LncRNA MACC1-AS1 induces gemcitabine resistance in pancreatic cancer cells through suppressing ferroptosis.

Pancreatic ductal adenocarcinoma (PDA) mortality is primarily attributed to metastasis and chemotherapy resistance. In this research, the long non-coding RNA MACC1-AS1 was studied, playing a significant role in regulating lipid oxidation processes. This regulation could further lead to the inhibition of ferroptosis induced by chemotherapeutic drugs, making it a contributing factor to gemcitabine resistance in PDA. In both gemcitabine-resistant PDA patients and mouse models, the elevated expression level of MACC1-AS1 in the tumors was noted. Additionally, overexpression of MACC1-AS1 in pancreatic cancer cells was found to enhance tolerance to gemcitabine and suppress ferroptosis. Proteomic analysis of drug-resistant pancreatic cells revealed that overexpressed MACC1-AS1 inhibited the ubiquitination degradation of residues in the protein kinase STK33 by MDM4. Furthermore, its accumulation in the cytoplasm activated STK33, further activating the ferroptosis-suppressing proteins GPX4, thereby counteracting gemcitabine-induced cellular oxidative damage. These findings suggested that the long non-coding RNA MACC1-AS1 could play a significant role in the ability of pancreatic cancer cells to evade iron-mediated ferroptosis induced by gemcitabine. This discovery holds promise for developing clinical therapeutic strategies to combat chemotherapy resistance in pancreatic cancer.

A multicenter clinical AI system study for detection and diagnosis of focal liver lesions.

Early and accurate diagnosis of focal liver lesions is crucial for effective treatment and prognosis. We developed and validated a fully automated diagnostic system named Liver Artificial Intelligence Diagnosis System (LiAIDS) based on a diverse sample of 12,610 patients from 18 hospitals, both retrospectively and prospectively. In this study, LiAIDS achieved an F1-score of 0.940 for benign and 0.692 for malignant lesions, outperforming junior radiologists (benign: 0.830-0.890, malignant: 0.230-0.360) and being on par with senior radiologists (benign: 0.920-0.950, malignant: 0.550-0.650). Furthermore, with the assistance of LiAIDS, the diagnostic accuracy of all radiologists improved. For benign and malignant lesions, junior radiologists' F1-scores improved to 0.936-0.946 and 0.667-0.680 respectively, while seniors improved to 0.950-0.961 and 0.679-0.753. Additionally, in a triage study of 13,192 consecutive patients, LiAIDS automatically classified 76.46% of patients as low risk with a high NPV of 99.0%. The evidence suggests that LiAIDS can serve as a routine diagnostic tool and enhance the diagnostic capabilities of radiologists for liver lesions.

Factors influencing the prevalence of frailty in older adults with fractures: the association of nutritional status with frailty.

BACKGROUND AND OBJECTIVES: To investigate the association between frailty, malnutrition, comorbid medical conditions and activities of daily living (ADL) in older adult patients with fractures, and to analyse the influential factors of frailty. METHODS AND STUDY DESIGN: The FRAIL scale including five components: fatigue, resistance, ambulation, illness, and loss of weight, was used to evaluate frailty. Participants were divided into frailty, pre-frailty and non-frailty groups. The ADL was assessed using the Barthel Index, while the nutrition risk screening tool, NRS-2002, was used to assess the nutritional risk, and the Global Leadership Initiative on Malnutrition diagnostic criteria were used to diagnose the nutritional status. Statistical analysis was performed using univariate and multivariate logistic regression to determine the factors associated with frailty. RESULTS: A total of 166 patients were included in the study, and the incidences of frailty, pre-frailty and non-frailty were 39.2%, 33.1% and 27.7%, respectively. The severe dependence rate (ADL scale of <40) in the frailty, pre-frailty and non-frailty groups was 49.2%, 20.0% and 6.52%, respectively. The prevalence of nutritional risk was 33.7% (56/166), including 56.9% (31/65) in the frailty group and 32.7% (18/55) in the pre-frailty group. Of the 166 patients, 45 (27.1%) were diagnosed with malnutrition, including 47.7% (31/65) in the frailty group and 23.6% (13/55) in the pre-frailty group. CONCLUSIONS: Frailty in older adult patients with fractures is widespread, and the prevalence of malnutrition is high. The occurrence of frailty may be related to an advanced age, increased medical comorbidity and impairment in ADL.

