Ligustrazine promotes hypoxia/reoxygenation-treated trophoblast cell proliferation and migration by regulating the microRNA-27a-3p/ATF3 axis.

OBJECTIVE: Preeclampsia (PE) is a pregnancy-specific syndrome. Ligustrazine (LSZ) is involved in hypoxia/reoxygenation (H/R)-treated trophoblast cell regulation, but its mechanism remains elusive. This study explored the mechanism of LSZ in H/R-treated trophoblast cells to provide a theoretical basis for the new treatment method development for PE. METHODS: H/R HTR8/SVneo cell model was established for PE simulation to some extent. Trophoblast cell proliferation, apoptosis rate, migration, and invasion were detected by MTT assay, flow cytometry, scratch test, and Transwell assay. miR-27a-3p expression in trophoblast cells was detected by RT-qPCR. Binding sites between miR-27a-3p and ATF3 were predicted using Starbase and verified by dual-luciferase reporter assay. Activating transcription factor 3 (ATF3), ß-catenin, Cyclin D1, and c-Myc protein levels were examined using Western blot. After LSZ treatment, H/R-induced HTR8/SVneo cells were delivered with miR-27a-3p mimic or ATF3 siRNA to verify their roles in HTR8/SVneo cells. RESULTS: LSZ facilitated the proliferation, migration, and invasion of trophoblast cells and inhibited apoptosis. miR-27a-3p was elevated in H/R-induced HTR8/SVneo cells and miR-27a-3p overexpression annulled the effect of LSZ on trophoblast cells. miR-27a-3p targeted ATF3. ATF3 silencing averted the property of LSZ on trophoblast cells. Wnt/ß-catenin pathway-related proteins were repressed in H/R-induced HTR8/SVneo cells, and LSZ activated the Wnt/ß-catenin pathway by promoting ATF3 expression. CONCLUSION: LSZ mediated the Wnt pathway by regulating the miR-27a-3p/ATF3 axis, thus promoting the proliferation and migration of trophoblast cells. The protective mechanism of LSZ showed the potential application value in the treatment of PE.

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.

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.

Gambogic acid induces autophagy and combines synergistically with chloroquine to suppress pancreatic cancer by increasing the accumulation of reactive oxygen species.

BACKGROUND: Gambogic acid is a natural component isolated from gamboge that possesses anticancer properties. Our previous study suggested that gambogic acid might be involved in autophagy; however, its role in pancreatic cancer remained unclear. METHODS: Cell viability and apoptosis of pancreatic cancer cell lines were determined using (4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan and flow cytometry. The effects of gambogic acid on autophagy was assessed by western blot, acridine orange staining, transmission electron microscopy, and measurement of autophagic flux through RFP-GFP-LC3 lentiviral transfection. The mitochondrial membrane potential was assessed by JC-1 staining. The production of reactive oxygen species was measured using CM-H2DCFDA staining. A xenograft tumor model of pancreatic cancer was created to determine the efficacy of gambogic acid and chloroquine. RESULTS: Gambogic acid induced the expression of LC3-II and Beclin-1 proteins in pancreatic cancer cells, whereas the expression of P62 showed a decline. Gambogic acid also increased the formation of both acidic vesicular organelles and autophagosomes, and increased autophagic flux. These findings indicated that gambogic acid induced the autophagic process. Furthermore, inhibition of autophagy by chloroquine or 3-methyladenine, or knockdown of Atg-7 all enhanced the cytotoxicity of gambogic acid, suggesting that gambogic acid-induced autophagy improves the survival of pancreatic cancer cells. Moreover, gambogic acid reduced the mitochondrial membrane potential and promoted ROS production, which contributed to the activation of autophagy. The inhibition of autophagy by chloroquine further reduced the mitochondrial membrane potential and increased the accumulation of ROS. This indicated that the inhibition of autophagy could mitigate the cellular protective effects induced by gambogic acid. The treatment combination of gambogic acid and chloroquine synergistically inhibited tumor growth in the xenograft tumor model. CONCLUSIONS: These results demonstrate that gambogic acid induces cytoprotective autophagy in pancreatic cancer cells. The inhibition of autophagy promotes the cytotoxicity of gambogic acid by increasing the accumulation of ROS in pancreatic cancer cells. Combining chloroquine and gambogic acid may be a promising treatment for pancreatic cancer.

