Machine learning and experimental validation identified autophagy signature in hepatic fibrosis.

Background: The molecular mechanisms of hepatic fibrosis (HF), closely related to autophagy, remain unclear. This study aimed to investigate autophagy characteristics in HF. Methods: Gene expression profiles (GSE6764, GSE49541 and GSE84044) were downloaded, normalized, and merged. Autophagy-related differentially expressed genes (ARDEGs) were determined using the limma R package and the Wilcoxon rank sum test and then analyzed by GO, KEGG, GSEA and GSVA. The infiltration of immune cells, molecular subtypes and immune types of healthy control (HC) and HF were analyzed. Machine learning was carried out with two methods, by which, core genes were obtained. Models of liver fibrosis in vivo and in vitro were constructed to verify the expression of core genes and corresponding immune cells. Results: A total of 69 ARDEGs were identified. Series functional cluster analysis showed that ARDEGs were significantly enriched in autophagy and immunity. Activated CD4 T cells, CD56bright natural killer cells, CD56dim natural killer cells, eosinophils, macrophages, mast cells, neutrophils, and type 17 T helper (Th17) cells showed significant differences in infiltration between HC and HF groups. Among ARDEGs, three core genes were identified, that were ATG5, RB1CC1, and PARK2. Considerable changes in the infiltration of immune cells were observed at different expression levels of the three core genes, among which the expression of RB1CC1 was significantly associated with the infiltration of macrophage, Th17 cell, natural killer cell and CD56dim natural killer cell. In the mouse liver fibrosis experiment, ATG5, RB1CC1, and PARK2 were at higher levels in HF group than those in HC group. Compared with HC group, HF group showed low positive area in F4/80, IL-17 and CD56, indicating decreased expression of macrophage, Th17 cell, natural killer cell and CD56dim natural killer cell. Meanwhile, knocking down RB1CC1 was found to inhibit the activation of hepatic stellate cells and alleviate liver fibrosis. Conclusion: ATG5, RB1CC1, and PARK2 are promising autophagy-related therapeutic biomarkers for HF. This is the first study to identify RB1CC1 in HF, which may promote the progression of liver fibrosis by regulating macrophage, Th17 cell, natural killer cell and CD56dim natural killer cell.

The Severity of CVB3-Induced Myocarditis Can Be Improved by Blocking the Orchestration of NLRP3 and Th17 in Balb/c Mice.

BACKGROUND: The functional characteristics of NLRP3 in the pathogenesis of coxsackievirus B3- (CVB3-) induced viral myocarditis (VMC) have not been fully elucidated, and the targeted therapeutic effect of NLRP3 or its related pathway in VMC has not been reported. METHOD: In this work, the change patterns of NLRP3- and Th17-related factors were detected during the pathological process of CVB3-induced VMC in Balb/c mice. The correlation between NLRP3 and Th17 cells during the VMC process was analyzed by Spearman test. The coculture system of spleen CD4+ T and bone marrow CD11c+ DC cells was set to explore the orchestration of NLRP3 and Th17 in the pathological development of VMC in vitro. Anti-IL-1ß antibody or NLRP3-/- Balb/c were used to block the NLRP3 pathway indirectly and directly to analyze the NLRP3-targeting therapeutic value. RESULTS: The change patterns of NLRP3- and Th17-related molecules in the whole pathological process of mouse CVB3-induced VMC were described. Through Spearman correlation analysis, it was confirmed that there was a close correlation between NLRP3 and Th17 cells in the whole pathological process of VMC. And the interaction mode between NLRP3 and Th17 was preliminarily explored in the cell experiment in vitro. Under the intervention of an anti-IL-1ß antibody or NLRP3 knockout, the survival rate of the intervention group was significantly improved, the degree of myocardial inflammation and fibrosis was significantly alleviated, and the content of myocardial IL-17 and spleen Th17 was also significantly decreased. CONCLUSION: Our findings demonstrated a key role of the NLRP3 inflammasome and its close relationship with Th17 in the pathological progression of CVB3-induced VMC and suggested a possible positive feedback-like mutual regulation mechanism between the NLRP3 inflammasome and Th17 in vitro and in the early stage of CVB3 infection. Taking NLRP3 as a new starting point, it provides a new target and idea for the prevention and treatment of CVB3-induced VMC.