A novel cuproptosis-related lncRNA signature predicts the prognosis and immunotherapy for hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most serious malignant tumors with a poor prognosis worldwide. Cuproptosis is a novel copper-dependent cell death form, involving mitochondrial respiration and lipoylated components of the tricarboxylic acid (TCA) cycle. Long non-coding RNAs (lncRNAs) have been demonstrated to affect the tumorigenesis, growth, and metastasis of HCC. OBJECTIVE: We explored the potential roles of cuproptosis-related lncRNAs in predicting the prognosis for HCC. METHODS: The RNA-seq transcriptome data, mutation data, and clinical information data of HCC patients were downloaded from The Cancer Genome Atlas (TCGA) database. The least absolute shrinkage and selection operator (LASSO) algorithm and Cox regression analyses were performed to identify a prognostic cuproptosis-related lncRNA signature. The receiver operating characteristic (ROC) analysis was used to evaluate the predictive value of the lncRNA signature for HCC. The enrichment pathways, immune functions, immune cell infiltration, tumor mutation burden, and drug sensitivity were also analyzed. RESULTS: We constructed a prognostic model consisting of 8 cuproptosis-related lncRNAs for HCC. The patients were divided into high-risk group and low-risk group according to the riskscore calculated using the model. Kaplan-Meier analysis revealed that the high-risk lncRNA signature was correlated with poor overall survival [hazard ratio (HR) =1.009, 95% confidence interval (CI) = 1.002-1.015; p= 0.010)] of HCC. A prognostic nomogram incorporated the lncRNA signature and clinicopathological features were constructed and showed favorable performance for predicting prognosis of HCC patients. In addition, the most immune-related functions were significantly different between the high-risk and low-risk groups. Tumor mutation burden (TMB) and immune checkpoints were also expressed differently between the two risk groups. Finally, HCC patients with low-risk score were more sensitive to several chemotherapy drugs. CONCLUSIONS: The novel cuproptosis-related lncRNA signature could be used to predict prognosis and evaluate the effect of chemotherapy for HCC.

Systems Network Pharmacology-Based Prediction and Analysis of Potential Targets and Pharmacological Mechanism of Actinidia chinensis Planch. Root Extract for Application in Hepatocellular Carcinoma.