Slow resolution of inflammation in severe adult dengue patients.

BACKGROUND: The pathogenesis of severe dengue has not been fully elucidated. The inflammatory response plays a critical role in the outcome of dengue disease. METHODS: In this study, we investigated the levels of 17 important inflammation mediators in plasma collected from mild or severe adult dengue patients at different time points to understand the contribution of inflammation to disease severity and to seek experimental evidence to optimize the existing clinical treatment strategies. Patients were simply classified as mild and severe dengue according to the 2009 WHO classification. Plasma was collected on day 3-5, 6-7, 8-10 and 14-17 of illness. Levels of 17 inflammation mediators including TNF-α, IL-1α, IFN-γ, IL-6, IFN-α, MIF, IL-10, IL-1RA, IL-8, IP-10, MCP-1, RANTES, GRO, eotaxin-1, sICAM-1 and sVCAM-1 were determined by a multiplex Luminex® system. Different trends of inflammation mediators throughout the disease were compared between mild and severe patients. RESULTS: Inflammation mediators including IL-1α, IFN-γ, IL-10, IL-8, IP-10, MCP-1 and sVCAM-1 displayed significant differences on day 8-10 of illness between mild and severe dengue patients. Their concentrations were higher in severe patients than mild ones at the same time points. Moreover, those cytokines decreased gradually in mild patients but not in severe patients. CONCLUSION: Our results revealed the coexistence of excessive inflammatory response and slow resolution of inflammation in severe adult dengue patients. Hence suppression and/or pro-resolution of inflammation could be a potential therapeutic approach for treatment of severe dengue.

Transduodenal ampullectomy provides a less invasive technique to cure early ampullary cancer.

BACKGROUND: The aim of this study was to evaluate the clinical efficiency of transduodenal ampullectomy (TDA) compared to conventional pancreatoduodenectomy (PD) in patients with early ampullary cancers. METHODS: We carried out a retrospective study by reviewing the medical records of 43 patients with early ampullary cancer who underwent either TDA or PD from January 2001 to December 2014. TDA and PD were performed on 22 patients and 21 patients, respectively. Clinical data, perioperative clinical outcomes and prognosis were evaluated. The median follow-up was 75 (range, 38-143) months. RESULTS: The sensitivity of intraoperative frozen resection was 100 % (4/4) and 94.9 % (37/39) in patients with pTis and pT1 tumors compared to final histologic diagnoses. The 5-year survival rate of patients with early ampullary cancer was 77.3 % in TDA group and 75.9 % in PD group (P = 0.927). Patients with lymph node metastasis presented a shorter 5-year survival rate (P = 0.014). TDA was associated with lower surgical morbidity (P = 0.033), estimated blood loss (P = 0.002), medical cost (P = 0.028) compared to PD. No pancreatic fistula and surgical mortality occurred in TDA group. CONCLUSIONS: TDA could produce satisfactory clinical efficiency in patients fulfilled the following criteria simultaneously: pTis or pT1 stage, tumor size ≤ 2 cm, without lymph node metastasis. To achieve favorable outcomes, intraoperative frozen section examinations should be reliable and resection margins should be negative.

Protective effects of ginsenoside Rg1 on astrocytes and cerebral ischemic-reperfusion mice.

Ginsenoside Rg1 (Rg1), one of the active ingredients in Panax ginseng, has been known to regulate many cellular processes. The purpose of this study was to investigate the protective effects of Rg1 on apoptosis in mouse cultured astrocytes in vitro and a mouse model of cerebral ischemia and reperfusion in vivo. The cell apoptosis was measured by fluorescence microplate reader and xCELLigence system and the Ca(2+) overload was recorded by confocal microscopy. The mitochondrial membrane potential and reactive oxygen species (ROS) were determined by flow cytometry. BALB/c mice were subjected to transient middle cerebral artery occlusion (MCAO) and randomly divided into four groups: Sham (sham-operated +0.9% saline), MCAO (MCAO+0.9% saline), Rg1-L (MCAO+Rg1 20 mg/kg) and Rg1-H (MCAO+Rg1 40 mg/kg). Neurological deficit scores, brain water content and infarct volume were evaluated at 24 h after reperfusion. The results showed that Rg1 significantly attenuated H2O2-induced apoptosis in astrocytes. Rg1 efficiently inhibited intracellular Ca(2+) overload, loss of mitochondrial membrane potential, and ROS production in astrocytes. In vivo study, it was also observed that Rg1 markedly reduced the neurological deficit scores, brain edema, and infarct volume in the model mice. These results suggest that Rg1 possesses significant neuroprotective effects, which might be related to the prevention of astrocytes from apoptosis.