The Value of a Seven-Autoantibody Panel Combined with the Mayo Model in the Differential Diagnosis of Pulmonary Nodules.

BACKGROUND: Identifying malignant pulmonary nodules and detecting early-stage lung cancer (LC) could reduce mortality. This study investigated the clinical value of a seven-autoantibody (7-AAB) panel in combination with the Mayo model for the early detection of LC and distinguishing benign from malignant pulmonary nodules (MPNs). METHODS: The concentrations of the elements of a 7-AAB panel were quantitated by enzyme-linked immunosorbent assay (ELISA) in 806 participants. The probability of MPNs was calculated using the Mayo predictive model. The performances of the 7-AAB panel and the Mayo model were analyzed by receiver operating characteristic (ROC) analyses, and the difference between groups was evaluated by chi-square tests (χ 2). RESULTS: The combined area under the ROC curve (AUC) for all 7 AABs was higher than that of a single one. The sensitivities of the 7-AAB panel were 67.5% in the stage I-II LC patients and 60.3% in the stage III-IV patients, with a specificity of 89.6% for the healthy controls and 83.1% for benign lung disease patients. The detection rate of the 7-AAB panel in the early-stage LC patients was higher than that of traditional tumor markers. The AUC of the 7-AAB panel in combination with the Mayo model was higher than that of the 7-AAB panel alone or the Mayo model alone in distinguishing MPN from benign nodules. For early-stage MPN, the sensitivity and specificity of the combination were 93.5% and 58.0%, respectively. For advanced-stage MPN, the sensitivity and specificity of the combination were 91.4% and 72.8%, respectively. The combination of the 7-AAB panel with the Mayo model significantly improved the detection rate of MPN, but the positive predictive value (PPV) and the specificity were not improved when compared with either the 7-AAB panel alone or the Mayo model alone. CONCLUSION: Our study confirmed the clinical value of the 7-AAB panel for the early detection of lung cancer and in combination with the Mayo model could be used to distinguish benign from malignant pulmonary nodules.

LncRNA TGFB2-AS1 regulates lung adenocarcinoma progression via act as a sponge for miR-340-5p to target EDNRB expression.

Long non-coding RNA TGFB2-antisense RNA1 (TGFB2-AS1) has been reported could regulate tumorigenesis. However, the roles of TGFB2-AS1 in lung adenocarcinoma (LUAD) remain largely unknown. In this work, we aimed to explore the expression levels of TGFB2-AS1 and mechanisms in regulating LUAD progression. Expression level of TGFB2-AS1 in LUAD tissues and normal tissues was analyzed at StarBase. Moreover, its expression in LUAD cells and normal cell was analyzed with quantitative real-time polymerase chain reaction method. Gain- and loss-of-function studies were conducted to analyze the biological roles of TGFB2-AS1 in LUAD. Results indicated TGFB2-AS1 was evidently downregulated in LUAD tissues and cells. Moreover, as analyzed by cell counting kit-8 assay, wound-healing and transwell invasion assays, results revealed TGFB2-AS1 overexpression could suppress proliferation, migration and invasion abilities of LUAD cells in vitro and tumor growth in vivo. In addition, LncBase V2.0 and TargetScan prediction tools showed TGFB2-AS1 and endothelin receptor type B (EDNRB) shares binding site in microRNA-340-5p (miR-340-5p). Furthermore, luciferase activity reporter assay and RT-qPCR assay validated these prediction results. Furthermore, we showed TGFB2-AS1 functions as sponge for miR-340-5p to regulate EDNRB expression. Collectively, our results indicated TGFB2-AS1/miR-340-5p/EDNRB axis plays crucial roles in regulating LUAD progression, indicating TGFB2-AS1 may be a novel therapeutic target for LUAD.

Lack of Efficacy of Combined Carbohydrate Antigen Markers for Lung Cancer Diagnosis.

