Multisignals Sensing Platform for Highly Sensitive, Accurate, and Rapid Detection of p-Aminophenol Based on Adsorption and Oxidation Effects Induced by Defective NH2-Ag-nMOFs.

Labile toxic pollutants detection remains a challenge due to the problem that a single method is prone to producing false-negative/-positive signals. The construction of a multisignal sensing platform with the advantages of different strategies is an effective way to solve this problem. Herein, a novel resonant light scattering (RLS), fluorescent and rapid visual multisignals sensing strategy for p-aminophenol (p-AP) detection was designed based on the adsorption and oxidation effects of defective amino-functionalized Ag-based nano metal-organic frameworks (NH2-Ag-nMOFs). In this reaction process, NH2-Ag-nMOFs with incomplete coordination oxidize H2O2 to produce singlet oxygen (1O2) which rapidly oxidizes p-AP, leading to the reduction of Ag+ to Ag0, thereby disrupting the structure of NH2-Ag-nMOFs and resulting in fluorescence quenching of NH2-Ag-nMOFs. Synchronously, owing to Ag0 aggregation and p-AP oxidation, the color of the system changed from colorless to purplish-red and pale brown within 20 s. The assay has realized the rapid naked-eye detection of 5 µM p-AP rapidly. Additionally, thanks to the intermolecular hydrogen bonding, NH2-Ag-nMOFs-p-AP aggregates formed, which enhanced the RLS signal. With the RLS signal, the designed multisignals sensing platform can analyze p-AP at a concentration as low as 11 nM and yield a wider dynamic response range than any single signal strategy reported before, which can quickly meet the measurement requirement of different actual samples. Overall, the proposed strategy without assembling various signal indicators presented an accurate, rapid, cost-effective, and sensitive multisignals sensing platform for p-AP analysis and has great prospects in labile toxic pollutants monitoring.

A clinically feasible circulating tumor cell sorting system for monitoring the progression of advanced hepatocellular carcinoma.

BACKGROUND: Hematogenous metastasis is essential for the progression of advanced hepatocellular carcinoma (HCC) and can occur even after patients receive multidisciplinary therapies, including immunotherapy and hepatectomy; circulating tumor cells (CTCs) are one of the dominant components of the metastatic cascade. However, the CTC capture efficiency for HCC is low due to the low sensitivity of the detection method. In this study, epithelial cell adhesion molecule (EpCAM)/vimentin/Glypican-3 (GPC3) antibody-modified lipid magnetic spheres (LMS) were used to capture tumor cells with epithelial phenotype, mesenchymal phenotype and GPC3 phenotype, respectively, in order to capture more CTCs with a more comprehensive phenotype for monitoring tumor metastasis. RESULTS: The novel CTC detection system of Ep-LMS/Vi-LMS/GPC3-LMS was characterized by low toxicity, strong specificity (96.94%), high sensitivity (98.12%) and high capture efficiency (98.64%) in vitro. A sudden increase in CTC counts accompanied by the occurrence of lung metastasis was found in vivo, which was further validated by a clinical study. During follow-up, the rapid increase in CTCs predicted tumor progression in HCC patients. Additionally, genetic testing results showed common genetic alterations in primary tumors, CTCs and metastatic tissues. The proportion of patients predicted to benefit from immunotherapy with the CTC detection method was higher than that for the tissue detection method (76.47% vs. 41.18%, P = 0.037), guiding the application of clinical individualized therapy. CONCLUSIONS: The Ep-LMS/Vi-LMS/GPC3-LMS sequential CTC capture system is convenient and feasible for the clinical prediction of HCC progression. CTCs captured by this system could be used as a suitable alternative to HCC tissue detection in guiding immunotherapy, supporting the clinical application of CTC liquid biopsy.

Single Nanoparticle Counting-Based Liquid Biopsy for Cancer Diagnosis.