Distribution of lymphocyte subpopulations in thyroid glands of human autoimmune thyroid disease.

BACKGROUND: The autoimmune thyroid disease (AITD) is an organ-specific autoimmune disease characterized by the breakdown of self-tolerance to thyroid antigens. Some lymphocytes have been identified to be related notably to the pathogenesis of AITD. This article evaluated the distribution of the lymphocytic subpopulation in thyroid glands in order to develop the immunospecific forms of therapy for AITD. METHODS: Damaged thyroid specimens were obtained from 18 Graves' disease (GD) and 17 Hashimoto's thyroiditis (HT) patients. Normal thyroid specimens were obtained from unaffected glands of 17 patients who underwent parathyroidectomy. We evaluated the distribution of lymphocytic subpopulation by analyzing the expression difference and correlationship among CD4+ T lymphocyte, CD8+ T lymphocyte, CD20+ B lymphocyte as well as regulatory T cells(Tregs)' marker FoxP3 in the thyroid tissues via immunohistochemistry. RESULTS: Our research uncovered that no distinct lymphocyte infiltrated in the normal thyroid specimens. Scarcely any lymphocyte infiltration could be found in half of the totally 18 GD thyroid specimens. For the rest 9 GD specimens, CD8+ T cells and CD20+ B cells were expressed more or less in all of them, FoxP3+ Tregs were detected in 7 of them and CD4+ T cells were weakly expressed in only 2 of them. For the 17 HT thyroid specimens, CD20+ B cells were stained strongly in all of them, CD4+, CD8+ T cells were expressed more or less in most of them and FoxP3+ Tregs could be detected in 9 of them. CONCLUSION: Based on CD20+ B cells predominantly infiltrating in all HT thyroid tissues we suggested CD20 antibody might be of help for HT treatment. Furthermore based on FoxP3+ Tregs abundantly infiltrating in some of the AITD thyroid specimens, we considered that activating the Tregs' function in comparison to increasing the Tregs' number only, may be a more effective approach to the treatment of AITD in some cases.

Salidroside prevents gestational hypertension-induced impairment of offspring learning and memory via Wnt/Skp2 pathway.

BACKGROUND: Salidroside (Sal) has been found to protect against multiple impairments caused by diabetes, and we designed this study to investigate the effect of Sal on gestational hypertension (GHP)-induced impairment of offspring learning and memory. METHODS: We established a GHP rat model by intraperitoneal injection of NG-nitro-L-arginine methyl ester (L-NAME), and treated with Sal by daily gavage. We used Morris Water Maze test to evaluate the learning and memory ability of offspring rats. HE staining was used to measured the pathological changes in hippocampus of offspring. Immunohistochemistry, cellular immunofluorescence and western blot were used to detect the protein expression. RESULTS: The learning and memory abilities of GHP offspring rats were significantly lower than those of normal rat offspring, while Sal treatment could significantly improve the learning and memory abilities of GHP offspring rats. HE staining did not reveal pathological differences in the hippocampus of normal rats, GHP rats and Sal-treated GHP offspring rats. However, Sal treatment can significantly increase the expression of Wnt1 and Skp2 protein, and decrease the expression of P27kiwf and P21waf1 protein in the hippocampus of GHP offspring rats. In vitro, Sal significantly promoted the proliferation and differentiation on neural stem cell, while Wnt1 knockdown could reverse these promotions by Sal. In the hippocampus of GHP offspring rats, Sal treatment significantly increased the expression of Tuj1, SOX2, Ki67 and DCX protein. CONCLUSION: Salidroside significantly improves the learning and memory impairment of offspring caused by GHP, and its mechanism may be related to the fact that Salidroside promotes the proliferation and differentiation of neural stem cells by activating the Wnt1/Skp2 signaling pathway.

