (MDR). In recent years, the anticancer effects of artesunate (ART), a Chinese medicine monomer have gained extensive attentions due to its characteristics of low toxicity, high potency, and reversal of MDR. In this study, we develop the artesunate-loaded solid lipid nanoparticles (SLNART) to overcome the poor water solubility and bioavailability of ART, further improving the efficiency of ART on ESCC treatment. Especially mentioned, SLNART is shown to present marked inhibitory effects on ESCC development based on the induction of ferroptosis by two pathways included upregulating TFR to increase Fe2+ ions and inhibiting the AKT/mTOR signaling to downregulate GPX4. Collectively, this study is the first to pave a promising approach for ESCC therapy based on a strategy of developing SLNART to induce ferroptosis by mediating Fe2+ ions and AKT/mTOR signaling.

NSG1 promotes glycolytic metabolism to enhance Esophageal squamous cell carcinoma EMT process by upregulating TGF-ß.

As a highly enriched endosomal protein within neuronal cells, NSG1 has been discovered to facilitate the process of epithelial-mesenchymal transition (EMT) in esophageal squamous cell carcinoma (ESCC). However, the precise mechanisms behind this phenomenon have yet to be elucidated. The pivotal role of transforming growth factor-ß (TGF-ß) in triggering the EMT and its significant contribution towards tumor metabolic reprogramming-responsible for EMT activation-has been robustly established. Nevertheless, the extent of TGF-ß involvement in the NSG1-mediated EMT within ESCC and the processes through which metabolic reprogramming participates remain ambiguous. We accessed an array of extensive public genome databases to analyze NSG1 expression in ESCC. Regulation of TGF-ß by NSG1 was analyzed by transcriptome sequencing, quantitative Real-Time PCR (qRT-PCR), co-immunoprecipitation (CO-IP), and immunofluorescence (IF). Additionally, cellular functional assays and western blot analyses were conducted to elucidate the effect of NSG1 on TGF-ß/Smad signaling pathway, as well as its role in ESCC cell metastasis and proliferation. We validated the influence of the NSG1/TGF-ß axis on metabolic reprogramming in ESCC by measuring extracellular acidification, glucose uptake, and lactate production. Our findings identify an oncogenic role for NSG1 in ESCC and show a correlation between high NSG1 expression and poor prognosis in ESCC patients. Additional research indicated TGF-ß's involvement in the NSG1-induced EMT process. From a mechanistic perspective, NSG1 upregulates TGF-ß, activating the TGF-ß/Smad signaling pathway and subsequently fostering the EMT process by inducing cell metabolic reprogramming-evident from elevated glycolysis levels. In conclusion, our study highlights the NSG1/TGF-ß axis as a promising therapeutic target for ESCC.

Construction and validation of a novel IGFBP3-related signature to predict prognosis and therapeutic decision making for Hepatocellular Carcinoma.

Background: IGFBP3 plays a pivotal role in carcinogenesis by being anomalously expressed in some malignancies. However, the clinical value of IGFBP3 and the role of IGFBP3-related signature in HCC remain unclear. Methods: Multiple bioinformatics methods were used to determine the expression and diagnostic values of IGFBP3. The expression level of IGFBP3 was validated by RT-qPCR and IHC. A IGFBP3-related risk score (IGRS) was built via correlation analysis and LASSO Cox regression analysis. Further analyses, including functional enrichment, immune status of risk groups were analyzed, and the role of IGRS in guiding clinical treatment was also evaluated. Results: IGFBP3 expression was significantly downregulated in HCC. IGFBP3 expression correlated with multiple clinicopathological characteristics and demonstrated a powerful diagnostic capability for HCC. In addition, a novel IGRS signature was developed in TCGA, which exhibited good performance for prognosis prediction and its role was further validated in GSE14520. In TCGA and GSE14520, Cox analysis also confirmed that the IGRS could serve as an independent prognostic factor for HCC. Moreover, a nomogram with good accuracy for predicting the survival of HCC was further formulated. Additionally, enrichment analysis showed that the high-IGRS group was enriched in cancer-related pathways and immune-related pathways. Additionally, patients with high IGRS exhibited an immunosuppressive phenotype. Therefore, patients with low IGRS scores may benefit from immunotherapy. Conclusions: IGFBP3 can act as a new diagnostic factor for HCC. IGRS signature represents a valuable predictive tool in the prognosis prediction and therapeutic decision making for Hepatocellular Carcinoma.