BACKGROUND: Lung cancer (LC) is top-ranked in cancer incidence and is the leading cause of cancer death globally. Combining serum biomarkers can improve the accuracy of LC diagnosis. The identification of the best potential combination of traditional tumor markers is essential for LC diagnosis. Patients and Methods. Blood samples were collected from 132 LC cases and 118 benign lung disease (BLD) controls. The expression levels of ten serum tumor markers (CYFR21, CEA, NSE, SCC, CA15-3, CA 19-9, CA 125, CA50, CA242, and CA724) were assayed, and that the expression in the levels of tumor markers were evaluated, isolated, and combined in different patients. The performance of the biomarkers was analyzed by receiver operating characteristic (ROC) analyses, and the difference between combinations of biomarkers was compared by Chi-square (χ 2) tests. RESULTS: As single markers, CYFR21 and CEA showed good diagnostic efficacy for nonsmall cell lung cancer (NSCLC) patients, while NSE and CEA were the most sensitive in the diagnosis of small cell lung cancer (SCLC). The area under the curve (AUC) value was 0.854 for the panel of four biomarkers (CYFR21, CEA, NSE, and SCC), 0.875 for the panel of six biomarkers (CYFR21, CEA, NSE, SCC, CA125, and CA15-3), and 0.884 for the panel of ten markers (CYFR21, CEA, NSE, SCC, CA125, CA15-3, CA19-9, CA50, CA242, and CA724). With a higher sensitivity and negative predictive value (NPV), the diagnostic accuracy of the three panels was better than that of any single biomarker, but there were no statistically significant differences among them (all P values > 0.05). However, the panel of six carbohydrate antigen (CA) biomarkers (CA125, CA15-3, CA19-9, CA50, CA242, and CA724) showed a lower diagnostic value (AUC: 0.776, sensitivity: 59.8%, specificity: 73.0%, and NPV: 60.4%) than the three panels (P value < 0.05). The performance was similar even when analyzed individually by LC subtypes. CONCLUSION: The biomarkers isolated are elevated for different types of lung cancer, and the panel of CYFR21, CEA, NSE, and SCC seems to be a promising serum biomarker for the diagnosis of lung cancer, while the combination with carbohydrate antigen markers does not improve the diagnostic efficacy.

Upregulation of lncRNA Sox2ot indicates a poor prognosis for patients with hepatocellular carcinoma and promotes cell invasion.

Long non-coding RNA Sox2 overlapping transcript (lncRNA Sox2ot) expression has been demonstrated to be upregulated in a number of types of tumor, and may act as an oncogene. The present study aimed to evaluate the clinical role of lncRNA Sox2ot and its association with the epithelial-mesenchymal transition in hepatocellular carcinoma (HCC). Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed to determine the expression of lncRNA Sox2ot in 86 cases of HCC tissues and adjacent non-tumor tissues. Kaplan-Meier and log-rank test was used to analyze the association between lncRNA Sox2ot expression and disease-free (DFS) or overall (OS) survival time. In addition, the capacity of HCC cells with lncRNA Sox2ot knockdown for invasion was evaluated via transwell cell invasion assays. RT-qPCR and western blot analyses were also performed to determine the mRNA and protein expression of Twist1, E-cadherin and N-cadherin in the HCC cells. It was indicated that lncRNA Sox2ot expression levels were significantly higher in HCC tissues compared with adjacent non-tumor tissues. Increased expression levels of lncRNA Sox2ot were associated with the tumor size, the tumor number and vein invasion in patients with HCC. An association was observed between lncRNA Sox2ot and the DFS and OS of patients with HCC. Furthermore, it was determined that cell invasion was inhibited following the siRNA knockdown of lncRNA Sox2ot in MHCC97H and SMCC-7721 cells via transwell cell invasion assays. Furthermore, it was demonstrated that knockdown of lncRNA Sox2ot downregulated the mRNA and protein expression of Twist1 and N-cadherin, but upregulated the E-cadherin expression levels in MHCC97H and SMCC-7721 cells. Thus, it was indicated that lncRNA Sox2ot may be a novel predictive biomarker and a potential therapeutic target for HCC.

Genetic polymorphism analysis of MICB gene in Jing ethnic minority of Southern China.

