Size-Optimized Layered Double Hydroxide Nanoparticles Promote Neural Progenitor Cells Differentiation of Embryonic Stem Cells Through the Regulation of M6A Methylation.

Purpose: The committed differentiation fate regulation has been a difficult problem in the fields of stem cell research, evidence showed that nanomaterials could promote the differentiation of stem cells into specific cell types. Layered double hydroxide (LDH) nanoparticles possess the regulation function of stem cell fate, while the underlying mechanism needs to be investigated. In this study, the process of embryonic stem cells (ESCs) differentiate to neural progenitor cells (NPCs) by magnesium aluminum LDH (MgAl-LDH) was investigated. Methods: MgAl-LDH with diameters of 30, 50, and 100 nm were synthesized and characterized, and their effects on the cytotoxicity and differentiation of NPCs were detected in vitro. Dot blot and MeRIP-qPCR were performed to detect the level of m6A RNA methylation in nanoparticles-treated cells. Results: Our work displayed that LDH nanoparticles of three different sizes were biocompatible with NPCs, and the addition of MgAl-LDH could significantly promote the process of ESCs differentiate to NPCs. 100 nm LDH has a stronger effect on promoting NPCs differentiation compared to 30 nm and 50 nm LDH. In addition, dot blot results indicated that the enhanced NPCs differentiation by MgAl-LDH was closely related to m6A RNA methylation process, and the major modification enzyme in LDH controlled NPCs differentiation may be the m6A RNA methyltransferase METTL3. The upregulated METTL3 by LDH increased the m6A level of Sox1 mRNA, enhancing its stability. Conclusion: This work reveals that MgAl-LDH nanoparticles can regulate the differentiation of ESCs into NPCs by increasing m6A RNA methylation modification of Sox1.

Application of Bioactive Materials for Osteogenic Function in Bone Tissue Engineering.

Bone tissue defects present a major challenge in orthopedic surgery. Bone tissue engineering using multiple versatile bioactive materials is a potential strategy for bone-defect repair and regeneration. Due to their unique physicochemical and mechanical properties, biofunctional materials can enhance cellular adhesion, proliferation, and osteogenic differentiation, thereby supporting and stimulating the formation of new bone tissue. 3D bioprinting and physical stimuli-responsive strategies have been employed in various studies on bone regeneration for the fabrication of desired multifunctional biomaterials with integrated bone tissue repair and regeneration properties. In this review, biomaterials applied to bone tissue engineering, emerging 3D bioprinting techniques, and physical stimuli-responsive strategies for the rational manufacturing of novel biomaterials with bone therapeutic and regenerative functions are summarized. Furthermore, the impact of biomaterials on the osteogenic differentiation of stem cells and the potential pathways associated with biomaterial-induced osteogenesis are discussed.

ScRNA-seq of gastric cancer tissues reveals differences in the immune microenvironment of primary tumors and metastases.

Gastric carcinoma (GC) is regarded as one of the deadliest cancer characterized by diversity and haste metastasis and suffers limited understanding of the spatial variation between primary and metastatic GC tumors. In this project, transcriptome analysis on 46 primary tumorous, adjacent non-tumorous, and metastatic GC tissues was performed. The results demonstrated that metastatic tumorous tissues had diminished CD8+ T cells compared to primary tumors, which is mechanistically attributed to being due to innate immunity differences represented by marked differences in macrophages between metastatic and primary tumors, particularly those expressing ApoE, where their abundance is linked to unfavorable prognoses. Examining variations in gene expression and interactions indicated possible strategies of immune evasion hindering the growth of CD8+ T cells in metastatic tumor tissues. More insights could be gained into the immune evasion mechanisms by portraying information about the GC ecosystem.

Deep whole-genome analysis of 494 hepatocellular carcinomas.