MiR-942-5p inhibits tumor migration and invasion through targeting CST1 in esophageal squamous cell carcinoma.

INTRODUCTION: Cysteine Protease Inhibitor 1 (CST1), a cystatin superfamily protein with the effect on the inhibition of cysteine protease activity, is reported to be involved in the development of many malignancies. MiR-942-5p has been demonstrated its regulatory effects on some malignancies. However, the roles of CST1 and miR-942-5p on esophageal squamous cell carcinoma (ESCC) are still unknown up to now. METHODS: The expression of CST1 in ESCC tissues was analyzed by TCGA database, immunohistochemistry, and RT-qPCR, respectively. Matrigel-uncoated or-coated transwell assay was used to determine the effect of CST1 on migration and invasion of ESCC cells. Regulatory effect of miR-942-5p on CST1 was detected by dual luciferase assay. RESULTS: CST1 was ectopically highly expressed in ESCC tissues, and had the effect on promoting the migration and invasion of ESCC cells by upregulating phosphorylated levels of key effectors including MEK1/2, ERK1/2, and CREB in MEK/ERK/CREB pathway. Dual-luciferase assay results showed that miR-942-5p had a regulatory effect on targeting CST1. CONCLUSIONS: CST1 plays a carcinogenic role on ESCC, and miR-942-5p can regulate the migration and invasion of ESCC cells by targeting CST1 to downregulate MEK/ERK/CREB signaling pathway, suggesting that miR-942-5p/CST1 axis might be a promising target for diagnosis and treatment of ESCC.

Aberrant NSG1 Expression Promotes Esophageal Squamous Cell Carcinoma Cell EMT by the Activation of ERK Signaling Pathway.

BACKGROUND: Neuron-specific gene family member 1 (NSG1) is a 21 kDa endosomal protein that is specifically expressed in neurons. AIMS: This study was to explore the expression of NSG1 and possible mechanism in Esophageal Squamous Cell Carcinoma (ESCC). METHODS: The Cancer Genome Atlas (TCGA) database was consulted to analyze the expression of NSG1 in ESCC. Immunohistochemistry (IHC) staining was used to evaluate NSG1 expression in ESCC cancerous tissues and matched paracancerous tissues. The CCK-8 assay, wound-healing assay, and transwell assay were used to detect the cell viability, migration, and invasion of ESCC cells. Western blot was used to assay epithelial-mesenchymal transition (EMT)-related proteins and ERK signaling pathway protein expression. RESULTS: The results showed that the expression of NSG1 in ESCC cancerous tissues was higher than noncancerous tissues. Compared with negative control (NC) group, cell viability, migration. and invasion significantly increased, the expression of p-ERK in ERK signaling pathway was significantly upregulated, the expressions of mesenchymal marker Vimentin and EMT-related transcription factors including snail and slug were significantly upregulated, and the expression of epithelial marker E-cadherin was significantly downregulated in KYSE-150 cells with NSG1 overexpression. However, these effects were reversed by the ERK signaling pathway inhibitor U0126. On the other hand, TE-1 cells with NSG1 knockdown had the changes contrary to that in KYSE-150 cells with NSG1 overexpression. CONCLUSION: NSG1 plays a potential carcinogenic role on the occurrence and progression of ESCC, and aberrant NSG1 expression might promote ESCC cell EMT by the activation of ERK signaling pathway.

Fucosylated exosomal miRNAs as promising biomarkers for the diagnosis of early lung adenocarcinoma.