Purpose: Traditional Chinese medicine (TCM) sometimes plays a crucial role in advanced cancer treatment. Despite the significant therapeutic efficacy in hepatocellular carcinoma (HCC) that Actinidia chinensis Planch root extract (acRoots) has proven, its complex composition and underlying mechanism have not been fully elucidated. Therefore, this study analyzed the multiple chemical compounds in acRoots and their targets via network pharmacology and bioinformatics analysis, with the overarching goal of revealing the potential mechanisms of the anti-HCC effect. Methods: The main ingredients contained in acRoots were initially screened from the traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP), and the candidate bioactive ingredient targets were identified using DrugBank and the UniProt public databases. Second, the biological processes of the targets of active molecules filtered from the ingredients of acRoots were evaluated using gene ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. Third, weighted gene coexpression network analysis (WGCNA) was performed to identify gene coexpression modules associated with HCC. The hub genes of acRoots in HCC were defined via contrasting the above module eigengenes with candidate target genes of acRoots. Furthermore, the target-pathway network was analyzed to explore the mechanism for anti-HCC effect of hub genes. Kaplan-Meier plotter database analysis was performed to validate the hub genes of acRoots correlation with prognostic values in HCC. In order to verify the results of the network pharmacological analysis, we performed a molecular docking approach on the active ingredients and key targets using the Discovery Studio software. The viability of SMMC-7721 and HL-7702 cells was determined by Cell counting kit-8 (CCK-8) after being treated with different concentrations of (+)-catechin (0, 50, 100, 150, 200, and 250 g/ml) for 24, 48, and 72 hours, respectively. Finally, qRT-PCR and Western blot involving human hepatocarcinoma cells were utilized to verify the impact of (+)-catechin on the hub genes associated with prognosis. Results: 6 out of 26 active ingredients extracted from TCMSP were deemed as the core ingredients of acRoots. 175 bioactive-ingredient targets of acRoots were obtained and a bioactive-ingredient targets network was established correspondingly. The biological processes (BP) of target genes mainly involved processes, such as toxic substance and wounding. The results of KEGG pathways indicated that the target genes were mainly enriched in pathways in cancer, AGE-RAGE signaling pathway in diabetic complications, IL-17 signaling pathway, and other pathways. Also, the two hub genes (i.e., ESR1 and CAT) were closely associated with the prognosis of HCC patients. As a consequence, we predicated a series of signaling pathways, including estrogen signaling pathway and longevity regulation pathway, through which acRoots could facilitate the treatment for HCC. The molecular docking experiment ascertained that ESR1 and CAT had an effective binding force with (+)-catechin, one of the core ingredients of acRoots. Furthermore, (+)-catechin inhibited SMMC-7721 cell growth in a dose-dependent manner and a time-dependent manner. Finally, we suggest that the expression level of ESR1 and CAT is positively related to the (+)-catechin concentrations in in-vitro experiments. Conclusion: The bioactive ingredients of acRoots, including quercetin, (+)-catechin, beta-sitosterol, and aloe-emodin, have synergistic interactions in reinforcing the anticancer effect in HCC. Evidently, acRoots took effect by regulating multitargets and multipathways through its active ingredients. Further, (+)-catechin, the possible paramount anti-HCC active ingredient in acRoots, helped improve the prognosis of HCC patients by increasing the expression of ESR1 and CAT. Additionally, the findings yielded provide a conceptual guidance for the clinical treatment of HCC and the methods adopted are potentially applicable in the future comprehensive analysis of the underlying mechanisms of TCMs.

METTL3-mediated m6A methylation of ASPM drives hepatocellular carcinoma cells growth and metastasis.

BACKGROUND: Abnormal spindle-like microcephaly (ASPM) has been proved to participate in tumor progression. However, the underlying mechanism of ASPM in liver hepatocellular carcinoma (LIHC) remains elusive. METHODS: The mRNA and protein expression were determined using Western blot and qRT-PCR, and the capacities of cells proliferation, migration, and invasion were evaluated by CCK-8, colony formation, wound healing, and transwell. MeRIP was performed to validate the interaction between ASPM and methyltransferase-like 3 (METTL3). RESULTS: Herein, we found that ASPM was significantly upregulated in LIHC, and the high expression of ASPM was associated with poor LIHC prognosis. Furthermore, ASPM knockdown could suppress LIHC cells proliferation, migration, and invasion, while ASPM overexpression exerted reverse effect. Mechanistically, we revealed that the N6-methyladenosine (m6A) modification of ASPM mRNA mediated by METTL3 promoted its expression in LIHC. More importantly, silencing METTL3 suppressed LIHC cells proliferation, migration, and invasion, which could be retained by ASPM overexpression. CONCLUSION: Collectively, our findings suggested that METTL3/ASPM axis could serve as a novel promising therapeutic candidate for LIHC.

GAS5 Regulates RECK Expression and Inhibits Invasion Potential of HCC Cells by Sponging miR-135b.

