LC-MS-based serum metabolomics analysis for the screening and monitoring of colorectal cancer.

Background: Colorectal Cancer (CRC) is a prevalent digestive system tumour with significant mortality and recurrence rates. Serum metabolomics, with its high sensitivity and high throughput, has shown potential as a tool to discover biomarkers for clinical screening and monitoring of the CRC patients. Methods: Serum metabolites of 61 sex and age-matched healthy controls and 62 CRC patients (before and after surgical intervention) were analyzed using a ultra-performance liquid chromatography-high resolution mass spectrometer (UPLC-MS). Statistical methods and pathway enrichment analysis were used to identify potential biomarkers and altered metabolic pathways. Results: Our analysis revealed a clear distinction in the serum metabolic profile between CRC patients and healthy controls (HCs). Pathway analysis indicated a significant association with arginine biosynthesis, pyrimidine metabolism, pantothenate, and CoA biosynthesis. Univariate and multivariate statistical analysis showed that 9 metabolites had significant diagnostic value for CRC, among them, Guanosine with Area Under the Curve (AUC) values of 0.951 for the training group and0.998 for the validation group. Furthermore, analysis of four specific metabolites (N-Phenylacetylasparticacid, Tyrosyl-Gamma-glutamate, Tyr-Ser and Sphingosine) in serum samples of CRC patients before and after surgery indicated a return to healthy levels after an intervention. Conclusion: Our results suggest that serum metabolomics may be a valuable tool for the screening and monitoring of CRC patients.

[Clinical Study of miRNAs Derived from Serum Exosomes in Multiple Myeloma].

OBJECTIVE: To investigate the expression level and prognostic value of miR-21, miR-18a, miR-146a, and Let-7b derived from serum exosomes in patients with multiple myeloma (MM). METHODS: Serum exosomes were extracted from 57 MM patients and 20 healthy persons using ExoQuick exosome precipitation solution kit, and the relative expression level of miR-21, miR-18a, miR-146a, and Let-7b derived from serum exosomes was measured by RT-qPCR. Correlations of the expression levels of all miRNAs mentioned above with routine laboratory parameters were analyzed by Spearman correlation analysis. The relationship between the expression level of miR-21, miR-18a, miR-146a, and Let-7b derived from serum exosomes and overall survival of patients with MM was analyzed using the Kaplan-Meier survival curve. RESULTS: The expression levels of miR-21, miR-18a, and Let-7b derived from serum exosomes in patients with MM were significantly lower than those in the normal control group (P<0.001), while the expression level of miR-146a between the two groups was not significantly different (P>0.05). The expression level of miR-21 was strongly negatively correlated with serum ß2-microglobulin concentration (r=-0.830), and weakly negatively correlated with serum creatinine, corrected serum calcium, and cystatin C (r=-0.488, -0.282, -0.627). The expression levels of Let-7b and miR-18a were also weakly negatively correlated with the corrected serum calcium, ß2-microglobulin, and cystatin C concentration (r=-0.305, -0.362, -0.461; -0.317, -0.542, -0.434). However, there was no significant correlation between the expression level of miR-146a and routine laboratory parameters in MM patients. The overall survival rate of MM patients with low expression level of miR-21, miR-18a, and Let-7b significantly decreased compared with high expression level group (P<0.05), however, the expression level of miR-146a was not related to the overall survival rate. CONCLUSION: Aberrant low expression levels of miR-21, miR-18a, and Let-7b derived from serum exosomes exist in patients with MM, which are associated with a worse overall survival rate.

Expression of CDK6 in Stomach Cancer and the Effect of CDK4/6 Inhibitor PD-0332991 on the Function of Stomach Cancer Cells.

