Detection of fucosylated extracellular vesicles miR-4732-5p related to diagnosis of early lung adenocarcinoma by the electrochemical biosensor.

Our preliminary investigation has identified the potential of serum fucosylated extracellular vesicles (EVs) miR-4732-5p in the early diagnosis of lung adenocarcinoma (LUAD) by a fucose-captured strategy utilizing lentil lectin (LCA)-magnetic beads and subsequent screening of high throughput sequencing and validation of real-time quantitative polymerase chain reaction (RT-qPCR). Considering the relatively complicated procedure, expensive equipment, and stringent laboratory condition, we have constructed an electrochemical biosensor assay for the detection of miR-4732-5p. miR-4732-5p is extremely low in serum, down to the fM level, so it needs to be detected by highly sensitive electrochemical methods based on the Mg2+-dependent DNAzyme splitting nucleic acid lock (NAL) cycle and hybridization chain reaction (HCR) signal amplification. In this study, signal amplification is achieved through the dual amplification reactions using NAL cycle in combination with HCR. In addition, hybridized DNA strands bind to a large number of methylene blue (MB) molecules to enhance signaling. Based on the above strategy, we further enhance our signal amplification strategies to improve detection sensitivity and accuracy. The implementation of this assay proceeded as follows: initially, miR-4732-5p was combined with NAL, and then Mg2+-dependent DNAzyme splitted NAL to release auxiliary DNA (S1) strands, which were subsequently captured by the immobilized capture probe DNA (C1) strands on the electrode surface. Following this, abundant quantities of DNA1 (H1) and DNA2 (H2) tandems were generated by HCR, and S1 strands then hybridized with the H1 and H2 tandems through base complementary pairing. Finally, MB was bonded to the H1 and H2 tandems through π-π stacking interaction, leading to the generation of a signal current upon the detection of a potential capable of inducing a redox change of MB by the electrode. Furthermore, we evaluated the performance of our developed electrochemical biosensor assay. The results demonstrated that our assay is a reliable approach, characterized by its high sensitivity (with a detection limit of 2.6 × 10-17 M), excellent specificity, good accuracy, reproducibility, and stability. Additionally, it is cost-effective, requires simple operation, and is portable, making it suitable for the detection of serum fucosylated extracellular vesicles miR-4732-5p. Ultimately, this development has the potential to enhance the diagnostic efficiency for patients with early-stage LUAD.

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.

Combined detection of serum IL-6 and CEA contributes to the diagnosis of lung adenocarcinoma in situ.

Background: Effective discrimination of lung adenocarcinoma (LUAD) in situ (AIS) from benign pulmonary nodules (BPN) is critical for the early diagnosis of AIS. Our pilot study in a small cohort of 90 serum samples has shown that serum interleukin 6 (IL-6) detection can distinguish AIS from BPN and health controls (HC). In this study, we intend to comprehensively define the diagnostic value of individual and combined detection of serum IL-6 related to the traditional tumor markers carcinoembryonic antigen (CEA) and cytokeratin 19 fragment (CYFRA21-1) for AIS. Methods: The diagnostic performance of serum IL-6 along with CEA and CYFRA21-1 were evaluated in a large cohort of 300 serum samples by a chemiluminescence immunoassay and an electrochemiluminescence immunoassay. A training set comprised of 65 AIS, 65 BPN, and 65 HC samples was used to develop the predictive model for AIS. Data obtained from an independent validation set was applied to evaluate and validate the predictive model. Results: In the training set, the levels of serum IL-6 and CEA in the AIS group were significantly higher than those in the BPN/HC group (P < 0.05). There was no significant difference in serum CYFRA21-1 levels between the AIS group and the BPN/HC group (P> 0.05). Serum IL-6 and CEA levels for AIS patients showed an area under the curve (AUC) of 0.622 with 23.1% sensitivity at 90.7% specificity, and an AUC of 0.672 with 24.6% sensitivity at 97.6% specificity, respectively. The combination of serum IL-6 and CEA presented an AUC of 0.739, with 60.0% sensitivity at 95.4% specificity. The combination of serum IL-6 and CEA showed an AUC of 0.767 for AIS patients, with 57.1% sensitivity at 91.4% specificity in the validation set. Conclusions: IL-6 shows potential as a prospective serum biomarker for the diagnosis of AIS, and the combination of serum IL-6 with CEA may contribute to increased accuracy in AIS diagnosis. However, it is worth noting that further research is still necessary to validate and optimize the diagnostic efficacy of these biomarkers and to address potential sensitivity limitations.