OBJECTIVES: Long noncoding RNA (LncRNA) growth arrest-specific 5 (GAS5) has been characterized as a tumor suppressor in numerous kinds of human cancers. Its anticancer function in hepatocellular carcinoma (HCC) includes repression of cell proliferation and metastasis, leaving the internal mechanisms unclear. In this study, we intended to examine the anti-invasion effects of GAS5 on HCC and explore the downstream regulatory mechanisms. METHODS: Expression of GAS5 and microRNA-135b (miR-135b) was analyzed by qRT-PCR in paired HCC tissue samples. Their correlation with HCC patients' survival was determined. Transwell assays were done to evaluate in vitro invasion ability. Targeting of GAS5 and RECK by miR-135b was confirmed by qRT-PCR, western blot, and luciferase reporter assays. RESULTS: Decreased GAS5 and increased miR-135b in HCC inversely correlate with each other and both correlate with poor prognosis of HCC patients. Functionally, GAS5 suppresses while miR-135b promotes HCC cell invasion capacities in vitro. Mechanistically, GAS5 is a target of miR-135b. Furthermore, GAS5 positively regulates expression of RECK, also a target of miR-135b, which further inhibits MMP-2 expression and contributes to invasion repression. CONCLUSION: GAS5 acted as a tumor suppressor in HCC invasion in a competing endogenous RNA manner. Our findings indicate that GAS5 is a promising therapeutic target for HCC treatment.

Antitumor effects of matrine on cancer stem like cells isolated from the human liver cancer SMMC-7721 cell line.

The existence of cancer stem cells (CSCs) or cancer stem-like cells (CSLCs) is regarded as the cause of tumor formation and recurrence. Matrine has been reported to exhibit antitumor effects in cancer cells. In the present study, a preliminary study was performed on the mechanisms of matrine on hepatocellular carcinoma (HCC) stem-like cells. The HCC SMMC-7721 cell line was cultured in tumor stem cell-specific medium to form spheres, and different concentrations (1, 2 and 5 mg/kg) of cisplatin were then used in order to purify the most drug-resistant cells, which were used as CSLCs. An MTT assay was performed to detect the inhibitory effects of matrine against CSLC proliferation. Quantitative polymerase chain reaction (qPCR) and western blot analysis were used to detect changes in cell adhesion regulating gene (CAR), E-cadherin, laminin and fibronectin. As a result, using tryptose sulfite cycloserine medium culture and cisplatin-resistance screening, CSLCs were successfully isolated from the SMMC-7721 cell line. Matrine inhibited the proliferation of CSLCs in vitro. The results of qPCR and western blot analysis demonstrated that matrine upregulated the expression of CAR, E-cadherin, laminin and fibronectin in CSLCs compared with the control treatment. A certain concentration of matrine exhibited antitumor effects on HCC stem like cells.

Folate receptor-targeted ultrasonic PFOB nanoparticles: Synthesis, characterization and application in tumor-targeted imaging.

In this study, we aimed to determine an effective strategy for the synthesis of folate receptor (FR) targeted-nanoparticles (FRNPs). The nanoparticles used as ultrasound contrast agents (UCAs) were composed of a liquid core of perfluorooctyl bromide (PFOB) liposome and a targeted shell chemically conjugated with folic acid (FA) and polyethylene glycol (PEG). This was done in order to avoid recognition and clearance by the mononuclear phagocyte system [also known as the reticuloendothelial system (RES)] and enhance the targeting capability of the nanoparticles to tumors overexpressing folate receptor (FR). The FRNPs exhibited an average particle size of 301±10.8 nm and surface potential of 39.1±0.43 mV. Subsequently, in vitro, FRNPs labeled with FITC fluorescence dye were visibly uptaken into the cytoplasm of FR-overexpressing cancer cells (Bel7402 and SW620 cells), whereas the A549 cells expressing relatively low levels of FR just bound with few FRNPs. These results demonstrated that FRNPs have a high affinity to FR-overexpressing cancer cells. Additionally, in in vivo experiments, FRNPs achieved a greater enhancement of tumor ultrasound imaging and a longer enhancement time in FR-overexpressing tumors and the Cy7-labeled FRNPs exhibited a relatively high tumor-targeted distribution in FR­overexpressing tumors. Targeted ultrasound and fluorescence imaging revealed that FRNPs have the ability to target FR-overexpressing tumors and ex vivo fluorescence imaging was then used to further verify and confirm the presence of FRNPs in tumor tissues with histological analysis of the tumor slices. On the whole, our data demonstrate that the FRNPs may prove to be a promising candidate for the early diagnosis for FR-overexpressing tumors at the molecular and cellular levels.