Objective: To study the expression and prognostic value of CDK6 in stomach cancer and the function of CDK4/6 inhibitor PD-0332991 on the proliferation of stomach cancer cells. Methods: Immunohistochemistry was used to detect the expression of CDK6 in stomach cancer tissues and adjacent normal tissues and to analyze the effect of CDK6 on clinicopathological parameters of stomach cancer patients. Kaplan-Meier plotter was employed to study the relationship between CDK6 and overall survival in stomach cancer. Western blot and RT-PCR were used to detect protein and gene expression of CDK6 in different cells. The effects of CDK4/6 inhibitor PD-0332991 on apoptosis and aging of stomach cancer cells were detected by flow cytometry and ß-galactosidase aging staining assay. The effects of CDK4/6 inhibitor PD-0332991 on the invasion and migration of stomach cancer cells were explored by the wound healing experiment and the Transwell experiment. The supernatant of stomach cancer cells was collected, and the effect of CDK4/6 inhibitor PD-0332991 on tumor markers of stomach cancer cells was detected by biochemical immunoassay. Results: (1) CDK6 was highly expressed in stomach cancer tissues and cells. (2) Abnormally elevated CDK6 expression results in shorter survival in stomach cancer patients. (3) CDK4/6 inhibitor PD-0332991 could block the proliferation of stomach cancer cells, but not stomach epithelial proliferation. PD-0332991 could inhibit the secretion of pro-GRP by MGC 823. (4) PD-0332991 could advance the development of the apoptosis and senescence of stomach cancer cells and suppressed the invasion and migration of stomach cancer cells. Conclusion: CDK6 expression is elevated in gastric cancer, and the CDK4/6 inhibitor PD-0332991 can remarkably promote apoptosis and senescence of stomach cancer cells and effectively inhibit the migration and invasion of stomach cancer cells.

Increased sensitivity of gp210 autoantibody detection using a newly designed gp210 antigen.

OBJECTIVES: The detection of autoantibody to glycoprotein 210 (gp210 Ab) against a 15 amino-acid peptide epitope by enzyme-linked immunosorbent assay (ELISA) has been widely used in the diagnosis of primary biliary cholangitis (PBC). However, this small peptide antigen presents spatial limitations for antibody access, which reduces the sensitivity of autoantibody detection. A recombinant gp210 antigen was constructed for increased sensitivity in antibody detection is described here. METHODS: The gp210 C terminal 18 amino acid coding sequence was ligated to the modified C-terminal 108 amino acid coding sequence of human serum albumin (mHSA108) and produced as a recombinant gp210 antigen mHSA108-gp210-C18. Measurements of gp210 Ab using the gp210 C-terminal 25 amino acid peptide (gp210-C25) and mHSA108-gp210-C18 by in-house ELISA were compared. ELISAs with mHSA108-gp210-C18 and commercial INOVA kit for gp210 Ab detection were also compared in PBC patients and healthy controls. The correlation between the two assays was analyzed and their efficiency in diagnosing was compared. RESULTS: Of 86 PBC samples, 35 (40.70%) and 44 (52.33%) positive samples were detected for anti-gp210 Ab using gp210-C25 and mHSA108-gp210-C18, respectively. Of 252 samples from PBC, 114 (45.24%) were positive for mHSA108-gp210-C18 ELISA whereas 94 (37.3%) for commercial ELISA (INOVA). All positive samples detected with commercial ELISA kit were also tested positive in mHSA108-gp210-C18 ELISA. Among 374 patients with other autoimmune diseases, anti-gp210 Ab were detected by mHSA108-gp210-C18 ELISA in 0.95% systemic lupus erythematosus (SLE) patients (2/210), 13.04% rheumatoid arthritis (RA) patients (13/97), and 1.47% of Sjögren's Syndrome (SS) patients (1/67). CONCLUSIONS: Compared to the gp210 peptide antigen, the sensitivity of the ELISA system using mHSA108-gp210-C18 antigen was improved. The novel gp210 antigen could be useful for screening patients known to be at increased risk of developing PBC.

Prevalence and genotype distribution of high-risk human papillomavirus in 34 420 cases in Yangzhou city, Jiangsu province, China.

We aimed to study the infection status and distribution of human papillomavirus (HPV) in Yangzhou City to provide precise guidance for the prevention and treatment of cervical cancer in this area. Reproductive tract secretions were collected from patients admitted at Subei People's Hospital over the past 3 years. Fifteen high-risk HPV (HR-HPV) genotypes were analyzed by fluorescent polymerase chain reaction. The positive rate of HR-HPV in 34 420 subjects was 23.56%. There was no significant difference in the rate of overall infection between males and females (χ 2 = 0.04; p = 0.952 > 0.05). The five genotypes with high infection rates in the population were HPV52, HPV58, HPV16, HPV51, and HPV39. Single infection was found to be dominant, primarily with the HPV52 genotype. The infection rate was higher in patients less than 20 years old and more than 60 years old. Most patients with cervical intraepithelial neoplasms 2/3 and cervical cancer were infected by HPV16, followed by those infected by HPV52 and HPV58. There was a significant difference in the infection rate of HPV16 among patients with different cervical lesions (χ 2 = 31.660; p < 0.01), and the infection rate of HPV16 was higher in patients with cervical cancer than in healthy individuals. Single infection was dominant among the study patients with HPV infection in Yangzhou city. There was no significant difference in infection rate and genotype distribution between males and females. The infection rate in young and old women was higher, and the rate increased with age (>20 years).