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.

Cysteine protease inhibitor 1 promotes metastasis by mediating an oxidative phosphorylation/MEK/ERK axis in esophageal squamous carcinoma cancer.

Cysteine protease inhibitor 1 (CST1) is a cystatin superfamily protein that inhibits cysteine protease activity and is reported to be involved in the development of many malignancies. Mitochondrial oxidative phosphorylation (OXPHOS) also plays an important role in cancer cell growth regulation. However, the relationship and roles of CST1 and OXPHOS in esophageal squamous cell carcinoma (ESCC) remains unclear. In our pilot study, CST1 was shown the potential of promoting ESCC migration and invasion by the activation of MEK/ERK pathway. Transcriptome sequencing analysis revealed that CST1 is closely associated with OXPHOS. Based on a real-time ATP rate assay, mitochondrial complex I enzyme activity assay, immunofluorescence, co-immunoprecipitation, and addition of the OXPHOS inhibitor Rotenone and MEK/ERK inhibitor PD98059, we determined that CST1 affects mitochondrial complex I enzyme activity by interacting with the GRIM19 protein to elevate OXPHOS levels, and a reciprocal regulatory relationship exists between OXPHOS and the MEK/ERK pathway in ESCC cells. Finally, an in vivo study demonstrated the potential of CST1 in ESCC metastasis through regulation of the OXPHOS and MEK/ERK pathways. This study is the first to reveal the oncogenic role of CST1 in ESCC development by enhancing mitochondrial respiratory chain complex I activity to activate the OXPHOS/MEK/ERK axis, and then promote ESCC metastasis, suggesting that CST1/OXPHOS is a promising target for ESCC treatment.

Combination of serum CST1 and HE4 for early diagnosis of endometrial cancer.

Purpose: Optimal serological biomarkers have been absent for the early diagnosis of endometrial cancer, to date. In this study, we aimed to define the diagnostic performances of individual and combined detection of serum cysteine protease inhibitor 1 (CST1) with traditional tumor markers, including glycated antigen 125 (CA125) and human epididymis protein 4 (HE4), in patients with early-stage endometrial cancer (EC). Methods: The performances of individual and combined detection of serum CST1, HE4, and CA125 were evaluated by enzyme-linked immunosorbent assay (ELISA) and chemiluminescent immunoassay, respectively. A training data set of 67 patients with early EC, 67 patients with endometrial benign lesion (EBL), and 67 healthy controls (HC) was used to develop a predictive model for early EC diagnosis, which was validated by an independent validation data set. Results: In the training data set, serum CST1 and HE4 levels in the early EC group were significantly higher than in EBL/HC groups (P < 0.05), while there was no significant difference of serum CA125 level between the early EC and EBL/HC groups (P > 0.05). Serum CST1 and HE4 exhibited areas under the curve (AUC) of 0.715 with 31.3% sensitivity at 90.3% specificity, and 0.706 with 23.9% sensitivity at 95.5% specificity, respectively. Combined detection of serum CST1 and HE4 exhibited an AUC of 0.788 with 49.3% sensitivity at 92.5% specificity. The combination of serum CST1 and HE4 showed promise in diagnosis. Conclusion: CST1 is a prospective serological biomarker for early EC diagnosis, and the combination of CST1 and HE4 contributes to the further improvement in the diagnosis of patients with early-stage EC.

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.

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.

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.