Cancer targeting gene-viro-therapy for pancreatic cancer using oncolytic adenovirus ZD55-IL-24 in immune-competent mice.

Cancer targeting gene-viro-therapy (CTGVT) may prove to be an effective treatment for pancreatic cancer (PC). This study was intended to explore the anti-tumor effect of ZD55-IL-24 (oncolytic adenovirus ZD55 harboring IL-24) on PC in immune-competent mice. The expression of gene harbored by oncolytic adenovirus ZD55 in PC cells was detected by reporter-gene assays. The in vitro anti PC ability of ZD55-IL-24 was tested by MTT, crystal violet staining and apoptosis assays. The in vivo anti PC effect of ZD55-IL-24 was further observed in an immune-competent mice model by detecting anti-tumor immunity and induction of apoptosis. The expression of gene harbored by ZD55 in PC cells was significantly higher than that harbored by the replicated-deficient adenovirus, and the amount of gene expression was time-dependent and dose-dependent. Both ZD55-IL-24 and ZD55 inhibited PC cells growth, but the anti-tumor effect of ZD55-IL-24 was significantly stronger than that of ZD55, and the ability of ZD55-IL-24 in inducing PC apoptosis was significantly stronger than that of ZD55. The tumor-forming rate of group ZD55-IL-24 was the lowest, and the tumor-growing rate was also significantly lower than that of group ZD55 in immune-competent PC models. Moreover, ZD55-IL-24 mediated more anti-cancer immunity effects by induction of stronger T-lymphocytes response to PC cells, higher levels of γ-IFN and IL-6 cytokines. ZD55-IL-24-mediated CTGVT could inhibit PC growth not only by inducing oncolysis and apoptosis but enhancing the anti-cancer immune effects by inducing T cell response to PC and up-regulating γ-IFN and IL-6 cytokine in immune-competent mice. This may serve as a candidate therapeutic approach for the treatment of PC.

[Effects of the Chinese herbal extract Songyou Yin on the residual hepatocellular carcinoma after chemotherapy in nude mice].

OBJECTIVE: To investigate the effects of a Chinese herbal extract Songyou Yin on residual hepatocellular carcinoma after chemotherapy in nude mice and the relevant mechanisms. METHODS: Orthotopic nude mouse models bearing residual hepatocellular carcinoma after chemotherapy was established using human liver carcinoma MHCC97L cells. Three different doses of Songyon Yin (2.1 g/kg, 4.2 g/kg and 8.4 g/kg) were administered to the mice in the trial groups by intragastric gavage, respectively. The mice in the control group were administered physiological saline. The tumor growth, metastasis and survival in the mice of each group were recorded. The corresponding mechanisms were studied. RESULTS: The pulmonary metastasis rates of the control group and 2.1g/kg, 4.2g/kg, 8.4g/kg Songyou Yin treatment group were 86.7%, 73.3%, 40.0%, and 20.0%, respectively, and the survivals of these groups were 53.83 ± 4.71, 56.50 ± 6.09, 66.67 ± 5.61, 81.17 ± 7.36 days, respectively. Compared with the mice in the control group, mice in the 4.2 g/kg, 8.4 g/kg Songyou Yin treatment groups had a lower pulmonary metastasis rate (P = 0.021 and P = 0.001, respectively) and longer survival (P = 0.002 and P = 0.001, respectively). A restoration of E-cadherin expression and a concomitant reduction of N-cadherin expression were detected in the tumors of the 4.2 g/kg and 8.4 g/kg Songyou Yin treatment groups. CONCLUSIONS: Songyou Yin effectively inhibits the invasion and metastasis of the residual hepatocellular carcinoma after chemotherapy in nude mice through attenuating the epithelia-mesenchymal transition and prolongs the survival. Songyon Yin may have potential to promote the efficacy of chemotherapy in hepatocellular carcinoma.

[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.

Bererine induces peripheral lymphocytes immune regulations to realize its neuroprotective effects in the cerebral ischemia/reperfusion mice.

