Novel DNA methylation biomarkers in stool and blood for early detection of colorectal cancer and precancerous lesions.

BACKGROUND: Early detection and prevention of precancerous lesions can significantly reduce the morbidity and mortality of colorectal cancer (CRC). Here, we developed new candidate CpG site biomarkers for CRC and evaluated the diagnostic value of their expression in blood and stool samples of CRC and precancerous lesions. METHODS: We analyzed 76 pairs of CRC and adjacent normal tissue samples, 348 stool samples, and 136 blood samples. Candidate biomarkers for CRC were screened using a bioinformatics database and identified using a quantitative methylation-specific PCR method. The methylation levels of the candidate biomarkers were validated using blood and stool samples. The divided stool samples were used to construct and validate a combined diagnostic model and to analyze the independent or combined diagnostic value of candidate biomarkers in stool samples of CRC and precancerous lesions. RESULTS: Two candidate CpG site biomarkers for CRC, cg13096260 and cg12993163, were identified. Although both biomarkers demonstrated diagnostic performance to a certain extent when using blood samples, they showed better diagnostic value for different stages of CRC and AA with stool samples. CONCLUSIONS: cg13096260 and cg12993163 detection in stool samples could be a promising approach for screening and early diagnosis of CRC and precancerous lesions.

Comprehensive Diagnostic Criteria for MELAS Syndrome; a Case Study Involving an Elderly Patient With MT-TWm.5541C>T Mutation.

INTRODUCTION: The mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome is a matrilineal hereditary multisystem disease caused by mutations in the mitochondrial DNA. Although the initial diagnostic criteria correlate with a range of clinical phenotypes, including clinical onset after the age of 40, there is still lack of a unified single diagnostic standard for MELAS. CASE REPORT: A 71-year-old female patient with recurrent stroke was reported. Magnetic resonance imaging showed a cerebral gyrus-like diffusion weighted imaging high signal lesion in the parietal-occipital lobe and the area of this lesion expanded with disease progression. The MRS result showed significantly inverted Lac/Lip peaks. The nucleic acid sequencing result displayed a MT-TWm.5541C>T mutation, and a 12.86% mutation rate in the blood sample. The patient had a 6-year history of type 2 diabetes. CONCLUSION: Patients with the MELAS syndrome may present with a variety of clinical manifestations. Our data demonstrated that, for patients with atypical cerebral infarction and suspected MELAS syndrome, gene sequencing and muscle biopsy should be performed in time. This case provides a reference for the diagnostic criteria of MELAS syndrome.

ATG16L2 inhibits NLRP3 inflammasome activation through promoting ATG5-12-16L1 complex assembly and autophagy.

NLRP3 inflammasome activation is regulated by autophagy, a process tightly controlled by the ATG16L family proteins. However, the inside mechanisms remain elusive. Although the autophagy-related protein ATG16L1 has been well characterized, regulation and biological functions of its close homolog ATG16L2 still remain elusive. Here we report that ATG16L2 deficiency attenuates LPS-induced autophagy flux in macrophages through mediating ATG5-12-16L1 complex assembly. Importantly, NLRP3 inflammasome activation is elevated in ATG16L2-deficient macrophages, which also have defects in mitochondrial integrity and respiration. Finally, ATG16l2 knockout mice are more susceptible to DSS-induced intestinal damage, which can be ameliorated by inhibition of NLRP3. Collectively, our data demonstrate that ATG16L2 positively regulates autophagy and ATG16L2 could be a potential target for manipulating aberrant NLRP3 inflammasome activation induced inflammatory diseases.