Liquid biopsy has become a sought-after technique for disease diagnosis because it provides real-time, dynamic, and comprehensive information that can alleviate the dilemmas faced by tissue biopsy. Multiplexed microRNAs (miRNAs) have been demonstrated to be promising biomarkers in liquid biopsy but fall short in providing simple and sensitive analytical methods. In this work, we established a novel nanoparticle counting strategy to accomplish simultaneous analysis of three breast cancer-associated miRNAs for breast cancer diagnosis. The frequency of nanoparticles not involved in the formation of sandwich structures was detected by single-particle inductively coupled plasma mass spectrometry (SP-ICPMS) to quantify the targets. The detection limits for miR-21, miR-155, and miR-16 were 49, 51, and 55 amol, respectively. The strategy was applied to clinical samples and successfully achieved the diagnosis between normal individuals and breast cancer patients. The area under the curve (AUC) for the combination of the relative expression of miR-21 and miR-155 was 0.818. The above results indicate that this strategy has potential and holds great promise for playing an essential role in cancer diagnosis.

Mass Spectrometric Multiplex Detection of MicroRNA and Protein Biomarkers for Liver Cancer.

The occurrence of cancers is often accompanied by the abnormal expression of several sorts of biomarkers (e.g., nucleic acids and proteins). The multiplex assessment of them would substantially aid in the early detection and precise diagnosis, which is often hampered by their different detection schemes, different reaction matrix and reagents, and spectral overlapping. Herein, we propose a simple and sensitive mass spectrometric method for the multiplex detection of nucleic acid and protein, in which liver cancer-related biomarkers miRNA 223 and alpha-fetoprotein (AFP) were selected as model analytes. The self-amplification effect of metal atom-based nanoparticle probes can provide high sensitivity in complex serum samples without any additional amplification procedure. The detection limits for the simultaneous detection of miRNA 223 and AFP were 103 (2.1 pM) and 219 amol (0.15 ng/mL), respectively, with high specificity and selectivity. The proposed method is potentially useful for the rapid screening of cancers.

Single-Nanoparticle Differential Immunoassay for Multiplexed Gastric Cancer Biomarker Monitoring.

Precision medicine demands the best application of multiple unambiguous biomarkers to bring uniform decisions in disease prognosis. The remarkable development of heterogeneous immunoassay greatly promotes precision medicine when combined with the biomarker combination strategy. Nevertheless, the cumbersome washing steps in heterogeneous immunoassay have inevitably compromised the accuracy because of the sample losses and nature change of the matrix, challenging the further exploration of a more facile and lower limit-of-detection analysis. The new methodologies with high throughputs and specificity are never out of date to provide simultaneous evaluations and uniform decisions on multiple analytes through a simple process. Herein, we propose a new wash-free immunoassay, named differential assay, for multiplexed biomarker monitoring. The method is based on counting the number difference of unbound nanoparticle tags before and after immunoreactions from a solid support (i.e., magnetic microsphere) by single-particle inductively coupled plasma mass spectrometry (sp-ICP-MS), discarding the tedious washing steps. We primarily explore the proof-of-concept proposal within two types (sandwich and competitive assay), demonstrating the good feasibility for further facile clinical practice. To provide efficient multiplexed evaluations, we synthesized PtNPs with four diameters and screened the most suitable size for efficient differential immunoassay. The wash-free strategy was successfully utilized in simultaneous serological biomarker (CA724, CA199, and CEA) evaluation, with results in good accordance with those measured by the clinical routine method. Potentially, the proposed differential bioassay can be regarded as a more facile and valuable tool in malignancy prognosis and cancer recurrence monitoring.

Multiplex Nucleic Acid Assay of SARS-CoV-2 via a Lanthanide Nanoparticle-Tagging Strategy.

Early diagnosis, early isolation, and early treatment are efficient solutions to control the COVID-19 pandemic. To achieve the accurate early diagnosis of SARS-CoV-2, a multiplex detection strategy is required for the cross-validation to solve the problem of "false negative" of the existing gold standard assay. Here, we present a multicomponent nucleic acid assay platform for SARS-CoV-2 detection based on lanthanide nanoparticle (LnNP)-tagging strategy. For targeting SARS-CoV-2's RNA fragments ORF1ab gene, RdRp gene, and E gene, three LnNP probes can be used simultaneously to identify three sites in one sample through elemental mass spectrometry detection with limits of detection of 1.2, 1.3, and 1.3 fmol, respectively. With the multisite cross-validation, we envision that this multiplex and sensitive detection platform may provide an effective strategy for SARS-CoV-2 fast screening with a high accuracy.