Previous studies have shown that Bererine (Ber) has significant neuroprotective effects and the present article aimed to investigate its mechanism from the aspect of peripheral immune system. We evaluated the effects of Ber 24 h after cerebral ischemia/reperfusion (I/R) on histologic injury in BALB/C mice and NOD-SCID (severe combined immunodeficient) mice lacking T and B cells. Infarct volume, neurological scores and brain water content were strikingly reduced by Ber in BALB/C mice versus NOD-SCID mice. Which suggested that Ber induces peripheral immune regulations to realize its neuroprotective effects in the cerebral I/R mice. Then we tested the lymphocytes from BALB/C lymph nodes (LNs) with their survival, activation, proliferation, cell cycle, apoptosis and differentiation induced by cytokine secretion to provide direct evidences that Ber realized its neuroprotective effects by regulating I/R-induced peripheral lymphocytes early immunoactivation and following immunotolerance and to better understand the importance of peripheral immune system following I/R insults.

Indoleamine 2,3-dioxygenase (IDO) downregulates the cell surface expression of the CD4 molecule.

Indoleamine 2,3-dioxygenase (IDO) has been implicated in preventing the fetus from undergoing maternal T cell-mediated immune responses, yet the mechanism underlying these kinds of IDO-mediated immune responses has not been fully elucidated. Since the CD4 molecule plays a central role in the onset and regulation of antigen-specific immune responses, and T cell is sensitive in the absence of tryptophan, we hypothesize that IDO may reduce cell surface CD4 expression. To test this hypothesis, an adenoviral vector-based construct IDO-EGFP was generated and the effect of IDO-EGFP on CD4 expression was determined on recombinant adenoviral infected C8166 and MT-2 cells, by flow cytometry and/or Western blot analysis. The results revealed a significant downregulation of cell membrane CD4 in pAd-IDOEGFP infected cells when compared to that of mock-infected cells or infection with empty vector pAd-EGFP. Further experiments disclosed that either an addition of tryptophan or IDO inhibitor could partly restore CD4 expression in pAd-IDOEGFP infected C8166 cells. Our findings suggest that downregulation of CD4 by IDO might be one of the mechanisms through which IDO regulates T cell-mediated immune responses.

Integrative Analysis Identifies Cell-Type-Specific Genes Within Tumor Microenvironment as Prognostic Indicators in Hepatocellular Carcinoma.

Background: Hepatocellular carcinoma (HCC) is a leading cause of cancer-related mortality worldwide, but effective early detection and prognostication methods are lacking. Methods: The Cox regression model was built to stratify the HCC patients. The single-cell RNA sequencing data analysis and gene set enrichment analysis were employed to investigate the biological function of identified markers. PLCB1 gain- or loss-of-function experiments were performed, and obtained HCC samples were analyzed using quantitative real-time PCR and immunohistochemistry assay to validate the biological function of identified markers. Results: In this study, we developed a model using optimized markers for HCC recurrence prediction. Specifically, we screened out 8 genes through a series of data analyses, and built a multivariable Cox model based on their expression. The risk stratifications using the Eight-Gene Cox (EGC) model were closely associated with the recurrence-free survivals (RFS) in both training and three validation cohorts. We further demonstrated that this risk stratification could serve as an independent predictor in predicting HCC recurrence, and that the EGC model could outperform other models. Moreover, we also investigated the cell-type-specific expression patterns of the eight recurrence-related genes in tumor microenvironment using single-cell RNA sequencing data, and interpreted their functional roles from correlation and gene set enrichment analyses, in vitro and in vivo experiments. Particularly, PLCB1 and SLC22A7 were predominantly expressed in malignant cells, and they were predicted to promote angiogenesis and to help maintain normal metabolism in liver, respectively. In contrast, both FASLG and IL2RB were specifically expressed in T cells, and were highly correlated with T cell marker genes, suggesting that these two genes might assist in maintaining normal function of T cell-mediated immune response in tumor tissues. Conclusion: In conclusion, the EGC model and eight identified marker genes could not only facilitate the accurate prediction of HCC recurrence, but also improve our understanding of the mechanisms behind HCC recurrence.

Neuromodulatory activities of CD4+CD25+ regulatory T cells in a murine model of HIV-1-associated neurodegeneration.