Over half of hepatocellular carcinoma (HCC) cases diagnosed worldwide are in China1-3. However, whole-genome analysis of hepatitis B virus (HBV)-associated HCC in Chinese individuals is limited4-8, with current analyses of HCC mainly from non-HBV-enriched populations9,10. Here we initiated the Chinese Liver Cancer Atlas (CLCA) project and performed deep whole-genome sequencing (average depth, 120×) of 494 HCC tumours. We identified 6 coding and 28 non-coding previously undescribed driver candidates. Five previously undescribed mutational signatures were found, including aristolochic-acid-associated indel and doublet base signatures, and a single-base-substitution signature that we termed SBS_H8. Pentanucleotide context analysis and experimental validation confirmed that SBS_H8 was distinct to the aristolochic-acid-associated SBS22. Notably, HBV integrations could take the form of extrachromosomal circular DNA, resulting in elevated copy numbers and gene expression. Our high-depth data also enabled us to characterize subclonal clustered alterations, including chromothripsis, chromoplexy and kataegis, suggesting that these catastrophic events could also occur in late stages of hepatocarcinogenesis. Pathway analysis of all classes of alterations further linked non-coding mutations to dysregulation of liver metabolism. Finally, we performed in vitro and in vivo assays to show that fibrinogen alpha chain (FGA), determined as both a candidate coding and non-coding driver, regulates HCC progression and metastasis. Our CLCA study depicts a detailed genomic landscape and evolutionary history of HCC in Chinese individuals, providing important clinical implications.

Integrated characterization of hepatobiliary tumor organoids provides a potential landscape of pharmacogenomic interactions.

Despite considerable efforts to identify human liver cancer genomic alterations that might unveil druggable targets, the systematic translation of multiomics data remains challenging. Here, we report success in long-term culture of 64 patient-derived hepatobiliary tumor organoids (PDHOs) from a Chinese population. A divergent response to 265 metabolism- and epigenetics-related chemicals and 36 anti-cancer drugs is observed. Integration of the whole genome, transcriptome, chromatin accessibility profiles, and drug sensitivity results of 64 clinically relevant drugs defines over 32,000 genome-drug interactions. RUNX1 promoter mutation is associated with an increase in chromatin accessibility and a concomitant gene expression increase, promoting a cluster of drugs preferentially sensitive in hepatobiliary tumors. These results not only provide an annotated PDHO biobank of human liver cancer but also suggest a systematic approach for obtaining a comprehensive understanding of the gene-regulatory network of liver cancer, advancing the applications of potential personalized medicine.

Development of a nomogram model for prediction of new adjacent vertebral compression fractures after vertebroplasty.

BACKGROUND: Vertebroplasty is the main minimally invasive operation for osteoporotic vertebral compression fracture (OVCF), which has the advantages of rapid pain relief and shorter recovery time. However, new adjacent vertebral compression fracture (AVCF) occurs frequently after vertebroplasty. The purpose of this study was to investigate the risk factors of AVCF and establish a clinical prediction model. METHODS: We retrospectively collected the clinical data of patients who underwent vertebroplasty in our hospital from June 2018 to December 2019. The patients were divided into a non-refracture group (289 cases) and a refracture group (43 cases) according to the occurrence of AVCF. The independent predictive factors for postoperative new AVCF were determined by univariate analysis, least absolute shrinkage and selection operator (LASSO) logistic regression, and multivariable logistic regression analysis. A nomogram clinical prediction model was established based on relevant risk factors, and the receiver operating characteristic curve (ROC), calibration curve, and decision curve analysis (DCA) were used to evaluate the prediction effect and clinical value of the model. After internal validation, patients who underwent vertebroplasty in our hospital from January 2020 to December 2020, including a non-refracture group (156 cases) and a refracture group (21 cases), were included as the validation cohort to evaluate the prediction model again. RESULTS: Three independent risk factors of low bone mass density (BMD), leakage of bone cement and "O" shaped distribution of bone cement were screened out by LASSO regression and logistic regression analysis. The area under the curve (AUC) of the model in the training cohort and the validation cohort was 0.848 (95%CI: 0.786-0.909) and 0.867 (95%CI: 0.796-0.939), respectively, showing good predictive ability. The calibration curves showed the correlation between prediction and actual status. The DCA showed that the prediction model was clinically useful within the whole threshold range. CONCLUSION: Low BMD, leakage of bone cement and "O" shaped distribution of bone cement are independent risk factors for AVCF after vertebroplasty. The nomogram prediction model has good predictive ability and clinical benefit.