Homogeneous Multiplex Immunoassay for One-Step Pancreatic Cancer Biomarker Evaluation.

Pancreatic cancer is one of the foremost malignant gastrointestinal tumors, with prognosis and postoperative prediction remaining challenging because of the lack of facile, sensitive diagnostic methods and a specific single biomarker. Combined-biomarker analysis which provides a promising strategy to conquer such dilemma still requires developments in methodologies to gain accurate and reliable outcomes with wash-/separation-free scenarios and minimal interferences. Herein, a multiplex single-particle homogeneous immunoassay was proposed by simultaneously evaluating three pancreatic cancer-related biomarkers. Owing to the excellent resolution and multielement detectors without mass spectra overlapping, single-particle ICP-MS simultaneously provided biomarkers (CA125, CEA, and CA199) with three to four order-of-magnitude linear ranges and low-level limits of detection from specific antibody-labeled noble metal nanoparticles (AuNPs, AgNPs, and PtNPs). By scrutinizing both intensity and frequency signals, the proposed method was successfully applied in patients' serological evaluation, with results correlating well with those measured by the clinical routine method. The proposed method provides a potential tool in risk assessment of disease recurrence and survival.

Self-Validated Homogeneous Immunoassay by Single Nanoparticle in-Depth Scrutinization.

The most convenient method for the clinical routine analysis of disease biomarkers is homogeneous immunoassay, which minimizes the requirements for automation and time-/lab-consumption. Despite great success, because sample constituents are not removed by a separation or washing step, a major challenge in conducting homogeneous immunoassays for the practical application is the matrix effect-related inaccuracy. Herein, to guarantee an accurate quantification, a self-validated homogeneous immunoassay was proposed, by simultaneously scrutinizing both frequency and intensity of single gold nanoparticles. The two analytical modes of single particle inductively coupled plasma mass spectrometry (ICPMS) correlated well with each other, resulting in a self-validation mechanism for the accurate immunoassay. Both two modes of the proposed method provided linear ranges of 2 orders of magnitude and LODs of pM level. Thanks to the self-validated strategy and the high tolerance of the matrix effect of ICPMS, the proposed homogeneous immunoassay was successfully demonstrated in a series of human serum samples, with results in good accordance with clinical routine methods.

The Evolution Characteristics of Systemic Risk in China's Stock Market Based on a Dynamic Complex Network.

The stock market is a complex system with unpredictable stock price fluctuations. When the positive feedback in the market amplifies, the systemic risk will increase rapidly. During the last 30 years of development, the mechanism and governance system of China's stock market have been constantly improving, but irrational shocks have still appeared suddenly in the last decade, making investment decisions risky. Therefore, based on the daily return of all a-shares in China, this paper constructs a dynamic complex network of individual stocks, and represents the systemic risk of the market using the average weighting degree, as well as the adjusted structural entropy, of the network. In order to eliminate the influence of disturbance factors, empirical mode decomposition (EMD) and grey relational analysis (GRA) are used to decompose and reconstruct the sequences to obtain the evolution trend and periodic fluctuation of systemic risk. The results show that the systemic risk of China's stock market as a whole shows a downward trend, and the periodic fluctuation of systemic risk has a long-term equilibrium relationship with the abnormal fluctuation of the stock market. Further, each rise of systemic risk corresponds to external factor shocks and internal structural problems.

Spillover effects of RMB exchange rate among B&R countries: Before and during COVID-19 event.

The proposal of "The Belt and Road" (The B&R) Initiative has promoted regional economic cooperation and financial integration. It is crucial to measure the volatility spillover effects among "The B&R" currency market. Results from the time-varying spillover model show that "The B&R" system spillover index reflects some sudden regional crises. Likewise, the spillover of RMB exchange rate is affected by internal financial reforms as well as external economic shocks. Further, the recent outbreak of the COVID-19 has disrupted this system and the influence of RMB.