ATG16L2 overexpression is associated with a good prognosis in colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) is a highly aggressive, high-incidence malignancy. Several biomarkers associated with the prognosis and metastasis of CRC have been identified. Our study aimed to evaluate the value of ATG16L2 protein as a new biomarker to predict the prognosis of patients with CRC. METHODS: One hundred and fifty-two pairs of paraffin-embedded tissue samples and 19 fresh tissue samples were collected from the Department of Pathology of Renji Hospital, Shanghai Jiao Tong University School of Medicine. All the patients had undergone surgery in the hospital's Department of Gastrointestinal Surgery between 2013 and 2014. The samples were arranged on two tissue microarrays of normal (n=152) and tumor (n=152) tissue. The tissues were immunostained and graded as low (<50%) or high (≥50%) according to the proportion of ATG16L2-positive cells. An overexpression plasmid was constructed and transfected into RKO cells, and the cell proliferation and migration ability were detected. Finally, Flag-ATG16L2 RKO cells subcutaneous injection into the skin of BALB/c nude mice to determine the effects of ATG16L2 on the growth of subcutaneously transplanted tumors. RESULTS: ATG16L2 expression was negatively correlated with lymph node metastasis (P<0.05) and tumor-node-metastasis stage (P<0.05). High ATG16L2 expression in tumor tissues was related to a good prognosis, with patients with a high expression of ATG16L2 displaying longer overall survival. In vitro, overexpression of ATG16L2 in a CRC cell line RKO cell led to a decrease in cell proliferation but had no obvious influence on cell migration. In vivo, the mice in the Flag-NC (as control) group exhibited faster tumor growth than those in the experiment group. CONCLUSIONS: ATG16L2 expression is positively associated with patient prognosis in CRC. Further, ATG16L2 can negatively affect CRC cell proliferation in vitro and in vivo.

Identification and development of a novel invasion-related gene signature for prognosis prediction in colon adenocarcinoma.

The overall survival of metastatic colon adenocarcinoma (COAD) remains poor, so it is important to explore the mechanisms of metastasis and invasion. This study aimed to identify invasion-related genetic markers for prognosis prediction in patients with COAD. Three molecular subtypes (C1, C2, and C3) were obtained based on 97 metastasis-related genes in 365 COAD samples from The Cancer Genome Atlas (TCGA). A total of 983 differentially expressed genes (DEGs) were identified among the different subtypes by using the limma package. A 6-gene signature (ITLN1, HOXD9, TSPAN11, GPRC5B, TIMP1, and CXCL13) was constructed via Lasso-Cox analysis. The signature showed strong robustness and could be used in the training, testing, and external validation (GSE17537) cohorts with stable predictive efficiency. Compared with other published signatures, our model showed better performance in predicting outcomes. Pan-cancer expression analysis results showed that ITLN1, TSPAN11, CXCL13, and GPRC5B were downregulated and TIMP1 was upregulated in most tumor samples, including COAD, which was consistent with the results of the TCGA and GEO cohorts. Western blot analysis and immunohistochemistry were performed to validate protein expression. Tumor immune infiltration analysis results showed that TSPAN11, GPRC5B, TIMP1, and CXCL13 protein levels were significantly positively correlated with CD4+ T cells, macrophages, neutrophils, and dendritic cells. Further, the TIMP1 and CXCL13 proteins were significantly related to the tumor immune infiltration of CD8+ T cells. We recommend using our signature as a molecular prognostic classifier to assess the prognostic risk of patients with COAD.

Longitudinal evaluation of serum periostin levels in patients after large-artery atherosclerotic stroke: A prospective observational study.

Increasing evidence supports the involvement of periostin in the pathophysiological processes of stroke and atherosclerosis. The aim of this study was to assess circulating periostin levels at different times after large-artery atherosclerotic (LAA) stroke and their association with stroke. Serum periostin levels were measured using enzyme-linked immunosorbent assay on day 1 in 162 patients with LAA stroke and in 108 age- and sex-matched controls, on day 6 after stroke in 134 patients, and during the 4th week after stroke in 46 of the 162 patients. Stroke severity was determined using the National Institutes of Health Stroke Scale (NIHSS), and the stroke volume was measured. Outcome at 3 months was measured using the modified Rankin Scale (mRS). Our results indicated that periostin levels increased significantly on day 6 after stroke, and this increasing trend persisted for at least 4 weeks after the event. In addition, the increase in periostin levels was positively correlated with the NIHSS scores and stroke volume, but not with the mRS scores after adjusting for the NIHSS scores. In conclusion, these findings suggest that the increase in serum periostin levels observed after stroke may be associated with the stroke severity in patients with LAA stroke.

Serum levels of endocan and endoglin are associated with large-artery atherosclerotic stroke.