Integrated Multi-Omics Landscape of Liver Metastases.

BACKGROUND & AIMS: Lack of thorough knowledge about the complicated immune microenvironment (IM) within a variety of liver metastases (LMs) leads to inappropriate treatment and unsatisfactory prognosis. We aimed to characterize IM subtypes and investigate potential mechanisms in LMs. METHODS: Mass cytometry was applied to characterize immune landscape of a primary liver cancers and liver metastases cohort. Transcriptomic and whole-exome sequencing were used to explore potential mechanisms across distinct IM subtypes. Single-cell transcriptomic sequencing, multiplex fluorescent immunohistochemistry, cell culture, mouse model, Western blot, quantitative polymerase chain reaction, and immunohistochemistry were used for validation. RESULTS: Five IM subtypes were revealed in 100 LMs and 50 primary liver cancers. Patients featured terminally exhausted (IM1) or rare T-cell-inflamed (IM2 and IM3) immune characteristics showed worse outcome. Increased intratumor heterogeneity, enriched somatic TP53, KRAS, APC, and PIK3CA mutations and hyperactivated hypoxia signaling accounted for the formation of vicious subtypes. SLC2A1 promoted immune suppression and desert via increasing proportion of Spp1+ macrophages and their inhibitory interactions with T cells in liver metastatic lesions. Furthermore, SLC2A1 promoted immune escape and LM through inducing regulatory T cells, including regulatory T cells and LAG3+CD4+ T cells in primary colorectal cancer. CONCLUSIONS: The study provided integrated multi-omics landscape of LM, uncovering potential mechanisms for vicious IM subtypes and confirming the roles of SLC2A1 in regulating tumor microenvironment remodeling in both primary tumor and LM lesions.

Single-cell atlas of diverse immune populations in the advanced biliary tract cancer microenvironment.

Immunotherapies have been explored in treating solid tumors, albeit with disparate clinical effects in distinct cancer types. Systematic interrogation of immune cells in the tumor microenvironment (TME) is vital to the prediction of immunotherapy response and the development of innovative immunotherapeutics. To comprehensively characterize the immune microenvironment in advanced biliary tract cancer (BTC), we utilized single-cell RNA sequencing in unselected viable cells from 16 matched samples, and identified nineteen cell subsets from a total of 45,851 cells, in which exhausted CD8+ T cells, macrophages, and dendritic cells (DCs) in BTC were shown to augment and communicate within the TME. Transcriptional profiles coupled with T cell receptor (TCR) sequences revealed that exhausted CD8+ T cells retained clonal expansion and high proliferation in the TME, and some of them highly expressed the endoplasmic reticulum stress (ER) response gene, XBP1, indicating the role of ER stress in remodeling TME. Functional assays demonstrated that XBP1 and common immune checkpoints (PD1, TIGIT) were significantly upregulated in CD8+ T cells cocultured within the TME of BTC cells (GBC-SD, HCCC-9810). When treating the coculture groups with the specific inhibitor of IRE1α-XBP1 (4µ8C), the downregulation of TIGIT was observed in the treatment group. Collectively, comprehensive transcriptome profiling provides deep insights into the immune atlas in advanced BTC, which might be instrumental in exploring innovative immunotherapy strategies.

Hepatobiliary Tumor Organoids Reveal HLA Class I Neoantigen Landscape and Antitumoral Activity of Neoantigen Peptide Enhanced with Immune Checkpoint Inhibitors.