Systematic analysis of gene expression profiles reveals prognostic stratification and underlying mechanisms for muscle-invasive bladder cancer.

BACKGROUND: Muscle-invasive bladder cancer (MIBC) is originated in the muscle wall of the bladder, and is the ninth most common malignancy worldwide. However, there are no reliable, accurate and robust gene signatures for MIBC prognosis prediction, which is of the importance in assisting oncologists to make a more accurate evaluation in clinical practice. METHODS: This study used univariable and multivariable Cox regression models to select gene signatures and build risk prediction model, respectively. The t-test and fold change methods were used to perform the differential expression analysis. The hypergeometric test was used to test the enrichment of the differentially expressed genes in GO terms or KEGG pathways. RESULTS: In the present study, we identified three prognostic genes, KLK6, TNS1, and TRIM56, as the best subset of genes for muscle-invasive bladder cancer (MIBC) risk prediction. The validation of this stratification method on two datasets demonstrated that the stratified patients exhibited significant difference in overall survival, and our stratification was superior to three other stratifications. Consistently, the high-risk group exhibited worse prognosis than low-risk group in samples with and without lymph node metastasis, distant metastasis, and radiation treatment. Moreover, the upregulated genes in high-risk MIBC were significantly enriched in several cancer-related pathways. Notably, PDGFRB, a receptor for platelet-derived growth factor of PI3K-Akt signaling pathway, and TUBA1A were identified as two targets of multiple drugs. In addition, the angiogenesis-related genes, as well as two marker genes of M2 macrophage, CD163 and MRC1, were highly upregulated in high-risk MIBC. CONCLUSIONS: In summary, this study investigated the underlying molecular mechanism and potential therapeutic targets associated with worse prognosis of high-risk MIBC, which could improve our understanding of progression of MIBC and provide new therapeutic strategies for the MIBC patients.

Thermoregulation-Independent Regulation of Sleep by Serotonin Revealed in Mice Defective in Serotonin Synthesis.

A role for 5-hydroxytryptamine (5-HT) or serotonin in sleep has been known for decades but was challenged by recent papers that concluded that the apparent sleep phenotype was secondary to defective thermoregulation. Those studies used mice lacking serotonergic neurons resulting from the loss of function mutations in the gene encoding the LIM homeobox transcription factor 1 (Lmx1b). Here we show that, while Lmx1b mutants failed to keep the physiologic body temperature, they exhibited more activities at the room and elevated temperatures. More importantly, we used mice deficient in the gene encoding tryptophan hydroxylase 2 (Tph2), which could not synthesize 5-HT in the brain. Tph2 mutants were capable of thermoregulation and keeping physiologic body temperature when the environmental temperature was reduced and exhibited significantly more activities at both the room and elevated temperatures. Electroencephalographic (EEG) recording also showed decreased sleep in Tph2-deficient mice. Our results indicate that 5-HT is important for sleep regulation but not thermoregulation.

Preparation of Rh/Ag bimetallic nanoparticles as effective catalyst for hydrogen generation from hydrolysis of KBH4.

ISOBAM-104 protected Rh/Ag bimetallic nanoparticles (NPs) with average diameter less than 3.0 nm were synthesized by a co-reduction method. Ultraviolet-visible spectroscopy, transmission electron microscopy (TEM), high-resolution TEM and x-ray photoelectron spectroscopy (XPS) were employed to characterize the structure, particle size, and electronic structure of the prepared bimetallic NPs. The catalytic activities of prepared bimetallic NPs for hydrogen generation from hydrolysis of a basic KBH4 solution were evaluated in detail. The results indicated that as-prepared Rh/Ag bimetallic NPs showed a higher catalytic activity than corresponding monometallic NPs. Among all the monometallic NPs and bimetallic NPs, Rh80Ag20 bimetallic NPs exhibited the highest catalytic activity with a value of 6010 mol-H2·h-1·mol-catalyst-1 at pH = 12 and 303 K. The high catalytic activities of Rh/Ag bimetallic NPs could be attributed to presence of negatively charged Rh atoms and positively charged Ag atoms, which is supported by the results of XPS and density functional theory calculation. Based on the kinetic study, the apparent activation energy for the hydrolysis reaction of the basic KBH4 solution catalyzed by Rh80Ag20 bimetallic NPs was about 47.0 ± 3.9 kJ mol-1.

