HepG2.2.15-derived exosomes facilitate the activation and fibrosis of hepatic stellate cells.

BACKGROUND: The role of exosomes derived from HepG2.2.15 cells, which express hepatitis B virus (HBV)-related proteins, in triggering the activation of LX2 liver stellate cells and promoting liver fibrosis and cell proliferation remains elusive. The focus was on comprehending the relationship and influence of differentially expressed microRNAs (DE-miRNAs) within these exosomes. AIM: To elucidate the effect of exosomes derived from HepG2.2.15 cells on the activation of hepatic stellate cell (HSC) LX2 and the progression of liver fibrosis. METHODS: Exosomes from HepG2.2.15 cells, which express HBV-related proteins, were isolated from parental HepG2 and WRL68 cells. Western blotting was used to confirm the presence of the exosomal marker protein CD9. The activation of HSCs was assessed using oil red staining, whereas DiI staining facilitated the observation of exosomal uptake by LX2 cells. Additionally, we evaluated LX2 cell proliferation and fibrosis marker expression using 5-ethynyl-2'-deoxyuracil staining and western blotting, respectively. DE-miRNAs were analyzed using DESeq2. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were used to annotate the target genes of DE-miRNAs. RESULTS: Exosomes from HepG2.2.15 cells were found to induced activation and enhanced proliferation and fibrosis in LX2 cells. A total of 27 miRNAs were differentially expressed in exosomes from HepG2.2.15 cells. GO analysis indicated that these DE-miRNA target genes were associated with cell differentiation, intracellular signal transduction, negative regulation of apoptosis, extracellular exosomes, and RNA binding. KEGG pathway analysis highlighted ubiquitin-mediated proteolysis, the MAPK signaling pathway, viral carcinogenesis, and the toll-like receptor signaling pathway, among others, as enriched in these targets. CONCLUSION: These findings suggest that exosomes from HepG2.2.15 cells play a substantial role in the activation, proliferation, and fibrosis of LX2 cells and that DE-miRNAs within these exosomes contribute to the underlying mechanisms.

Methylated SEPT9 combined with AFP and PIVKA-II is effective for the detection of HCC in high-risk population.

BACKGROUND: The methylation SEPT9 (mSEPT9) appeared to be effective for hepatocellular carcinoma (HCC) detection. However, its performance in high-risk population has not been validated. We designed a pilot study and aimed to investigate the performance of mSEPT9, AFP, PIVKA-II and their combination in hepatic cirrhosis (HC) population. METHODS: A training cohort was established including 103 HCC and 114 HC patients. 10 ml blood was collected from each patient with K2EDTA tubes, and 3-4 ml plasma was extracted for subsequent tests. The performance of mSEPT9, AFP, PIVKA-II and their combination was optimized by the training cohort. Test performance was prospectively validated with a validation cohort, including 51 HCC and 121 HC patients. RESULTS: At the optimal thresholds in the training cohort, the sensitivity, specificity and area under curve (AUC) was 72.82%, 89.47%, 0.84, and 48.57%, 89.92%, 0.79, and 63.64%, 95.95%, 0.79 for mSEPT9, AFP and PIVKA-II, respectively. The combined test significantly increased the sensitivity to 84.47% (P < 0.05) at the specificity of 86.84% with an AUC of 0.91. Stage-dependent performance was observed with all single markers and their combination in plasma marker levels, positive detection rate (PDR) and AUC. Moderate correlation was found between mSEPT9 and AFP plasma levels (r = 0.527, P < 0.0001). Good complementarity was found between any two of the three markers, providing optimal sensitivity in HCC detection when used in combination. Subsequent validation achieved a sensitivity, specificity and AUC of 65.31%, 92.86%, 0.80, and 44.24%, 89.26%, 0.75, and 62.22%, 95.27%, 0.78 for mSEPT9, AFP and PIVKA-II, respectively. The combined test yielded a significantly increased sensitivity of 84.00% (P < 0.05) at 85.57% specificity, with an AUC at 0.89. CONCLUSIONS: The performance was optimal by the combination of mSEPT9, AFP, PIVKA-II compared with any single marker, and the combination may be effective for HCC opportunistic screening in HC population.