Neoantigen-directed therapy lacks preclinical models recapitulating neoantigen characteristics of original tumors. It is urgent to develop a platform to assess T cell response for neoantigen screening. Here, immunogenic potential of neoantigen-peptides of tumor tissues and matched organoids (n = 27 pairs) are analyzed by Score tools with whole genome sequencing (WGS)-based human leukocyte antigen (HLA)-class-I algorithms. The comparisons between 9203 predicted neoantigen-peptides from 2449 mutations of tumor tissues and 9991 ones from 2637 mutations of matched organoids demonstrate that organoids preserved majority of genetic features, HLA alleles, and similar neoantigen landscape of original tumors. Higher neoantigen load is observed in tumors with early stage. Multiomics analysis combining WGS, RNA-seq, single-cell RNA-seq, mass spectrometry filters out 93 candidate neoantigen-peptides with strong immunogenic potential for functional validation in five organoids. Immunogenic peptides are defined by inducing increased CD107aCD137IFN-γ expressions and IFN-γ secretion of CD8 cells in flow cytometry and enzyme-linked immunosorbent assay assays. Nine immunogenic peptides shared by at least two individuals are validated, including peptide from TP53R90S . Organoid killing assay confirms the antitumor activity of validated immunogenic peptide-reactive CD8 cells, which is further enhanced in the presence of immune checkpoint inhibitors. The study characterizes HLA-class-I neoantigen landscape in hepatobiliary tumor, providing practical strategy with tumor organoid model for neoantigen-peptide identification in personalized immunotherapy.

Comprehensive analysis of spatial architecture in primary liver cancer.

Heterogeneity is the major challenge for cancer prevention and therapy. Here, we first constructed high-resolution spatial transcriptomes of primary liver cancers (PLCs) containing 84,823 spots within 21 tissues from seven patients. The progressive comparison of spatial tumor microenvironment (TME) characteristics from nontumor to leading-edge to tumor regions revealed that the tumor capsule potentially affects intratumor spatial cluster continuity, transcriptome diversity, and immune cell infiltration. Locally, we found that the bidirectional ligand-receptor interactions at the 100-µm-wide cluster-cluster boundary contribute to maintaining intratumor architecture and the PROM1+ and CD47+ cancer stem cell niches are related to TME remodeling and tumor metastasis. Last, we proposed a TLS-50 signature to accurately locate tertiary lymphoid structures (TLSs) spatially and unveiled that the distinct composition of TLSs is shaped by their distance to tumor cells. Our study provides previous unknown insights into the diverse tumor ecosystem of PLCs and has potential benefits for cancer intervention.

Discovery of a Carbamoyl Phosphate Synthetase 1-Deficient HCC Subtype With Therapeutic Potential Through Integrative Genomic and Experimental Analysis.

BACKGROUND AND AIMS: Metabolic reprogramming plays an important role in tumorigenesis. However, the metabolic types of different tumors are diverse and lack in-depth study. Here, through analysis of big databases and clinical samples, we identified a carbamoyl phosphate synthetase 1 (CPS1)-deficient hepatocellular carcinoma (HCC) subtype, explored tumorigenesis mechanism of this HCC subtype, and aimed to investigate metabolic reprogramming as a target for HCC prevention. APPROACH AND RESULTS: A pan-cancer study involving differentially expressed metabolic genes of 7,764 tumor samples in 16 cancer types provided by The Cancer Genome Atlas (TCGA) demonstrated that urea cycle (UC) was liver-specific and was down-regulated in HCC. A large-scale gene expression data analysis including 2,596 HCC cases in 7 HCC cohorts from Database of HCC Expression Atlas and 17,444 HCC cases from in-house hepatectomy cohort identified a specific CPS1-deficent HCC subtype with poor clinical prognosis. In vitro and in vivo validation confirmed the crucial role of CPS1 in HCC. Liquid chromatography-mass spectrometry assay and Seahorse analysis revealed that UC disorder (UCD) led to the deceleration of the tricarboxylic acid cycle, whereas excess ammonia caused by CPS1 deficiency activated fatty acid oxidation (FAO) through phosphorylated adenosine monophosphate-activated protein kinase. Mechanistically, FAO provided sufficient ATP for cell proliferation and enhanced chemoresistance of HCC cells by activating forkhead box protein M1. Subcutaneous xenograft tumor models and patient-derived organoids were employed to identify that blocking FAO by etomoxir may provide therapeutic benefit to HCC patients with CPS1 deficiency. CONCLUSIONS: In conclusion, our results prove a direct link between UCD and cancer stemness in HCC, define a CPS1-deficient HCC subtype through big-data mining, and provide insights for therapeutics for this type of HCC through targeting FAO.