In the present study, the polymorphism in the 5'-upstream regulation region (5'-URR), coding region (exons 2-4), and the 3'-untranslated region (3'-UTR) of MICB gene were investigated for 150 healthy unrelated Jing individuals in Guangxi Zhuang Autonomous Region, by using PCR-SBT method. A total of 14 variation sites in the 5'-URR, 9 in coding region, and 6 in the 3'-UTR were detected in the Jing population. The MICB gene seems to present two different lineages showing functional variations mainly in nucleotides of the promoter region. Nineteen different MICB extended haplotypes (EHs) encompassing the 5'-URR, exons 2-4, and 3'-UTR were found in this population, and the most frequent was EH2 (20.33%). The findings here are of importance for future studies on the potential role of regulation region of MICB gene in disease association, transplantation, viral infection, and tumor progression among Jing population.

Diagnosis, prognosis and bioinformatics analysis of lncRNAs in hepatocellular carcinoma.

This study aims to comprehensively analyze the diagnosis and prognosis of lncRNAs in hepatocellular carcinoma (HCC). From the Gene Expression Omnibus database, we screened out and analyzed the differently expressed lncRNAs and related mRNAs using bioinformatics methods. The expressions of lncRNAs were validated in tumor tissues, cell lines and the Cancer Genome Atlas database. At the same time, we also conducted an exploratory analysis on the diagnostic and prognostic ability of lncRNAs in HCC. In this study, we found that most of the targeted mRNAs promote the biological process of cell division, cellular component of nucleoplasm, molecular function of protein binding, which were significantly associated with 12 KEGG pathways. LncRNA CRNDE and LINC01419 also had significant diagnostic value in HCC. In particular, the sensitivity, specificity and area under the curve of CRNDE were 71.0%, 87.1% and 0.701 (95% CI: 0.543-0.860), respectively. In addition, the high expression of CRNDE and GBAP1 predicated poor prognosis, while the high expression of LINC01093 suggested the opposite outcome. Through the comprehensive analysis of lncRNAs, it provided an important reference for the early diagnosis, prognosis evaluation, pathogenesis and targeted therapy of HCC.

Enhanced proliferation and differentiation effects of a CGRP- and Sr-enriched calcium phosphate cement on bone mesenchymal stem cells.

INTRODUCTION: Because of its good osteoconductivity, strontium (Sr) ranelate has been extensively used as a bone substitute for the treatment of bone disorders. To facilitate treatment, Sr is also incorporated into calcium phosphate cement (Sr-CPC); however, the Sr from Sr-CPC is not sufficient to induce a significant increase of bone mass in an ovariectomized rat model. To improve the efficiency of Sr-CPC, we developed a calcitonin gene-related peptide (CGRP)- and Sr-enriched CPC (CGRP-Sr-CPC). METHODS: We used X-ray diffraction and Fourier transform infrared spectroscopy to measure properties of CGRP-Sr-CPC. We also employed a cell proliferation assay, alkaline phosphatase (ALP) assay and real-time PCR to assess the effects of CPC implants on proliferation and differentiation of bone mesenchymal stem cells (BMSCs) from an ovariectomized rat model. RESULTS: CGRP did not change the composition, pore sizes and compressive strength of the cement body as compared with Sr-CPC. Meanwhile, CGRP-Sr-CPC did not show cell cytotoxicity to BMSCs. Further, CGRP and Sr released from CGRP-Sr-CPC significantly enhanced the cell proliferation of BMSCs and increased the activity of ALP during differentiation of BMSCs, compared with CGRP- or Sr-CPC. Moreover, CGRP-Sr-CPC significantly up-regulated the expression levels of osteogenic differentiation-related genes including Alp, Bmp2, Osteonectin and Runx2 during differentiation. CONCLUSIONS: These findings demonstrate the optimized effects of CGRP- and Sr-enriched CPC in promoting proliferation and osteogenic differentiation of BMSCs, suggesting the potential ability of this novel cement to assist the formation of new bone during osteoporosis-induced bone disorders.

The HLA-DRB1 allele polymorphisms and nasopharyngeal carcinoma.