Analysis of angiogenesis-related subtypes of hepatocellular carcinoma and tumor microenvironment infiltration feature in hepatocellular carcinoma

Purpose Hepatocellular carcinoma (HCC) is a highly vascularized tumor, and angiogenesis plays an important role in its progression. However, the role of angiogenesis in cell infiltration in the tumor microenvironment (TME) remains unclear. Methods We evaluated the associations of 35 angiogenesis-related genes (ARGs) with the clinicopathological features of 816 HCC patients. In addition, we assessed the associations between the ARGs and TME cell infiltration. A nomogram was constructed to determine the prognostic value of ARGs for HCC. The ARG score was used to distinguish angiogenic subtypes of HCC, and its usefulness for predicting the prognosis and treatment response of HCC patients was evaluated. Results We distinguished three ARG clusters differing in terms of TME cell infiltration, immune cell activation status, clinicopathological features, and clinical outcomes. There were significant associations of ARG expression with tumor immunity, the epithelial–mesenchymal transition (EMT), and transforming growth factor-β expression. An ARG score model was constructed to generate a risk score for each patient based on differentially expressed genes between clusters. Furthermore, a high ARG score was associated with high expression of CTLA-4 and PD-L1/PD-1, and a low Tumor Immune Dysfunction and Exclusion score, indicating the usefulness of the ARG score for selecting patients for immunotherapy. Considering the relationship between ARGs and tumor immunity, immunotherapy combined with vascular-targeted therapy may be the best treatment for HCC. Conclusions ARGs play an important role in TME diversity and complexity in HCC patients. The ARG score of HCC predicts TME invasion and can guide immunotherapy (AU)

miR-21-5p Inhibits Ferroptosis in Hepatocellular Carcinoma Cells by Regulating the AKT/mTOR Signaling Pathway through MELK.

Background: Hepatocellular carcinoma (HCC) is one of the most prevalent cancers, and its incidence rate is increasing worldwide. At present, there is no ideal treatment for HCC. In recent years, molecular-targeted therapy has shown significant therapeutic benefits for patients. Ferroptosis is a modality of regulated cell death, and previous studies have found that inducing ferroptosis in liver cancer cells can inhibit the progression of liver cancer. The aim of this study is to investigate the regulatory mechanism of miR-21-5p in regulating ferroptosis in HCC cells. Methods: CCK-8 was used to measure cell viability, EdU and colony formation were used to measure cell proliferation, and Transwell assays were used to measure cell migration and invasion. RT-qPCR was used to detect the level of miR-21-5p, Western blotting was used to detect the protein expression level, a dual-luciferase reporter gene assay was used to determine the targeting relationship between miR-21-5p and MELK, and coimmunoprecipitation was used to determine the interaction between MELK and AKT. Results: Overexpression of miR-21-5p and MELK facilitated the viability, proliferation, colony formation, invasion, and migration of HCC cells. Downregulation of miR-21-5p suppressed the level of MELK and the progression of HCC. MELK regulated the AKT/mTOR signaling pathway, causing changes in the levels of GPX4, GSH, FTH1, xCT, heme oxygenase 1(HO-1), reactive oxygen species, and Fe2+ to regulate the ferroptosis of hepatoma cells. Erastin, an inducer of ferroptosis, attenuated the repressive influence of miR-21-5p on ferroptosis in HCC cells. Conclusion: In summary, this study demonstrates that miR-21-5p inhibits the ferroptosis of HCC cells by regulating the AKT/mTOR signaling pathway through MELK.

Analysis of angiogenesis-related subtypes of hepatocellular carcinoma and tumor microenvironment infiltration feature in hepatocellular carcinoma.