BACKGROUND: Accumulating evidence has suggested that endocan and endoglin may play important roles in cardiovascular disease. However, no previous study has focused on these circulating levels in patients with large-artery atherosclerotic (LAA) stroke. METHODS: Serum levels of endocan and endoglin in 114 patients with LAA stroke and 114 age- and sex-matched controls were measured by ELISA. Serum samples from patients were available on day 1, day 6 and in the 4th week after ischaemic stroke(IS). Stroke severity was determined based on the NIHSS score and the stroke volume. An unfavourable outcome was defined as a mRS score>2 on day 90 after IS. RESULTS: The endocan levels were significantly higher in patients with LAA stroke compared with the controls (p=0.001), and after adjustment for other factors (p=0.001). In addition, higher endocan levels were independently associated with unfavourable outcomes on both day 1 and day 6 after IS (p=0.018 and p=0.011). Endoglin levels were decreased on day 6 (p=0.002) and then recovered in the 4th week after IS. No correlation was found between endocan or endoglin and stroke severity. CONCLUSIONS: Endocan levels are higher in patients with LAA stroke and can help in predicting the short-term unfavourable outcome. Endoglin levels are changed after stroke.

Effects of RNA silencing of matrix metalloproteinase-2 on the growth of esophageal carcinoma cells in vivo.

Esophageal carcinoma is one of the most common malignancies in China. Previous studies reported that matrix metalloproteinases (MMPs) have important roles in the progression and invasion of numerous types of solid tumors. Among the MMPs, MMP-2 has been closely associated with tumor growth and invasion. In the present study, a short hairpin RNA (shRNA) lentiviral expression vector targeting the MMP-2 gene was constructed in order to observe the inhibitory effect of MMP-2 gene silencing on the growth of the KYSE150 esophageal carcinoma cell line in vivo. Three small hairpin RNA sequences targeting MMP-2 were designed and cloned into lentiviral vectors. Following transfection of the lentiviral vectors into KTSE150 cells, MMP-2 mRNA and protein expression levels were examined by reverse transcription-quantitative polymerase chain reaction and western blotting, and the growth rate of cells was analyzed by MTT assays. Subsequently, tumor growth was assessed in nude mice. Lentivirus-mediated RNA interference effectively inhibited the expression of MMP-2 mRNA and protein in KYSE150 esophageal carcinoma cells, and suppressed the growth of esophageal carcinoma cells in vivo. The results of the present study suggested that lentivirus-mediated gene therapy targeting MMP-2 may be an attractive strategy for the treatment of esophageal carcinoma and justifies the performance of further studies on the application of lentivirus vectors to cancer gene therapy.

Serum levels of galectin-1, galectin-3, and galectin-9 are associated with large artery atherosclerotic stroke.

The aim of this study was to assess the expression patterns of serum galectin-1 (Gal-1), galectin-3 (Gal-3), galectin-9 (Gal-9), and galectin-3 binding protein (Gal-3BP) and their associations with stroke outcome in large artery atherosclerotic (LAA) stroke. The serum levels of Gal-1, Gal-3, Gal-9, and Gal-3BP were measured by ELISA in 130 patients with LAA stroke and 130 age- and sex-matched controls. Serum samples were collected from the patients on day 1, day 6, and in the 4th week after ischaemic stroke (IS). An unfavourable outcome was defined as a modified Rankin Scale score of >2 on day 90 after IS. Our results indicated that the Gal-3 and Gal-9 levels were higher in patients with LAA stroke than in controls. A higher Gal-3 level was independently associated with an unfavourable outcome both on day 1 and day 6 after IS. In addition, Gal-9 and Gal-1 levels were upregulated on day 6 and in the 4th week after IS, respectively. For Gal-3BP, no difference was detected between patients and controls and no predictive value was found in patients. In conclusion, these findings suggest that the serum levels of Gal-1, Gal-3, and Gal-9 may be associated with LAA stroke.

Video-assisted thoracoscopic surgery for adult Bochdalek hernia: a case report.

BACKGROUND: Bochdalek hernia is a type of congenital diaphragmatic hernia that typically presents in childhood, while this diseases is extremely rare in adults. CASE PRESENTATION: We review a case of a 63-year-old man with a left-sided Bochdalek hernia who was experiencing occasional pain at the left side of his chest for 8 months. The diagnosis of Bochdalek hernia was made by chest computed tomography. A part of the retroperitoneal adipose tissue was herniated into the left thoracic cavity through the diaphragmatic defect. The hernia was treated via video-assisted thoracoscopic surgery and he made an uneventful recovery. CONCLUSIONS: We report a rare case of a left-sided Bochdalek hernia for which our patient was treated successfully via video-assisted thoracoscopic surgery. Even though rare, this disorder should be recognised, examined and treated appropriately to avoid complications.

Angiopoietin-like protein 4 serum levels and gene polymorphisms are associated with large artery atherosclerotic stroke.