Midterm Follow-up of Coronary Artery Bypass Grafting with 64-Slice Multi-detector Computed Tomography: Identification of Risk Factors Affecting Graft Patency.

To identify the risk factors that are associated with the midterm coronary artery bypass grafting (CABG) functionality by assessing patency of left internal mammary artery (LIMA) graft and saphenous vein (SV) graft with 64-slice multi-detector computed tomography (64-MDCT).Methods Patients who underwent CABG operation and postoperative 64-MDCT follow-up examinations from August 2012 to December 2015 were included. The graft patent status was classified into patent and poor patent according to MDCT findings predominantly on 3D reconstructed images by two radiologists. The clinical data and imaging findings of the patients were collected and compared between the patent group and poor patent group. Univariate analysis and the multivariate logistic regression analysis were performed to identify the risk factors that affect graft patency.Results Among 341 patients in the study, there were 330 LIMA grafts [326 anastomosed to the left anterior descending artery (LAD), 4 to right coronary artery (RCA)] and 564 SV grafts (SVG) [100 anastomosed to the diagonal branch (D), 226 to the obtuse marginal branch (OM), and 238 to the RCA territory]. The approximal vessel stenosis exceeding 90% occurred in 268 of 292 patent LIMA grafts, and in 1 of 34 poor patent grafts (χ 2=167, P<0.001). The patency rate was higher when SVG was anastomosed to OM (85.4%) or RCA territory (81.9%) than to D (69.0%) (χ 2=15.471, P=0.004). The proximal target vessel stenosis < 90% (OR= 0.015, 95% CI: 0.01-0.14, P=0.000) was independently associated with the closure risk of LIMA grafts, the dyslipidemia (OR= 1.52, 95% CI: 1.0-2.5, P=0.048), history of diabetes (OR = 1.28, 95% CI : 0.90-2.26, P=0.045) and typical angina symptoms (OR=1.81, 95% CI :1.33-4.15, P=0.003) were independently associated with the closure risk of SVG. Conclusions The proximal LAD stenosis less than 90% was adversely associated with graft patency in LIMA recipients; dyslipidemia, diabetes and angina symptoms were associated with the midterm failure in SVG recipients. The choice of the target anastomosis sites may affect the patency of SVG.

Value of Multi-detector CT in Detection of Isolated Spontaneous Superior Mesenteric Artery Dissection.

Objective To investigate the role of multi-detector computed tomography (CT) in the diagnosis and classification of isolated spontaneous superior mesenteric artery dissection (ISSMAD). Methods From July 2012 to December 2016, 30 consecutive patients with ISSMAD underwent CT scan at least two times. We retrospectively summarized the clinical characteristics and CT findings of them. The stenosis ratio of true lumen was compared between the patients without bowel ischemia and ones with bowel ischemia. Results There were 5 cases of type I ISSMAD, 14 cases of type 2, 1 case of type 3, 7 cases of type 4 and 3 cases of type V. Intestinal ischemia occurred in 5 patients. The stenosis ratio of true lumen in the patients without bowel ischemia was lower than that with bowel ischemia (45.6% vs. 76.0%, t=-14.5, P=0.000). Five patients with intestinal ischemia underwent superior mesenteric artery stenting and others received conservative therapy. The abdominal pain was alleviated for all the patients after treatment. Follow-up was complete in 30 cases. Follow-up CT angiography of superior mesenteric artery showed dissection remodeling in 12 patients. Conclusion Multi-detector CT is a valuable method in diagnosis and classification of ISSMAD and monitoring the changes of dissection.

Feasibility, Safety, and Efficacy of Accurate Uterine Fibroid Ablation Using Magnetic Resonance Imaging-Guided High-Intensity Focused Ultrasound With Shot Sonication.