Human leukocyte antigen (HLA)-DRB1 has been reported to influence individual's susceptibility to nasopharyngeal carcinoma (NPC) by many studies in recent years; however, these studies provided controversial results. The meta-analysis was thus conducted here to estimate the relationship between HLA-DRB1 polymorphisms and NPC. After an extensive review of journals from various databases (PubMed, the Web of Science, Embase, China National Knowledge Internet (CNKI), and Wanfang Database), 8 out of 69 case-control studies, including 778 cases and 1148 controls, were extracted. The results showed that 4 of 13 polymorphisms allele are statistically significantly associated with NPC, among them, HLA-DRB1*3, HLA-DRB1*9, and HLA-DRB1*10 may increase the risk of NPC while HLA-DRB1*01 has the opposite effect. The pooled odds ratio and 95 % confidence interval (CI) were 1.702 [95 % CI (1.047, 2.765)], 1.363 [95 % CI (1.029, 1.806)], 1.989 [95 % CI (1.042, 3.799)], and 0.461 [95 % CI (0.315, 0.676)], respectively. In a further ethnicity-based subgroup analysis, HLA-DRB1*08, HLA-DRB1*11, and HLA-DRB1*16 were found to be linked with NPC in Asian, Tunisian, and Caucasian, respectively. In Asian, HLA-DRB1*03, 08, and 10 may elevate the risk whereas HLA-DRB1*09 could lower it. In Tunisian, HLA-DRB1*01 and 11 are the protective factors while HLA-DRB1*03 is the only risk factor. In Caucasian, HLA-DRB1*01 and 03 increase the risk and HLA-DRB1*16 lowers it. The most frequent statistically associated gene is found to be HLA-DRB1*03 which has protective influence on Asian and Tunisian. In conclusion, HLA-DRB1*01, DRB1*03, DRB1*09, and DRB1*10 are related with NPC susceptibility, and the association of HLA-DRB1*08, DRB1*11, and DRB1*16 with NPC risk are significantly different in different ethnicities.

Calcitonin gene-related peptide stimulates proliferation and osteogenic differentiation of osteoporotic rat-derived bone mesenchymal stem cells.

Osteoporosis, a systemic bone disorder, is prevalent in postmenopausal woman. Bone mesenchymal stem cells (BMSCs), precursors of osteogenic cells, may contribute to prevention or treatment of bone frustrate in osteoporosis. Recently, two studies suggested a role of calcitonin gene-related peptide (CGRP) in promoting osteogenesis of BMSCs under physiological conditions. However, the role of CGRP on BMSCs, which are derived from osteoporotic tissues, is unclear. Here, we investigated the role of CGRP on BMSCs isolated from female osteoporotic rats. Data showed that CGRP stimulated cell proliferation and inhibited cell apoptosis for short-term culture of BMSCs. Instead, CGRP induced BMSCs differentiation into the osteoblasts and promoted formation of calcified nodules after long-term culture. Moreover, CGRP gradually up-regulated expression levels of osteoporotic differentiation-related genes including alkaline phosphatase, Collagen type I, Bmp2, Osteonectin, and Runx2 during osteogenic differentiation. In conclusion, CGRP promoted proliferation and induced osteogenic differentiation and mineralization during female osteoporotic rat-derived BMSC differentiation. These findings support a potential role of CGRP on the prevention or treatment of osteoporotic fracture.

Hec1 inhibition alters spindle morphology and chromosome alignment in porcine oocytes.

Aneuploidy is caused by incorrect chromosome segregation and can result in cancer or birth defects. The spindle assembly checkpoint (SAC) guarantees proper cell cycle progression. Highly Expressed in Cancer protein 1 (Hec1, also called Ndc80) is the core component of the Ndc80 complex and is involved in regulating both kinetochore-microtubule interactions and the SAC during mitosis in multiple cell types. However, its involvement in pig oocyte meiotic maturation remains uncertain. Thus, we investigated Hec1 expression, localization, and possible functions during porcine oocyte meiosis. Immunofluorescent staining showed that Hec1 was expressed in porcine oocytes and was associated with centromeres at both the metaphase I and metaphase II stages. Disrupting Hec1 function with its inhibitor INH1 resulted in polar body extrusion defects in porcine oocytes. Moreover, inhibiting Hec1 activity also resulted in severe chromosome misalignments and aberrant spindle morphology. Our results showed a unique localization pattern for Hec1 in porcine oocytes and suggested that Hec1 was required for chromosome alignment and spindle organization. Thus, Hec1 might regulate spindle checkpoint activity during mammalian oocyte meiosis.

Identification of biomarkers that distinguish chemical contaminants based on gene expression profiles.