BACKGROUND: Angiopoietin-like protein 4 (ANGPTL4) is a central player in lipid metabolism and atherosclerosis and may thus be involved in ischaemic stroke. However, no study in humans has investigated the association of ANGPTL4 gene polymorphisms or serum levels with ischaemic stroke. METHODS: We investigated the influence of the tagged single nucleotide polymorphisms (tSNPs) rs4076317 (c.207C>G) and rs1044250 (c.797C>T; T266M) of the ANGPTL4 gene on ischaemic stroke risk in a large group of 712 large artery atherosclerotic (LAA) stroke patients and 828 controls. In addition, we examined the association of the serum ANGPTL4 levels with lipid metabolism, LAA stroke severity and ischaemic volume in a sample of 302 LAA stroke patients and 307 controls. RESULTS: The findings reveal that rs4076317 exerts a co-dominant effect on lower serum TG levels compared with common homozygotes. Fewer stroke cases were homozygous for variants of rs4076317 compared with the controls (7.0% vs. 10.9%). The serum ANGPTL4 levels in patients were significantly higher than those in the controls in a univariate manner (P=0.001) and after adjustment for other risk factors (1.463 [1.215-1.835]; P<0.001). Consistently, the ANGPTL4 levels were statistically correlated with higher NIHSS scores (r=0.172, P=0.003) and larger lesion volumes (r=0.124, P=0.031). CONCLUSION: We concluded that the tagged SNPs and high serum levels of ANGPTL4 are associated with LAA stroke and the lipid characteristics.

Esophageal squamous cell carcinoma: assessing tumor angiogenesis using multi-slice CT perfusion imaging.

OBJECTIVES: The purpose of this study was to investigate the correlation between multi-slice computed tomographic perfusion imaging (CTPI) parameters and immunohistologic markers of angiogenesis in esophageal squamous cell carcinoma (ESCC). METHODS: Fifty patients with histologically proven esophageal squamous cell carcinoma were enrolled in this study. All subjects underwent multi-slice CT perfusion scan. The hemodynamic parameters of vascular tumor, including blood volume (BV), blood flow (BF), mean transit time (MTT) and permeability surface (PS) were generated. All the ESCC specimens were stained immunohistochemically to identify CD31 for quantification of microvessel density (MVD). CTPI parameters were correlated with MVD by using Pearson correlation analysis. RESULTS: The value of CT perfusion parameters of ESCC were as follows: BF 116.71 ± 47.59 ml/100 g/min, BV 6.74 ± 2.70 ml/100 g, MTT 6.42 ± 2.84 s, PS 13.82 ± 6.25 ml/100 g/min. The mean MVD of all 50 tumor specimens was 34.44 ± 19.75. The PS values were significantly higher in ESCC patients with involvement of lymph node than those without involvement of lymph node (p < 0.01). Blood volume and permeability surface were positively correlated with MVD (p < 0.01), whereas no significant correlation was observed between MVD and BF or between MVD and MTT. CONCLUSIONS: Blood volume and permeability surface were positively correlated with MVD. CTPI could reflect the angiogenesis in ESCC.

Effects of RNAi-mediated matrix metalloproteinase-2 gene silencing on the invasiveness and adhesion of esophageal carcinoma cells, KYSE150.

BACKGROUND: Esophageal carcinoma is one of the main malignancies in China. Previous studies indicated that matrix metalloproteinases (MMPs) play important roles in the process of tumor invasion and metastasis in several types of solid tumors. Among all of the MMPs, MMP-2 is one of the MMPs closely associated with tumor invasion. In this study, we suppressed MMP-2 expression with RNA interference and then observed inhibitory effects on the invasion and migration of the esophageal carcinoma cell line KYSE150. METHODS: Three target sequences were selected and siRNA against MMP-2 mRNA were synthesized. After being transfected by the transfection complexes, the MMP-2 expression of KYSE150 cells, which overexpresses MMP-2, were examined by Western blot analysis and real-time polymerase chain reaction (PCR). Cell migration and invasion were measured with migration assay and Boyden chamber assays, respectively. RESULTS: RNAi against MMP-2 successfully inhibited the mRNA and protein expression of MMP-2 in the esophageal carcinoma cell line KYSE150. MMP-2 knockdown inhibited the invasion and migration of esophageal carcinoma cell line KYSE150. CONCLUSIONS: These findings suggested that the RNAi approach towards MMP-2 may be a potentially effective therapeutic method for the treatment of esophageal carcinoma.