The aim of this study was to investigate the feasibility, safety, and efficacy of uterine fibroid treatment using magnetic resonance imaging (MRI)-guided high-intensity focused ultrasound (US) with shot sonication for accurate ablation. Forty-three patients with 51 symptomatic uterine fibroids were treated with MRI-guided high-intensity focused US with shot sonication, which was a small acoustic focus of higher intensity with a shorter time (2 seconds) of US exposure and a shorter cooling time (2-3 seconds). The treatment efficacy and adverse events were analyzed, and the changes in the severity of symptoms and the reduction in fibroid volume were assessed 3 and 6 months after the procedure. All patients were successfully treated in a single session, without major complications, and the mean nonperfused volume ratio ± SD was 84.3% ± 15.7% (range, 33.8%-100%).Complete ablation was achieved in 13 T2-hypointense fibroids from 10 patients, and partial ablation was achieved in 38 fibroids from 33 patients. The overall mean treatment time was 135.0 ± 50.9 minutes (2.2 ± 0.8 hours). The transformed symptom severity scores and mean fibroid volumes decreased significantly after treatment (P < .05). In conclusion, MRI-guided high-intensity focused US with shot sonication is a feasible, safe, and effective technique for ablation of uterine fibroids and complete ablation of T2-hypointense fibroids.

Highly sensitive detection of aflatoxin B1 byCRISPR/Cas12a-assisted single nanoparticle counting.

CRISPR (clustered regularly interspaced short palindromic repeats) and CRISPR-associated protein (Cas) have gained extensive applications in bioassays. However, CRISPR-based detection platforms are often hampered by limited analytical sensitivity, while nucleic acid-based amplification strategies are usually indispensable for additional signal enhancement with potential risks of amplification leakages. To address these challenges, an amplification-free CRISPR-based bioassay of aflatoxin B1 (AFB1) was proposed by applying single nanoparticle counting. Single-particle mode inductively coupled plasma mass spectrometry (Sp-ICPMS) has been regarded as a sensitive tool for nanoparticle counting since one nanoparticle can generate considerable signals above backgrounds. With AFB1, activator strands were introduced to initiate the trans-cleavage of CRISPR/Cas12a for cutting the nanoparticles-tagged-magnetic beads, which were transduced to nanoparticle count signals after separation. Finally, a pico-mole level limit-of-detections (LODs) with moderate selectivity was achieved. Certified reference materials (CRMs) analysis and recovery tests were conducted with promising results. To our best knowledge, this is the first report of the single particle counting-based CRISPR/Cas12a biosensing study.

[Establishment of a high metastatic potential human hepatocellular carcinoma orthotopic transplantation model with palliative liver resection in nude mice].

OBJECTIVE: To construct a high metastatic potential human hepatocellular carcinoma (HCC) orthotopic transplantation model with palliative liver resection in nude mice. METHODS: A human HCC orthotopic nude mice model was established by administering a single inoculation of the highly metastatic MHCC97H tumor tissue (size 2 mm * 2 mm * 2 mm) into the left liver lobe. At day 14 post-inoculation, a random group of the mice received palliative liver resection; the unresected mice served as controls. Changes in expression levels of 113 genes with metastasis-related functions were evaluated in the residual HCC tissues. At day 35 post-resection, a random group of the mice were sacrificed by cervical dislocation and a comprehensive metastases examination was performed. The remaining mice were used to observe life span. All statistical analyses were performed by the SPSS v17.0 software, and significance was defined as P less than 0.05. RESULTS: The nude mouse model of highly metastatic HCC with palliative liver resection was successfully established. Incidences of intrahepatic and abdominal metastases were higher in the palliative resected group (vs. unresected group: 11.7+/-4.7 vs. 6.3+/-2.8, t = -2.412, P less than 0.05 and 9.8+/-3.4 vs. 5.2+/-2.6, t = -2.641, P less than 0.05 respectively). In addition, the palliative resected group showed significantly enhanced pulmonary metastasis (vs. unresected group: 14.3+/-4.7 vs. 8.7+/-4.7, t = -2.348, P less than 0.05). Differential gene expression levels were found for MTSS1, TGFbl, SMAD2, IL-1b, and MMP7, and were situated in the central position of gene function net of residual HCC. The life-span of the palliative resected group was significantly longer than that of the unresected group (60.8+/-2.7 vs. 51.3+/-1.4 days, x2 = 12.850, P less than 0.01). CONCLUSION: The highly metastatic human HCC nude mouse model with palliative liver resection that was successfully constructed in this study represents a useful investigational tool to assess the biological characteristics of residual cancer and to screen therapeutic strategies.