The prognostic value of circRNAs for gastric cancer: A systematic review and meta-analysis.

Gastric cancer is the third leading cause of cancer-related deaths worldwide. Novel biomarkers circRNAs can play an important role in the development of gastric cancer as oncogenes or tumor suppressor genes. The purpose of this study was to clarify the relationship between the abnormal expression of multiple circRNAs and their prognostic value in gastric cancer patients through a meta-analysis. We researched articles reporting the relationship between circRNAs and the prognosis of gastric cancer published in PubMed, Cochrane, Embase, Web of Science, Wanfang, CNKI, and VIP databases before 31 December 2019. Thirty-five articles were selected for the meta-analysis, involving 3135 gastric cancer patients. The total HR values (95% CI) of OS and DFS related to highly expressed circRNAs that indicated worse prognosis were 1.83 (1.64-2.03; p < 0.001) and 1.66 (1.33-2.07; p < 0.001), respectively. The total HR (95% CI) of OS and DFS related to highly expressed circRNAs that indicated better prognosis was 0.54 (0.45-0.66; p < 0.001) and 0.58 (0.43-0.78; p < 0.001), respectively. Two panels of five circRNAs predicted a more considerable HR value (circ_0009910, hsa_circ_0000467, hsa_circ_0065149, hsa_circ_0081143, and circDLST; and circSMARCA5, circLMTK2, hsa_circ_0001017, hsa_circ_0061276, and circ-KIAA1244). The results of the meta-analysis were 2.63 (2.08-3.33; p < 0.001) and 0.39 (0.27-0.59; p < 0.001) for OS, respectively. The two panels of dysregulated circRNAs can be considered as more suitable potential prognostic tumor biomarkers in patients with gastric cancer because of their larger HR values.

Identification of nine key genes by bioinformatics analysis for predicting poor prognosis in smoking-induced lung adenocarcinoma.

AIM: To screen and identify key genes related to the development of smoking-induced lung adenocarcinoma (LUAD). MATERIALS & METHODS: We obtained data from the GEO chip dataset GSE31210. The differentially expressed genes were screened by GEO2R. The protein interaction network of differentially expressed genes was constructed by STRING and Cytoscape. Finally, core genes were screened. The overall survival time of patients with the core genes was analyzed by Kaplan-Meier method. Gene ontology and Kyoto encyclopedia of genes and genomes bioaccumulation was calculated by DAVID. RESULTS: Functional enrichment analysis indicated that nine key genes were actively involved in the biological process of smoking-related LUAD. CONCLUSION: 23 core genes and nine key genes among them were correlated with adverse prognosis of LUAD induced by smoking.

Risk factors and prognosis of bone metastases in newly diagnosed gastric cancer.

Aim: To predict the occurrence of bone metastases and prognosis among patients with gastric cancer on a population level. Materials & methods: Data were obtained from the SEER database (2010-2016). Multivariable logistic regression and multivariable Cox regression were used to determine factors that predict the occurrence of bone metastasis and prognosis. Results: Cardia cancer, younger age, white race, poor differentiation grade, higher N stage, diffuse-type were positively associated with the presence of bone metastasis. For gastric cancer patients with bone metastasis, the median survival time was longer (9.0 months) among patients with surgery of primary site compared with those without surgery (3.0 months). Conclusion: According to the results of risk assessment, clinical efforts should be targeted to focus on screening high-risk patients.

Fine mapping of the MHC region identifies major independent variants associated with Han Chinese primary biliary cholangitis.

The genetic association of primary biliary cholangitis with major histocompatibility complex (MHC) has been widely confirmed among different ethnicities. To map specific MHC region variants associated with PBC in a Han Chinese cohort, we imputed HLA antigens and amino acids (AA) in 1126 PBC cases and 1770 healthy control subjects using a Han-MHC reference database. We demonstrate that HLA-DRB1 and/or HLA-DQB1 contributed the strongest signals, and that HLA-DPB1 was a separate independent locus. Regression analyses with classical HLA alleles indicate that HLA-DQB1*03:01 or HLA-DQß1-Pro55, HLA-DPB1*17:01 or HLA-DPß1-Asp84 and HLA-DRB1*08:03 could largely explain MHC association with PBC. Forward stepwise regression analyses with HLA amino acid variants localize the major signals to HLA-DRß1-Ala74, HLA-DQß1-Pro55 and HLA-DPß1-Asp84. Electrostatic potential calculations implicated AA variations at HLA-DQß1 position 55 and HLA-DPß1 position 84 as critical to peptide binding properties. Furthermore, although several critical Han Chinese AA variants differed from those shown in European populations, the predicted effects on antigen binding are likely to be very similar or identical and underlie the major component of MHC association with PBC.