Preparation and Characterization of Novel Perfluorooctyl Bromide Nanoparticle as Ultrasound Contrast Agent via Layer-by-Layer Self-Assembly for Folate-Receptor-Mediated Tumor Imaging.

A folate-polyethylene glycol-chitosan derivative was synthesized and its structure was characterized. An optimal perfluorooctyl bromide nanocore template was obtained via utilizing the ultrasonic emulsification method combining with orthogonal design. The targeted nanoparticles containing targeted shell of folate-polyethylene glycol-chitosan derivative and perfluorooctyl bromide nanocore template of ultrasound imaging were prepared successfully by exploiting layer-by-layer self-assembly as contrast agent for ultrasound. Properties of the novel perfluorooctyl bromide nanoparticle were extensively studied by Dynamic Light Scattering and Transmission Electron Microscopy. The targeted nanoparticle diameter, polydispersity, and zeta potential are around 229.5 nm, 0.205, and 44.7 ± 0.6 mV, respectively. The study revealed that spherical core-shell morphology was preserved. Excellent stability of targeted nanoparticle is evidenced by two weeks of room temperature stability tests. The results of the cell viability assay and the hemolysis test confirmed that the targeted nanoparticle has an excellent biocompatibility for using in cell studies and ultrasound imaging in vivo. Most importantly, in vitro cell experiments demonstrated that an increased amount of targeted nanoparticles was accumulated in hepatocellular carcinoma cell line Bel7402 relative to hepatoma cell line L02. And targeted nanoparticles had also shown better ultrasound imaging abilities in vitro. The data suggest that the novel targeted nanoparticle may be applicable to ultrasonic molecular imaging of folate-receptor overexpressed tumor.

[A new method for purification and identification of hepatocellular carcinoma stem cell of SMMC-7721].

OBJECTIVE: To explore a new efficient purification method of hepatocellular carcinoma (HCC) stem cells and identify their features. METHODS: Human hepatocellular carcinoma cell line SMMC-7721 was cultured in sphere-culture system of polyhedra-treated dish and tumor stem cell specific medium. Upon the formation of cellular sphere, the cells were inoculated subcutaneously into immunocompromised mice and received the interventions of different concentrations of cisplatin. Then the drug-resistant cells were purified and re-cultured in TSC medium. Finally the stem cell markers and tumor stem cell markers were determined through real-time polymerase chain reaction (PCR), immunofluorescence method and flow cytometry. RESULTS: Through the double filter of TSC medium and cisplatin-resistance, SMMC-7721 stem cells could be grown in a suspended form and formed spheres in TSC medium. The stem cell markers (NANOG, OCT-4, SOX-2 and Notch) and tumor stem cell markers (CD24, 90.0%; CD133, 6.1%; CD90, 4.8%) were all over-expressed in purified cancer stem cells as compared with ordinary cells. And the over-expression of CD24 was the most obvious. CONCLUSIONS: The combination of in vitro cell culture with TSC medium, in vivo proliferation and cisplatin resistance test is a new efficient method of purifying hepatocellular carcinoma stem cells. Tumor stem cell with stem cell characteristics and an over-expression of CD24 may be cloned from SMMC-7721.