Comprehensive Genomic Profiling Identifies FAT1 as a Negative Regulator of EMT, CTCs, and Metastasis of Hepatocellular Carcinoma.

Background: FAT atypical cadherin 1 (FAT1) acts as a tumor suppressor or oncogene, which regulates cell adherence, proliferation, motility, and actin kinetics. FAT1 gene expression is closely related to hepatocarcinogenesis; however, the function and mechanism of FAT1 in hepatocellular carcinoma (HCC) remain unclear. Methods: Here, we screened for the FAT1, which is intimately linked to the development and progression of HCC, both in circulating tumor cells (CTCs) and tumor tissues using next generation sequencing (NGS). Immunohistochemical staining was performed to detect FAT1 protein expression. To determine the impact of FAT1 on epithelial-mesenchymal transition (EMT), migration and invasion of HCC, an in vitro transwell assay and Western blot were performed. Moreover, Gene Set Enrichment Analysis was carried out to discover the underlying mechanism. Finally, animal experiments were conducted to confirm the effects of FAT1 on HCC metastasis and tumorigenicity. Results: Our results showed that FAT1 expression was decreased in HCC tissues, while in vitro and in vivo, the FAT1 knockdown facilitated invasion, cell motility, colony formation, and proliferation. FAT1 knockdown also resulted in decreased expression of E-cadherin and markedly elevated expression of N-cadherin, vimentin, and snail. We also confirmed our hypothesis from the analysis of group differences in the CTC phenotype and lung metastasis in nude mice. Conclusion: Our findings illustrated that FAT1 played a negative regulatory role in the HCC EMT and metastasis, providing further evidence for the role played by FAT1 in the formation and progression of HCC.

Exosomal proteomics identifies RAB13 as a potential regulator of metastasis for HCC.

BACKGROUND: Exosomal proteins from cancer cells are becoming new biomarkers for cancer monitoring and efficacy evaluation. However, their biological function and molecular mechanism underlying tumor metastasis are largely unknown. METHODS: Bioinformatic methods such as bulk gene expression analysis, single-cell RNA sequencing data analysis, and gene set enrichment analysis were employed to identify metastasis-associated proteins. The in vitro and in vivo experiments were used to investigate the function of RAB13 in HCC metastasis. RESULTS: We identified RAB13 as one of the critical regulators of metastasis in HCC-derived exosomes for the first time. In vitro, the invasiveness of HCC cell lines could be attenuated by RAB13 silence. In vivo, tumor size and proportion of high-grade lung metastatic nodule could be reduced in the mice with orthotopic transplantation of tumors and intravenously injected with exosomes derived from MHCC97H cell with RAB13 silence (si-RAB13-Exo), as compared with those without RAB13 silence (si-NC-Exo). Moreover, in si-RAB13-Exo group, circulating tumor cell counts were decreased at the third, fourth, and fifth weeks after orthotopic transplantation of tumors, and MMP2 (matrix metalloproteinase 2)/TIMP2 (tissue inhibitor of metalloproteinases 2) ratio was also significantly decreased. In addition, RAB13 expression was also associated with VEGF levels, microvessel density, and tube formation of vascular endothelial cells by both in vitro and in vivo models, indicating that RAB13 was associated with angiogenesis in HCC. CONCLUSIONS: We have demonstrated exosomal RAB13 as a potential regulator of metastasis for HCC by in silico, in vitro, and in vivo methods, which greatly improve our understanding of the functional impact of exosomal proteins on HCC metastasis.