PURPOSE: Hepatocellular carcinoma (HCC) is a highly vascularized tumor, and angiogenesis plays an important role in its progression. However, the role of angiogenesis in cell infiltration in the tumor microenvironment (TME) remains unclear. METHODS: We evaluated the associations of 35 angiogenesis-related genes (ARGs) with the clinicopathological features of 816 HCC patients. In addition, we assessed the associations between the ARGs and TME cell infiltration. A nomogram was constructed to determine the prognostic value of ARGs for HCC. The ARG score was used to distinguish angiogenic subtypes of HCC, and its usefulness for predicting the prognosis and treatment response of HCC patients was evaluated. RESULTS: We distinguished three ARG clusters differing in terms of TME cell infiltration, immune cell activation status, clinicopathological features, and clinical outcomes. There were significant associations of ARG expression with tumor immunity, the epithelial-mesenchymal transition (EMT), and transforming growth factor-ß expression. An ARG score model was constructed to generate a risk score for each patient based on differentially expressed genes between clusters. Furthermore, a high ARG score was associated with high expression of CTLA-4 and PD-L1/PD-1, and a low Tumor Immune Dysfunction and Exclusion score, indicating the usefulness of the ARG score for selecting patients for immunotherapy. Considering the relationship between ARGs and tumor immunity, immunotherapy combined with vascular-targeted therapy may be the best treatment for HCC. CONCLUSIONS: ARGs play an important role in TME diversity and complexity in HCC patients. The ARG score of HCC predicts TME invasion and can guide immunotherapy.

Integrated Bioinformatics Analysis for the Screening of Hub Genes and Therapeutic Drugs in Hepatocellular Carcinoma.

BACKGROUND: Liver cancer is a major medical problem because of its high morbidity and mortality. Hepatocellular carcinoma (HCC) is the most common type of liver cancer. Currently, the mechanism of HCC is unclear, and the prognosis is poor with limited treatment. OBJECTIVE: The purpose of this study is to identify hub genes and potential therapeutic drugs for HCC. METHODS: We used the GEO2R algorithm to analyze the differential expression of each gene in 4 gene expression profiles (GSE101685, GSE62232, GSE46408, and GSE45627) between HCC and normal hepatic tissues. Next, we screened out the differentially expressed genes (DEGs) by corresponding calculation data according to adjusted P-value < 0.05 and | log fold change (FC) | > 1.0. Subsequently, we used the DAVID software to analyze the DEGs by GO and KEGG enrichment analysis. Then, we carried out the protein-protein interaction (PPI) network analysis of DEGs using the STRING tool, and the PPI network was constructed by Cytoscape software. MCODE plugin was used for module analysis, and the hub genes were screened out by the Cyto- Hubba plugin. Meanwhile, we used The Kaplan-Meier plotter, GEPIA2 and HPA databases to exert survival analysis and verify the expression alternation of hub genes. Furthermore, we used ENCORI, TargetScan, miRDB and miRWalk database to predict the upstream regulated miRNA of hub genes and construct a miRNA-hub genes network by Cytoscape software. Finally, we selected potential therapeutic drugs for HCC through DGIdb databases. RESULTS: A total of 415 DEGs were screened in HCC, including 196 up-regulated DEGs and 219 down-regulated DEGs. The results of KEGG pathway analysis suggested that the up-regulated DEGs can regulate the cell cycle, and DNA replication signal pathway, while the down-regulated DEGs were associated with metabolic pathways. In this study, we identified 11 hub genes (AURKA, BUB1B, TOP2A, MAD2L1, CCNA2, CCNB1, BUB1, KIF11, CDK1, CCNB2 and TPX2), which were independent risk factors of HCCand all up-regulated DEGs. We verified the expression difference of hub genes through the GEPIA2 and HPA database, which was consistent with the results of GEO data. We found that those hub genes were mutations in HCC according to the cBioPortal database. Finally, we used the DGIdb database to select 32 potential therapeutic targeting drugs for hub genes. CONCLUSION: In summary, our study provided a new perspective for researching the molecular mechanism of HCC. Hub genes, miRNAs, and candidate drugs provide a new direction for the early diagnosis and treatment of HCC.

CD97 serves as a novel biomarker of immune cell infiltration in hepatocellular carcinoma.