M6A Demethylase ALKBH5 Regulates PD-L1 Expression and Tumor Immunoenvironment in Intrahepatic Cholangiocarcinoma.

N6-methyladenosine (m6A) has been reported as an important mechanism of posttranscriptional regulation. Programmed death-ligand 1 (PD-L1) is a primary immune inhibitory molecule expressed on tumor cells that promotes immune evasion. Here we report ALKBH5 as an important m6A demethylase that orchestrates PD-L1 expression in intrahepatic cholangiocarcinoma (ICC). Regulation of PD-L1 expression by ALKBH5 was confirmed in human ICC cell lines. Sequencing of the m6A methylome identified PD-L1 mRNA as a direct target of m6A modification whose levels were regulated by ALKBH5. Furthermore, ALKBH5 and PD-L1 mRNA were shown to interact. ALKBH5 deficiency enriched m6A modification in the 3'UTR region of PD-L1 mRNA, thereby promoting its degradation in a YTHDF2-dependent manner. In vitro and in vivo, tumor-intrinsic ALKBH5 inhibited the expansion and cytotoxicity of T cells by sustaining tumor cell PD-L1 expression. The ALKBH5-PD-L1-regulating axis was further confirmed in human ICC specimens. Single-cell mass cytometry analysis unveiled a complex role of ALKBH5 in the tumor immune microenvironment by promoting the expression of PD-L1 on monocytes/macrophages and decreasing the infiltration of myeloid-derived suppressor-like cells. Analysis of specimens from patients receiving anti-PD1 immunotherapy suggested that tumors with strong nuclear expression patterns of ALKBH5 are more sensitive to anti-PD1 immunotherapy. Collectively, these results describe a new regulatory mechanism of PD-L1 by mRNA epigenetic modification by ALKBH5 and the potential role of ALKBH5 in immunotherapy response, which might provide insights for cancer immunotherapies. SIGNIFICANCE: This study identifies PD-L1 mRNA as a target of ALKBH5 and reveals a role for ALKBH5 in regulating the tumor immune microenvironment and immunotherapy efficacy.

Single-Cell Transcriptome Analysis Uncovers Intratumoral Heterogeneity and Underlying Mechanisms for Drug Resistance in Hepatobiliary Tumor Organoids.

Molecular heterogeneity of hepatobiliary tumor including intertumoral and intratumoral disparity always leads to drug resistance. Here, seven hepatobiliary tumor organoids are generated to explore heterogeneity and evolution via single-cell RNA sequencing. HCC272 with high status of epithelia-mesenchymal transition proves broad-spectrum drug resistance. By examining the expression pattern of cancer stem cells markers (e.g., PROM1, CD44, and EPCAM), it is found that CD44 positive population may render drug resistance in HCC272. UMAP and pseudo-time analysis identify the intratumoral heterogeneity and distinct evolutionary trajectories, of which catenin beta-1 (CTNNB1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and nuclear paraspeckle assembly transcript 1 (NEAT1) advantage expression clusters are commonly shared across hepatobiliary organoids. CellphoneDB analysis further implies that metabolism advantage organoids with enrichment of hypoxia signal upregulate NEAT1 expression in CD44 subgroup and mediate drug resistance that relies on Jak-STAT pathway. Moreover, metabolism advantage clusters shared in several organoids have similar characteristic genes (GAPDH, NDRG1 (N-Myc downstream regulated 1), ALDOA, and CA9). The combination of GAPDH and NDRG1 is an independent risk factor and predictor for patient survival. This study delineates heterogeneity of hepatobiliary tumor organoids and proposes that the collaboration of intratumoral heterogenic subpopulations renders malignant phenotypes and drug resistance.

MgFe-LDH Nanoparticles: A Promising Leukemia Inhibitory Factor Replacement for Self-Renewal and Pluripotency Maintenance in Cultured Mouse Embryonic Stem Cells.