Identification of specific key Hub genes in young hepatocellular carcinoma patients based on bioinformatical analysis and its clinical significance / 中国肿瘤生物治疗杂志

@#Objective: To identify the specific Hub genes in young hepatocellular carcinoma (HCC) patients, and to explore their biological and clinical significance by using bioinformatic methods. Methods: The data information of HCC and normal tissues of young (≤40 years old at diagnosis) and old (＞40 years old at diagnosis) HCC patients were obtained from GEO chip data set GSE45267. The differentially expressed genes (DEGs) in HCC tissues as comparing to normal tissues in the two groups were screened by using GEO2R and Venn chart software. The Protein-Protein Interaction (PPI) network of the specific DEGs in young group was constructed by bioinformatics tools STRING and Cytoscape to screen the Hub genes and significant modules. The Hub genes were verified by GEPIA database, and the overall survival time was analyzed by Kaplan-Meier. Finally, Gene Ontology (GO) Enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were used to analyze the DEGs specific to young group and the common DEGs of the two groups by DAVID. Results: Finally, 117 up-regulated and 179 down-regulated DEGs specific to the young group were screened out, and PPI network screened 10 most connected genes as Hub genes, among which 7 Hub genes were concentrated in the first module. Six up-regulated Hub genes, including TYMS, CDC6, BUB1, TPX2, OIP5 and KIF23, were indicated to associate with the poor prognosis in young HCC patients by GEPIA and Kaplan-Meier analysis. GO function and KEGG pathway analyses showed that the DEGs specific to young HCC patients were mainly involved in biological processes such as ATP binding, and were mainly enriched in S phase of cell cycle; while the common DEGs of two groups were mainly involved in biological processes such as cyclooxygenase P450 and cell division, and were mainly enriched in the G2/M phase of the cell cycle. Conclusion: In this study, 6 up-regulated DEGs specific to young group that suggested poor prognosis were identified, which may be the potential therapeutic and prognostic targets for young patients with HCC.

Genome-wide Association Studies of Specific Antinuclear Autoantibody Subphenotypes in Primary Biliary Cholangitis.

Anti-nuclear antibodies to speckled 100 kDa (sp100) and glycoprotein 210 (gp210) are specific serologic markers of primary biliary cholangitis (PBC) of uncertain/controversial clinical or prognostic significance. To study the genetic determinants associated with sp100 and gp210 autoantibody subphenotypes, we performed a genome-wide association analysis of 930 PBC cases based on their autoantibody status, followed by a replication study in 1,252 PBC cases. We confirmed single-nucleotide polymorphisms rs492899 (P = 3.27 × 10-22 ; odds ratio [OR], 2.90; 95% confidence interval [CI], 2.34-3.66) and rs1794280 (P = 5.78 × 10-28 ; OR, 3.89; 95% CI, 3.05-4.96) in the human major histocompatibility complex (MHC) region associated with the sp100 autoantibody. However, no genetic variant was identified as being associated with the gp210 autoantibody. To further define specific classical human leukocyte antigen (HLA) alleles or amino acids associated with the sp100 autoantibody, we imputed 922 PBC cases (211 anti-sp100-positive versus 711 negative cases) using a Han Chinese MHC reference database. Conditional analysis identified that HLA-DRß1-Asn77/Arg74, DRß1-Ser37, and DPß1-Lys65 were major determinants for sp100 production. For the classical HLA alleles, the strongest association was with DRB1*03:01 (P = 1.51 × 10-9 ; OR, 2.97; 95% CI, 2.06-4.29). Regression analysis with classical HLA alleles identified DRB1*03:01, DRB1*15:01, DRB1*01, and DPB1*03:01 alleles can explain most of the HLA association with sp100 autoantibody. Conclusion: This study indicated significant genetic predisposition to the sp100 autoantibody, but not the gp210 autoantibody, subphenotype in PBC patients. Additional studies will be necessary to determine if these findings have clinical significance to PBC pathogenesis and/or therapeutics.

Spreading of Pandemic Vibrio parahaemolyticus O3:K6 and Its Serovariants: A Re-analysis of Strains Isolated from Multiple Studies.