BACKGROUND: High throughput transcriptomics profiles such as those generated using microarrays have been useful in identifying biomarkers for different classification and toxicity prediction purposes. Here, we investigated the use of microarrays to predict chemical toxicants and their possible mechanisms of action. RESULTS: In this study, in vitro cultures of primary rat hepatocytes were exposed to 105 chemicals and vehicle controls, representing 14 compound classes. We comprehensively compared various normalization of gene expression profiles, feature selection and classification algorithms for the classification of these 105 chemicals into14 compound classes. We found that normalization had little effect on the averaged classification accuracy. Two support vector machine (SVM) methods, LibSVM and sequential minimal optimization, had better classification performance than other methods. SVM recursive feature selection (SVM-RFE) had the highest overfitting rate when an independent dataset was used for a prediction. Therefore, we developed a new feature selection algorithm called gradient method that had a relatively high training classification as well as prediction accuracy with the lowest overfitting rate of the methods tested. Analysis of biomarkers that distinguished the 14 classes of compounds identified a group of genes principally involved in cell cycle function that were significantly downregulated by metal and inflammatory compounds, but were induced by anti-microbial, cancer related drugs, pesticides, and PXR mediators. CONCLUSIONS: Our results indicate that using microarrays and a supervised machine learning approach to predict chemical toxicants, their potential toxicity and mechanisms of action is practical and efficient. Choosing the right feature and classification algorithms for this multiple category classification and prediction is critical.

HSP70 interacts with TRAF2 and differentially regulates TNFalpha signalling in human colon cancer cells.

Members of tumour necrosis factor (TNF) family usually trigger both survival and apoptotic signals in various cell types. Heat shock proteins (HSPs) are conserved proteins implicated in protection of cells from stress stimuli. However, the mechanisms of HSPs in TNFalpha-induced signalling pathway have not been fully elucidated. We report here that HSP70 over-expression in human colon cancer cells can inhibit TNFalpha-induced NFkappaB activation but promote TNFalpha-induced activation of c-Jun N-terminal kinase (JNK) through interaction with TNF receptor (TNFR)-associated factor 2 (TRAF2). We provide evidence that HSP70 over-expression can sequester TRAF2 in detergent-soluble fractions possibly through interacting with TRAF2, leading to reduced recruitment of receptor-interacting protein (RIP1) and IkappaB alpha kinase (IKK) signalosome to the TNFR1-TRADD complex and inhibited NFkappaB activation after TNFalpha stimuli. In addition, we found that HSP70-TRAF2 interaction can promote TNFalpha-induced JNK activation. Therefore, our study suggests that HSP70 may differentially regulate TNFalpha-induced activation of NFkappaB and JNK through interaction with TRAF2, contributing to the pro-apoptotic roles of HSP70 in TNFalpha-induced apoptosis of human colon cancer cells.

Phase I clinical trial of autologous ascites-derived exosomes combined with GM-CSF for colorectal cancer.

Exosomes are small membrane vesicles that are secreted by a multitude of cell types. The exosomes derived from dendritic cells (Dex), tumor cells (Tex), and malignant effusions demonstrate immunomodulatory functions, and are even under clinical trial for cancer treatments. In this study we report the phase I clinical trial of the ascites-derived exosomes (Aex) in combination with the granulocyte-macrophage colony-stimulating factor (GM-CSF) in the immunotherapy of colorectal cancer (CRC). The Aex isolated by sucrose/D(2)O density gradient ultracentrifugation are 60-90-nm vesicles that contain the diverse immunomodulatory markers of exosomes and tumor-associated carcinoembryonic antigen (CEA). Totally 40 patients (HLA-A0201(+)CEA(+)) with advanced CRC were enrolled in the study, and randomly assigned to treatments with Aex alone or Aex plus GM-CSF. Patients in both groups received a total of four subcutaneous immunizations at weekly intervals. We found that both therapies were safe and well tolerated, and that Aex plus GM-CSF but not Aex alone can induce beneficial tumor-specific antitumor cytotoxic T lymphocyte (CTL) response. Therefore, our study suggests that the immunotherapy of CRC with Aex in combination with GM-CSF is feasible and safe, and thus can serve as an alternative choice in the immunotherapy of advanced CRC.