BACKGROUND: CD97 is the most widely expressed G protein-coupled receptor in the epidermal growth factor seven-span transmembrane family. It plays a vital role in cell adhesion, migration, and cell connection regulation. We explored the role of CD97 in hepatocellular carcinoma (HCC). METHODS: We evaluated CD97 mRNA expression in HCC using TNMplot and the Gene Expression Omnibus database. The clinical prognostic significance of CD97 in HCC patients was evaluated by gene expression profiling interactive analysis, the Kaplan-Meier plotter, and the UALCAN database. The Tumor Immune Estimation Resource (TIMER) and CIBERSORT databases were used to analyze the relationships among CD97, genes positively related with CD97, and tumor-infiltrating immune cells. RESULTS: CD97 was highly expressed in HCC tissues and was associated with an adverse prognosis. CD97 and genes positively related with CD97 were positively correlated with the abundance of tumor-infiltrating immune cells and strongly correlated with tumor-infiltrating macrophages (all r ≥ 0.513, P < 0.001). CD97 was positively correlated with M2 macrophage and tumor-associated macrophage markers (both r ≥ 0.464, P < 0.001). CD97 was found to be an immune-related gene in HCC and positively correlated with the TOX, PD-L1, PD-L2, CTLA4, and PD-1 immune checkpoint genes. CD97 copy number alterations affect the level of immune cell infiltration and mRNA expression. CONCLUSIONS: CD97 can be used as a potential molecular marker of prognosis in HCC, which is associated with immune cell infiltration.

An injectable pH neutral bioactive glass-based bone cement with suitable bone regeneration ability.

Background: As a class of promising bone augmentation materials, bone cements have attracted particular attention. Due to various limitations, the current bone cements are still imperfect. In this study, an injectable pH neutral bioactive bone cement (PSC/CSC) was developed by mixing phosphosilicate bioactive glass (PSC) and α-calcium sulfate hemihydrate (CSH), with the goal of optimizing bone defects repairs. Methods: A range of compositions (PSC/CSC: 10P/90C, 30P/70C, 50P/50C) were developed and their physicochemical properties evaluated. Their bone regeneration ability was compared to those of two widely used bone cements as controls (calcium phosphate cement (CPC) and Genex®) in rabbit femoral condyle bone defect models for 4, 8 and 12 weeks. Based on physicochemical properties and in vivo bone regeneration ability, the PSC/CSC exhibited the best outcomes was selected. Then, in vitro, the effects of selected PSC/CSC, CPC and Genex® extracts on MC3T3-E1 cell proliferation, migration and osteogenesis as well as angiogenesis of HUVECs were examined. Results: Based on physicochemical properties, the 30P/70C formula exhibited suitable operability and compressive strength (3.5 ±â€¯0.3 MPa), which fulfilled the requirements for cancellous bone substitutes. In vivo, findings from micro-CT and histological analyses showed that the 30P/70C formula better promoted bone regeneration, compared to 10P/90C, 50P/50C, CPC and Genex®. Hence, 30P/70C was selected as the ideal PSC-based cement. In vitro, the 30P/70C extracts showed better promotion of cell viability, alkaline phosphatase (ALP) activity, calcium mineral deposition, mRNA and protein expression levels of osteogenesis in MC3T3-E1 cells, further supporting its superiority. Meanwhile, the 30P/70C extracts also showed better stimulation of HUVECs proliferation and angiogenesis. Conclusion: The new composite cement, 30P/70C, is a favorable bioactive glass-based bone cement with suitable operability, compressive strength and bone regeneration ability. The translational potential of this article: Clinically, treatment of large bone defects is still a major challenge for orthopaedic trauma. We showed that 30P/70C has the potential to be clinically used as an injectable cement for rapid bone repairs and reconstruction of critical sized bone defects.

mTORC1 coordinates NF-κB signaling pathway to promote chondrogenic differentiation of tendon cells in heterotopic ossification.

Heterotopic ossification (HO) is a pathological bone formation based on endochondral ossification distinguished by ossification within muscles, tendons, or other soft tissues. There has been growing studies focusing on the treatment with rapamycin to inhibit HO, but the mechanism of mTORC1 on HO remains unclear. Tendon cells (TDs) are the first cells to form during tendon heterotopic ossification. Here, we used an in vivo model of HO and an in vitro model of chondrogenesis induction to elucidate the effect and underlying mechanism of mTORC1 in HO. The current study highlights the effect of rapamycin on murine Achilles tenotomy-induced HO and the role of mTORC1 signaling pathway on TDs. Our result showed that mTORC1 was activation in the early stage of HO, whereas the mTORC1 maintained low expression in the mature ectopic cartilage tissue and the ectopic bone formation sites. The use of mTORC1-specific inhibitor (rapamycin) immediately after Achilles tendon injury could suppress the formation of HO; once ectopic cartilage and bone had formed, treatment with rapamycin could not significantly inhibit the progression of HO. Mechanistically, mTORC1 stimulation by silencing of TSC1 promoted the expression of the chondrogenic markers in TDs. In TDs, treated with mTORC1 stimulation by silencing of TSC1, mTORC1 increased the activation of the NF-κB signaling pathway. NF-κB selective inhibitor BAY11-7082 significantly suppressed the chondrogenesis of TDs that treated with mTORC1 stimulation by silencing of TSC1. Together, our findings demonstrated that mTORC1 promoted HO by regulating TDs chondrogenesis partly through the NF-κB signaling pathway; and rapamycin could be a viable HO therapeutic regimen.