Leukemia inhibitory factor (LIF), an indispensable bioactive protein that sustains self-renewal and pluripotency in stem cells, is vital for mouse embryonic stem cell (mESC) culture. Extensive research is conducted on reliable alternatives for LIF as its clinical application in stable culture and large-scale expansion of ESCs is limited by its instability and high cost. However, few studies have sought to replace LIF with nanoparticles to provide a xeno-free culture condition. MgAl-LDH (layered double hydroxide) nanoparticles can partially replace LIF in maintaining pluripotency of mESCs; however, the requirement and tolerance for aluminum ions in mice are far lesser than those of iron ions. Hence, MgFe-LDH nanoparticles are selected for this study. MgFe-LDH is superior to MgAl-LDH in maintaining self-renewal and pluripotency of mESCs, in the absence of LIF and mouse embryonic fibroblast. Furthermore, combined transcriptomic and proteomic analysis confirms that MgFe-LDH can activate the LIF receptor (LIFR)/phosphatidylinositol 3-kinase (PI3K)/protein kinase B(AKT), LIFR/JAK/janus kinase (JAK)/signal transducer and activator of transcription 3(STAT3), and phospho-signal transducer and activator of transcription 3(p-STAT3)/ten-eleven translocation (TET) signaling pathways, while the extra Fe2+ provided by MgFe-LDH would also enhance TET1/2 abundance thus affecting the TET1/2 regulated pluripotency related marker expression and TET1/2 meditated DNA demethylation. These results suggest that MgFe-LDH nanoparticles can thus be used as an affordable and efficient replacement for LIF in mESC cultivation.

The identification and functional analysis of CD8+PD-1+CD161+ T cells in hepatocellular carcinoma.

Immunotherapy is a powerful therapeutic strategy for end-stage hepatocellular carcinoma (HCC). It is well known that T cells, including CD8+PD-1+ T cells, play important roles involving tumor development. However, their underlying phenotypic and functional differences of T cell subsets remain unclear. We constructed single-cell immune contexture involving approximate 20,000,000 immune cells from 15 pairs of HCC tumor and non-tumor adjacent tissues and 10 blood samples (including five of HCCs and five of healthy controls) by mass cytometry. scRNA-seq and functional analysis were applied to explore the function of cells. Multi-color fluorescence staining and tissue micro-arrays were used to identify the pathological distribution of CD8+PD-1+CD161 +/- T cells and their potential clinical implication. The differential distribution of CD8+ T cells subgroups was identified in tumor and non-tumor adjacent tissues. The proportion of CD8+PD1+CD161+ T cells was significantly decreased in tumor tissues, whereas the ratio of CD8+PD1+CD161- T cells was much lower in non-tumor adjacent tissues. Diffusion analysis revealed the distinct evolutionary trajectory of CD8+PD1+CD161+ and CD8+PD1+CD161- T cells. scRNA-seq and functional study further revealed the stronger immune activity of CD8+PD1+CD161+ T cells independent of MHC class II molecules expression. Interestingly, a similar change in the ratio of CD8+CD161+/ CD8+CD161- T cells was also found in peripheral blood samples collected from HCC cases, indicating their potential usage clinically. We here identified different distribution, function, and trajectory of CD8+PD-1+CD161+ and CD8+PD-1+CD161- T cells in tumor lesions, which provided new insights for the heterogeneity of immune environment in HCCs and also shed light on the potential target for immunotherapy.

Trajectory and Functional Analysis of PD-1high CD4+CD8+ T Cells in Hepatocellular Carcinoma by Single-Cell Cytometry and Transcriptome Sequencing.