Background: Considering the absence of apparent symptoms at the early stage, most patients with lung adenocarcinoma (LUAD) present at an advanced stage, leading to a dismal 5-year survival rate of <20%. Thus, finding perspective non-invasive biomarkers for early LUAD is very essential. Methods: We developed a fucose-captured strategy based on lentil lectin-magnetic beads to isolate fucosylated exosomes from serum. Then, a prospective study was conducted to define the diagnostic value of serum exosomal miRNAs for early LUAD. A total of 310 participants were enrolled, including 146 LUAD, 98 benign pulmonary nodules (BPNs), and 66 healthy controls (HCs). Firstly, exosome miRNAs in the discovery cohort (n = 24) were profiled by small RNA sequencing. Secondly, 12 differentially expressed miRNAs (DEmiRs) were selected for further screening in a screening cohort (n = 64) by qRT-PCR. Finally, four candidate miRNAs were selected for further validation in a validating cohort (n = 222). Results: This study demonstrated the feasibility of a fucose-captured strategy for the isolation of fucosylated exosomes from serum, evidenced with exosomal characteristics identified by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and Western blotting, as well as rapid and convenient operation of <10 min. Furthermore, a miRNA panel for early LUAD composed of miR4732-5p, miR451a, miR486-5p, and miR139-3p was defined with an AUC of 0.8554 at 91.07% sensitivity and 66.36% specificity. Conclusions: The fucose-captured strategy provides a reliable, as well as rapid and convenient, approach for the isolation of tumor-derived exosomes from serum. A four-fucosylated exosomal miRNA panel presents good performance for early LUAD diagnosis.

Combination of serum CST4 and DR-70 contributes to early diagnosis of colorectal cancer.

BACKGROUND: Early diagnosis is of great significance for the prognosis of colorectal cancer (CRC) patients. Either serum cystatin S (CST4) or DR-70 has been demonstrated to play an important role on the diagnosis for CRC, however, the diagnostic performances of individual and combined detection of serum CST4 and DR-70 for the patients with CRC at early stage have still not been clarified up to now. METHODS: The performances of CST4 and DR-70 were evaluated by ELISA for the early diagnosis of CRC with 288 retrospective serum samples. A training set comprised of 64 patients with early CRC, 64 patients with colorectal benign lesions (CBL), and 64 healthy controls (HC) was used to develop the predictive model for early CRC. And then, data obtained from an independent validation set was applied to evaluate and validate the predictive model. RESULTS: In the training set, the levels of CST4 and DR-70 in early CRC group were significantly higher than that in CBL group/HC group (P < 0.001); serum CST4 presented the AUC of 0.927 for early CRC patients, with 57.8% sensitivity at 95.3% specificity; serum DR-70 presented the AUC of 0.725 for early CRC patients, with 29.7% sensitivity at 95.3% specificity; combination of serum CST4 and DR-70 presented the AUC of 0.941, with 68.8% sensitivity at 93.8% specificity. Additionally, the combination of serum CST4 and DR-70 showed the AUC of 0.940 for early CRC patients, with 71.9 % sensitivity at 89.1% specificity in the validation set. CONCLUSIONS: Both serum CST4 and DR-70 present the diagnostic value for the patients with early CRC, and the combination of CST4 and DR-70 contributes to the further improvement of the early diagnosis for CRC.

Simple, low-cost and sensitive electrochemical sensing of antineoplastic drug amethopterin based on a nanocarbon black modified electrode.

Various methods have been proposed currently to detect amethopterin (ATP, a widely used anticancer drug that is also called methotrexate); however, a simple and low-cost electrochemical method coupled with high sensitivity is scarce. In this study, by using low-cost and readily available nanocarbon black (NCB), which has excellent conductivity and stable dispersion in water as well as large surface area, as electrode materials to modify a glassy carbon electrode (GCE), a simple, inexpensive and highly-sensitive electrochemical sensor was constructed based on NCB/GCE. The electrochemical behaviors of ATP at both NCB/GCE and GCE were studied; the results show that the peak current of ATP at NCB/GCE is extremely higher than that at the bare GCE. For sensing ATP with high sensitivity, various control conditions including accumulation time, pH values of the phosphate buffer solution and NCB amount were optimized. The quantitative testing results show that NCB/GCE presents excellent sensing performances with a wide linearity range from 0.01 to 10.0 µM and low limit of detection (4.0 nM) towards ATP. Moreover, the investigation in the reproducibility and stability as well as selectivity of NCB/GCE demonstrated that the related results are also satisfactory. It is thus simple and effective method for ATP analysis and has important applications.