Analysis and identification of potential key genes in hepatic ischemia-reperfusion injury.

Background: Hepatic ischemia-reperfusion injury (HIRI) is an unavoidable surgical complication after liver transplantation, but current HIRI treatments cannot achieve satisfactory clinical outcomes. Thus, safer and more effective prevention and treatment methods need to be explored. Methods: Transcriptome messenger ribonucleic acid (mRNA) and long non-coding RNA (lncRNA) sequencing data were obtained from male Sprague-Dawley rats, and these data were used to identify the differentially expressed genes (DEGs) and differentially expressed lncRNAs (DE-lncRNAs) between the HIRI and control samples. A protein-protein interaction (PPI) network was also constructed for the DE-mRNAs to identify candidate genes, and the receiver operating characteristic curves of the 21 candidate genes were plotted to evaluate the diagnostic value of the candidate genes for HIRI. A random forest (RF) model, support vector machine model and generalized linear model were constructed based on the candidate genes. A gene set enrichment analysis (GSEA) of the key genes was conducted to determine the enriched pathways in the high expression groups. The miRWalk and miRanda database were used to constructed the lncRNA-miRNA-mRNA network. Finally, the expressions of the key genes were verified by quantitative real-time polymerase chain reaction (qRT-PCR). Results: A total of 256 DEGs and 67 DE-lncRNAs were identified in the HIRI and control samples. To explore the interactions between the DE-mRNAs, a PPI network of 130 DEGs was constructed. Further, 21 genes were selected as the candidate genes. Subsequently, 6 genes [i.e., Keratin-14 (Krt14), Uroplakin 3B (Upk3b), Keratin 7 (Krt7), Cadherin 3 (Cdh3), mesothelin (Msln), and Glypican 3 (Gpc3)] in the RF model were defined as the key genes. The GSEA results indicated that these key genes were enriched in the terms of extracellular structure organization, and extracellular matrix organization. Moreover, a lncRNA-miRNA-mRNA network was constructed with 4 lncRNAs, 5 mRNAs, and 11 miRNAs. Finally, the results indicated that the expression of Krt14, Upk3b, Msln, and Gpc3 were more highly expressed in the control samples than the HIRI samples. Conclusions: A total of 6 key genes (i.e., Krt14, Upk3b, Krt7, Cdh3, Msln, and Gpc3) were identified. Our findings provide novel ideas for the diagnosis and treatment of HIRI.

Minimally invasive far lateral debridement combined with posterior instrumentation for thoracic and lumbar tuberculosis without severe kyphosis.