The spatial heterogeneity of immune microenvironment in hepatocellular carcinoma (HCC) remains elusive. Here, a single-cell study involving 17 432 600 immune cells of 39 matched HCC (T), nontumor (N), and leading-edge (L) specimens by mass cytometry is conducted. The tumor-associated CD4/CD8 double-positive T (DPT) cells are found enriched in L regions with synergetic expression of PD-1/HLA-DR/ICOS/CD45RO and exhibit a higher level of IFN-γ, TNF-α, and PD-1 upon stimulation. The enrichment of DPT and PD-1+DPT in L regions indicates favorable prognosis. These tumor-associated DPT cells with similar phenotype are also verified in other tumors and HCC animal models. Single-cell RNA-seq further characterizes the molecular features of DPT cells and uncovers 11 clusters with different cytotoxicity, exhaustion, and activation scores. TCR-based trajectory analysis reveals that tumor-associated DPT clusters share separated ancestries with local CD4+ or CD8+SPT cells rather than CD3+PBMC cells. TCR clones with frequency above 10 are mainly found coexisting in DPT and CD8+SPT cells. Specifically, PD-1highDPT cluster (TDPT_10) shares the same ancestry with exhausted CD8+SPT cluster (TCD8T_2) and shows higher expression similarity and closer pathological location to PD-1+CD8+ than PD-1+CD4+T cells. Together, this study systematically characterizes the unique distribution of PD-1+DPTs in HCC and puts forward new insights for the function and origin of tumor-associated DPT cells.

Solid Lipid Nanoparticles Enhanced the Neuroprotective Role of Curcumin against Epilepsy through Activation of Bcl-2 Family and P38 MAPK Pathways.

Oxidative stress of neurons caused by a series of complex neuropathological processes will induce certain neurodegenerative disorders including epilepsy. Curcumin (Cur) is an effective natural antioxidant compound; however, the poor bioavailability obstructs its neural protective applications. In this study, Cur is encapsulated in solid lipid nanoparticles (SLNs) for better neuroprotective efficacy. In vitro study certified that Cur-SLNs functioned obviously better against neuronal apoptosis than Cur, by significantly decreasing the level of free radical and reversing mitochondrial function through the activation of the Bcl-2 family. In vivo experiments showed that SLNs transported Cur through the blood-brain barrier (BBB). The behavioral performance of epileptic mice was improved by Cur-SLNs, with more NeuN but less TUNEL positive cells observed in hippocampus. The in vivo mechanism was also explored. Cur-SLNs reduced neuronal apoptosis through Bcl2 family and P38 MAPK pathways. Overall, Cur-SLNs have better protective effects toward oxidative stress in neurons than free Cur both in vitro and in vivo, which suggests they may be a promising agent against neurodegenerative disorders including epilepsy.

Targeted sequencing reveals the mutational landscape responsible for sorafenib therapy in advanced hepatocellular carcinoma.

Rationale: The existence of primary and acquired drug resistance is the main obstacle for the effect of multi-kinase inhibitor sorafenib and regorafenib in advanced hepatocellular carcinoma (HCC). However, plenty of patients did not significantly benefit from sorafenib treatment and little is known about the mechanism of drug resistance. Methods: Laser capture microdissection was used to acquire matched normal liver and tumor tissues on formalin-fixed paraffin-embedded specimens collected before sorafenib therapy from the first surgery of 119 HCC patients. Ultra-deep sequencing (~1000×) targeting whole exons of 440 genes in microdissected specimens and siRNA screen in 7 cell lines were performed to find mutations associated with differential responses to sorafenib. Patient-derived xenograft models were employed to determine the role of TP53 in response to sorafenib. Lentiviruses harboring wild-type and c.G52C-mutant OCT4 were applied to explore the function of OCT4 in resistance to sorafenib. ChIP-PCR assay for analysis of OCT4 transcriptional activity was performed to explore the affinity with the KITLG promoter. Statistical analyses were used to associate levels of p53 and OCT4 with tumor features and patient outcomes. Results: Total 1,050 somatic mutations and 26 significant driver genes were identified. SiRNA screening in 7 HCC cell lines was further performed to identify mutations associated with differential responses to sorafenib. A recurrent nonsynonymous mutation c.G52C in OCT4 (OCT4mut) was strongly associated with good response to sorafenib, whereas the stop-gain mutation in TP53 showed the opposite outcome both in vitro and in vivo. OCT4wt-induced stem cell factor (encoded by KITLG gene, SCF) expression and cross-activation of c-KIT/FLT3-BRAF signals were identified indispensably for sorafenib resistance, which could be reversed by the combination of c-KIT tyrosine kinase inhibitors or neutralizing antibody against SCF. Mechanistically, an OCT4 binding site in upstream of KITLG promoter was identified with a higher affinity to wildtype of OCT4 rather than G52C-mutant form, which is indispensable for OCT4-induced expression of KITLG and sorafenib resistance. Conclusion: Our study reported a novel somatic mutation in OCT4 (c.G52C) responsible for the sorafenib effect, and also shed new light on the treatment of HCC through the combination of specific tyrosine kinase inhibitors according to individual genetic patterns.