Development and Evaluation of Serum CST1 Detection for Early Diagnosis of Esophageal Squamous Cell Carcinoma.

PURPOSE: Our pilot study has shown that cystatin SN (CST1) protein is highly expressed in esophageal squamous cell carcinoma (ESCC) tissues. We intend to develop a chemiluminescent enzyme immunoassay (CLEIA) available for serum CST1 detection and define the diagnostic value of CST1 detection for early ESCC patients, and establish a panel of CST1 with traditional tumor markers to improve the diagnostic sensitivity for early ESCC. METHODS: Detection performance of CLEIA for CST1 was evaluated by linearity, detection limit, accuracy, precision, anti-interference and stability. Diagnostic performance of CST1 for early ESCC was evaluated by detecting CST1 of 112 early ESCC, 107 esophageal benign lesions (EBL), and 151 healthy controls (HC). CEA, CYFRA21-1 and SCC-Ag were detected by chemiluminescence immunoassay (CLIA). RESULTS: The linear range and detection limit of CLEIA for CST1 were 6.25-400 pg/mL and 1.35 pg/mL, respectively; the average recovery rate was 102.65%; CVs of intra-batch precision and inter-batch precision were <1/4 TEa and <1/3 TEa, respectively; 8 interferents including 7 common interferents and CST4 had no interference on CST1 detection; stability evaluation showed good sample and reagent stability. The level and positive rate of CST1 in early ESCC group were significantly higher than those in EBL/HC groups (P<0.05). The diagnostic sensitivity of CST1 for early ESCC was 31.25% (specificity 92.64%, AUC 0.654). The diagnostic sensitivity of traditional tumor markers ranged from 16.07% to 28.57%, at >93.0% specificity, and SCC-Ag showed the highest AUC (0.709). Combination of CST1 and CEA, SCC-Ag exhibited the highest AUC up to 0.736 (sensitivity 49.11%, specificity 89.53%). CONCLUSION: CLEIA has excellent detection performance for CST1. CST1 might be a prospective serological biomarker for early diagnosis of ESCC, while combination of CST1 and CEA, SCC-Ag might improve the early diagnostic performance.

Development of a panel of autoantibody against NSG1 with CEA, CYFRA21-1, and SCC-Ag for the diagnosis of esophageal squamous cell carcinoma.

OBJECTIVE: Development of a panel of serum autoantibody against Neuron specific gene family member 1 (NSG1) with traditional tumor biomarkers of esophageal squamous cell carcinoma (ESCC) to further improve the diagnostic efficiency for ESCC patients. METHODS: Immunohistochemistry (IHC) staining was used to detect the expression of NSG1 protein in 40 pairs of ESCC tissues and matched paracancerous tissues. Serum anti-NSG1 levels of 203 patients with early ESCC, 103 patients with advanced ESCC, 135 patients with esophageal benign lesion (EBL), and 155 healthy controls (HCs) were detected by ELISA. The diagnostic performances of all possible combinations of serum anti-NSG1with CEA, CYFRA21-1 and SCC-Ag were assessed to develop an optimal panel for ESCC diagnosis. RESULTS: NSG1 protein expression in ESCC tissues was significantly higher than that in matched paracancerous tissues (p < 0.001). Serum anti-NSG1 expression in ESCC group was significantly higher than that in EBL group and HC group (p < 0.001). The AUC of serum anti-NSG1 for ESCC was 0.706, with 49.7% sensitivity at 93.5% specificity, superior to that of CEA, CYFRA21-1 and SCC-Ag. Of all possible combinations, serum anti-NSG1 combined with CEA, CYFRA21-1 and SCC-Ag showed the highest AUC of 0.758 and 67.3% sensitivity at 88.3% specificity for ESCC, with the highest NPV of 71.9% and the lowest NLR of 0.37. CONCLUSION: Aberrant NSG1 protein expression in ESCC tissues might be responsible for massive releases of autoantibody anginst NSG1 in sera of ESCC. A panel of anti-NSG1 with CEA, CYFRA21-1 and SCC-Ag contributes to further improving the diagnostic efficiency for ESCC.