BACKGROUND: Anti-tuberculous therapy (ATT) alone cannot easily cure spine tuberculosis (STB) though it is the most essential treatment. Many studies have confirmed the efficacy of the surgical treatment of STB through anterior, anterolateral, posterior debridement, and intervertebral fusion or combined with internal fixation. However, the conventional surgical approach requires extensive exposure of the affected areas with high rates of morbidity and mortality. Recently, minimally invasive surgery has come into use to reduce iatrogenic trauma and relevant complications. Here, we introduced a novel technique for the treatment of thoracic and lumbar spine tuberculosis: minimally invasive far lateral debridement and posterior instrumentation (MI-FLDPI). In this study, we evaluated the technical feasibility, the clinical outcomes, and the postoperative complications. METHODS: We did a prospective, non-randomized study on this new technique. Twenty three patients (13 males) with thoracic or lumbar spine tuberculosis who underwent minimally invasive far lateral debridement and posterior instrumentation were included in the study. The preoperative comorbidities, operation duration, intra-operative hemorrhage, Cobb's angles, and postoperative complications were recorded and analyzed. Clinical outcomes were evaluated by Visual Analog Scale (VAS), Oswestry Disability Index (ODI), neurological recovery, and eradication of tuberculosis. Radiological outcomes were evaluated by changes in Cobb's angle and fusion status of the affected segments. RESULTS: The patients were followed for an average of 19 months (ranging from 12 to 36 months). At the final follow-up, CRP and ESR of all patients were normal. The VAS and ODI were significantly improved compared with preoperative values (P < 0.05). No evident progression of the kyphotic deformity was found after surgery. Twenty two patients showed spontaneous peripheral interbody fusion 1 year after surgery. There were no failure of the instrumentation even though a young female with drug-resistant tuberculosis showed no sign of interbody fusion at the third year follow-up. All the patients with preoperative neurological deficit showed complete recovery at the final follow-up. CONCLUSIONS: MI-FLDPI using expandable tubular retractor could be recommended to treat thoracic and lumbar spine tuberculosis for the advantages of less trauma, earlier recovery, and less complications. Spontaneous peripheral interbody fusion was observed in nearly all the cases even without bone grafting.

Prolonged warm ischemia aggravates hepatic mitochondria damage and apoptosis in DCD liver by regulating Ca2+/CaM/CaMKII signaling pathway.

This study was conducted to investigate the effect of warm ischemia duration on hepatocyte mitochondrial damage after liver transplantation, and confirm the role of CaMKIIγ in this process. Rat donation after cardiac death (DCD) liver transplantation model was established by exposing donor liver to 0 (W0 group), 15 (W15 group), and 30 (W30 group) min warm ischemia. Some rats in W15 group were transfected with CaMKIIγ and CaMKIIγ-shRNA lentivirus. On day 1, 3, and 7 post-transplantation, a series of experiments, including HE staining, TEM observation, ALT and AST measurement, flow cytometry analysis, qRT-PCR, and Western blotting were performed to evaluate the extent of hepatic and mitochondria damage. Within 7 days post-transplantation, prolonged ischemia led to an obvious deterioration of hepatic and mitochondria damage, presenting with a marked increase of apoptotic hepatocytes, ALT and AST levels, cells with low MMP, and AIF and Cyt C expression. CaMKIIγ overexpression caused the significant ultrastructural damage of hepatic cells, increase of cells with low MMP, enhancement of AIF and Cyt C expression, and augmented Ca2+/CaM/CaMKIIγ, while blocking CaMKIIγ showed an opposite result. In conclusion, ischemia duration is proportional to the extent of hepatic mitochondria damage, and CaMKIIγ plays a negative regulatory role in this process by regulating the Ca2+/CaM/CaMKII signaling pathway.

Repair of rabbit femoral condyle bone defects with injectable nanohydroxyapatite/chitosan composites.

Repair of massive bone loss remains a challenge to the orthopaedic surgeons. Autologous and allogenic bone grafts are choice for bone reconstructive surgery, but limited availability, risks of transmittable diseases and inconsistent clinical performances have prompted the development of tissue engineering. In the present work, the bone regeneration potential of nanohydroxyapatite/chitosan composite scaffolds were compared with pure chitosan scaffolds when implanted into segmental bone defects in rabbits. Critical size bone defects (6 mm diameter, 10 mm length) were created in the left femoral condyles of 43 adult New Zealand white rabbits. The femoral condyle bone defects were repaired by nanohydroxyapatite/chitosan compositions, pure chitosan or left empty separately. Defect-bridging was detected by plain radiograph and quantitative computer tomography at eight and 12 weeks after surgery. Tissue samples were collected for gross view and histological examination to determine the extent of new bone formation. Eight weeks after surgery, more irregular osteon formation was observed in the group treated with nanohydroxyapatite/chitosan composites compared with those treated with pure chitosan. 12 weeks after surgery, complete healing of the segmental bone defect was observed in the nanohydroxyapatite/chitosan-group, while the defect was still visible in the chitosan-group, although the depth of the defect had diminished. These observations suggest that the injectable nanohydroxyapatite/chitosan scaffolds are potential candidate materials for regeneration of bone loss.