LncRNA MALAT1 inhibits hypoxia/reoxygenation-induced human umbilical vein endothelial cell injury via targeting the microRNA-320a/RAC1 axis.

Angiogenesis is believed to protect against hypoxia/reoxygenation (H/R)-induced cell injury. MALAT1 and microRNA-320a (miR-320a) are involved in cancer angiogenesis. To investigate the function of the MALAT1/miR-320a axis in H/R-induced cell injury, human umbilical vein endothelial cell (HUVEC) angiogenesis was detected using the Cell Counting Kit-8 (CCK-8), Transwell migration, cell adhesion and tube formation assays. The expression of MALAT1 and miR-320a was revealed by quantitative reverse transcription polymerase chain reaction (qRT-PCR). The direct binding relationship between miR-320a and MALAT1 was detected by RNA immunoprecipitation (RIP) and dual luciferase reporter assays. The data indicated that H/R induces angiogenesis injury and that the expression of MALAT1 was augmented in H/R-stimulated HUVECs. Overexpression of MALAT1 alleviated H/R-stimulated HUVEC dysfunction, whereas silencing of MALAT1 exerted the opposite effects. MALAT1 also reduced miR-320a levels in HUVECs. Overexpression of miR-320a repressed the function of MALAT1 on H/R-stimulated HUVECs, whereas inhibition of miR-320a exerted the opposite effect. Additionally, miR-320a inhibition alleviated H/R-stimulated HUVEC injury via RAC1. Taken together, this investigation concluded that MALAT1 represses H/R-stimulated HUVEC injury by targeting the miR-320a/RAC1 axis.

Integrative molecular analysis of metastatic hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is the major type of primary liver cancer. Intrahepatic metastasis, such as portal vein tumor thrombosis (PVTT), strongly indicates poor prognosis of HCC. But now, there are limited understandings of the molecular features and mechanisms of those metastatic HCCs. METHODS: To characterize the molecular alterations of the metastatic HCCs, we implemented an integrative analysis of the copy number variations (CNVs), DNA methylations and transcriptomes of matched adjacent normal, primary tumor and PVTT samples from 19 HCC patients. RESULTS: CNV analysis identified a frequently amplified focal region chr11q13.3 and a novel deletion peak chr19q13.41 containing three miRNAs. The integrative analysis with RNA-seq data suggests that CNVs and differential promoter methylations regulate distinct oncogenic processes. Then, we used individualized differential analysis to identify the differentially expressed genes between matched primary tumor and PVTT of each patient. Results show that 5 out of 19 studied patients acquire evidential progressive alterations of gene expressions (more than 1000 differentially expressed genes were identified in each patient). While, another subset of eight patients have nearly identical gene expressions between the corresponding matched primary tumor and PVTT. Twenty genes were found to be recurrently and progressively differentially expressed in multiple patients. These genes are mainly associated with focal adhesion, xenobiotics metabolism by cytochrome P450 and amino acid metabolism. For several differentially expressed genes in metabolic pathways, their expressions are significantly associated with overall survivals and vascular invasions of HCC patients. The following transwell assay experiments validate that they can regulate invasive phenotypes of HCC cells. CONCLUSIONS: The metastatic HCCs with PVTTs have significant molecular alterations comparing with adjacent normal tissues. The recurrent alteration patterns are similar to several previously published general HCC cohorts, but usually with higher severity. By an individualized differential analysis strategy, the progressively differentially expressed genes between the primary tumor and PVTT were identified for each patient. A few patients aquire evidential progressive alterations of gene expressions. And, experiments show that several recurrently differentially expressed genes can strongly regulate HCC cell invasions.