HIV-1-associated neurocognitive impairments are intrinsically linked to microglial immune activation, persistent viral infection, and inflammation. In the era of antiretroviral therapy, more subtle cognitive impairments occur without adaptive immune compromise. We posit that adaptive immunity is neuroprotective, serving in both the elimination of infected cells through CD8(+) cytotoxic T cell activities and the regulation of neuroinflammatory responses of activated microglia. For the latter, little is known. Thus, we studied the neuromodulatory effects of CD4(+) regulatory T cells (Treg; CD4(+)CD25(+)) or effector T cells in HIV-1-associated neurodegeneration. A newly developed HIV-1 encephalitis mouse model was used wherein murine bone marrow-derived macrophages are infected with a full-length HIV-1(YU2)/vesicular stomatitis viral pseudotype and injected into basal ganglia of syngeneic immunocompetent mice. Adoptive transfer of CD3-activated Treg attenuated astrogliosis and microglia inflammation with concomitant neuroprotection. Moreover, Treg-mediated anti-inflammatory activities and neuroprotection were associated with up-regulation of brain-derived neurotrophic factor and glial cell-derived neurotrophic factor expression and down-regulation of proinflammatory cytokines, oxidative stress, and viral replication. Effector T cells showed contrary effects. These results, taken together, demonstrate the importance of Treg in disease control and raise the possibility of their utility for therapeutic strategies.

Multiple Omics Integration Reveals Key Circular RNAs in Hepatocellular Carcinoma.

BACKGROUND: HCC is one of the most common malignancies with an increasing incidence worldwide, especially in Asian countries. However, even though targeted cancer therapy drugs such as sorafenib and regorafenib are available, the overall outcome of HCC remains unsatisfactory. Thus, it is urgent to investigate the molecular mechanisms of HCC progression, so as to provide accurate diagnostic criteria and therapeutic targets. METHODS: RNA-seq data was used to identify and quantify circular RNAs (circRNAs). DESeq2 was used to identify the differentially expressed circRNAs. miRNA binding sites within circRNAs were identified by miRanda. Gene set enrichment analysis (GSEA) was conducted to predict the biological function of circRNAs. RESULTS: The differential expression analysis identified 107 upregulated and 95 downregulated circRNAs in HCC tissues. We observed that a differentially expressed circRNA (DE-circRNA), hsa_circ_0141900 was highly negatively correlated with its parental gene RAB1A (PCC < -0.6), which was also closely associated with mTOR signaling pathway. Moreover, we also constructed competing endogenous RNA (ceRNA) network to identify key circRNAs involved in HCC. Notably, hsa_circ_0002130 and hsa_circ_0008774 were highly correlated with the genes involved in gluconeogenesis and HNF3A pathway via the target genes, GOT2 and AR, suggesting that the two circRNAs might regulate these pathways, respectively. Survival analysis revealed that GOT2 was associated with favorable prognosis. Furthermore, high expression of hsa_circ_0002130 was found to inhibit tumor cell growth and promotes GOT2 expression. CONCLUSION: In summary, the circRNAs highlighted by the integrative analysis greatly improved our understanding of the underlying mechanism of circRNAs in HCC.

Functional proteomic analysis for regulatory T cell surveillance of the HIV-1-infected macrophage.

Regulatory T cells (Treg) induce robust neuroprotection in murine models of neuroAIDS, in part, through eliciting anti-inflammatory responses for HIV-1-infected brain mononuclear phagocytes (MP; macrophage and microglia). Herein, using both murine and human primary cell cultures in proteomic and cell biologic tests, we report that Treg promotes such neuroprotection by an even broader range of mechanisms than previously seen including inhibition of virus release, killing infected MP, and inducing phenotypic cell switches. Changes in individual Treg-induced macrophage proteins were quantified by iTRAQ labeling followed by mass spectrometry identifications. Reduction in virus release paralleled the upregulation of interferon-stimulated gene 15, an ubiquitin-like protein involved in interferon-mediated antiviral immunity. Treg killed virus-infected macrophages through caspase-3 and granzyme and perforin pathways. Independently, Treg transformed virus-infected macrophages from an M1 to an M2 phenotype by down- and up- regulation of inducible nitric oxide synthase and arginase 1, respectively. Taken together, Treg affects a range of virus-infected MP functions. The observations made serve to challenge the dogma of solitary Treg immune suppressor functions and provides novel insights into how Treg affects adaptive immunosurveillance for control of end organ diseases, notably neurocognitive disorders associated with advanced viral infection.