In China, V. parahaemolyticus has been a leading cause of foodborne outbreaks and bacterial infectious diarrhea since the 1990s, and most infections have been associated with the pandemic V. parahaemolyticus O3:K6 and its serovariants. However, a comprehensive overview of the sero-prevalence and genetic diversity of the pandemic V. parahaemolyticus clone in China is lacking. To compensate for this deficiency, pandemic isolates in both clinical and environmental Chinese samples collected from multiple studies were analyzed in this study. Surprisingly, as many as 27 clinical pandemic serovariants were identified and were widely distributed across nine coastal provinces and two inland provinces (Beijing and Sichuan). O3:K6, O4:K68, and O1:KUT represented the predominant clinical serovars. Only four environmental pandemic serovariants had previously been reported, and they were spread throughout Shanghai (O1:KUT, O3:K6), Jiangsu (O3:K6, O4:K48), Zhejiang (O3:K6), and Guangdong (O4:K9). Notably, 24 pandemic serovariants were detected within a short time frame (from 2006 to 2012). The pandemic isolates were divided into 15 sequence types (STs), 10 of which fell within clonal complex (CC) 3. Only three STs (ST3, ST192, and ST305) were identified in environmental isolates. Substantial serotypic diversity was mainly observed among isolates within pandemic ST3, which comprised 21 combinations of O/K antigens. The pandemic O3:K6 serotype showed a high level of sequence diversity, which was shared by eight different STs (ST3, ST227, ST431, ST435, ST487, ST489, ST526, and ST672). Antimicrobial susceptibility testing revealed that most isolates shared similar antibiotic susceptibility profiles. They were resistant to ampicillin but sensitive to most other drugs that were tested. In conclusion, the high levels of serotypic and genetic diversity of the pandemic clone suggest that the involved regions are becoming important reservoirs for the emergence of novel pandemic strains. We underscore the need for routine monitoring to prevent pandemic V. parahaemolyticus infection, which includes monitoring antimicrobial responses to avoid excessive misuse of antibiotics. Further investigations are also needed to delineate the specific mechanisms underlying the possible seroconversion of pandemic isolates.

A genome-wide association study identifies six novel risk loci for primary biliary cholangitis.

Primary biliary cholangitis (PBC) is an autoimmune liver disease with a strong hereditary component. Here, we report a genome-wide association study that included 1,122 PBC cases and 4,036 controls of Han Chinese descent, with subsequent replication in a separate cohort of 907 PBC cases and 2,127 controls. Our results show genome-wide association of 14 PBC risk loci including previously identified 6p21 (HLA-DRA and DPB1), 17q12 (ORMDL3), 3q13.33 (CD80), 2q32.3 (STAT1/STAT4), 3q25.33 (IL12A), 4q24 (NF-κB) and 22q13.1 (RPL3/SYNGR1). We also identified variants in IL21, IL21R, CD28/CTLA4/ICOS, CD58, ARID3A and IL16 as novel PBC risk loci. These new findings and histochemical studies showing enhanced expression of IL21 and IL21R in PBC livers (particularly in the hepatic portal tracks) support a disease mechanism in which the deregulation of the IL21 signalling pathway, in addition to CD4 T-cell activation and T-cell co-stimulation are critical components in the development of PBC.

High expression of miR-16 and miR-451 predicating better prognosis in patients with gastric cancer.

PURPOSE: To investigate the expression pattern of miR-16 and miR-451 and evaluate their prognostic value in 180 GC patients undergoing surgery. METHODS: In our previous study, a panel of five circulating miRNAs (miR-16, miR-25, miR-92a, miR-451 and miR-486-5p) can be used as a potential biomarker for detecting of early-stage gastric carcinoma (GC). Tissue microarrays were constructed from 180 patients with GC after surgery. MiR-16 and miR-451 expression was detected by miRNA-locked nucleic acid in situ hybridization, and their relationship with clinicopathological parameters and overall survival was analyzed. RESULTS: MiR-16 expression was decreased in 30.6 % (55/180) of GC, increased in 54.4 % (98/180) and unchanged in 15.0 % (27/180), compared with paracancerous normal tissue (P < 0.001). MiR-451 expression was decreased in 17.8 % (32/180), increased in 62.8 % (113/180) and unchanged in 19.4 % (35/180) of GC, compared with paracancerous normal tissue (P < 0.001).Univariate analysis indicated that low miR-16 and miR-451 expression, tumor stage, tumor status, node status and tumor size were significant negative prognostic predictors for overall survival in patients with GC (P < 0.001, P < 0.001, P = 0.002, P < 0.001 and P = 0.001, respectively). Multivariate regression analysis demonstrated that stage [hazard ratio (HR) 1.80; 95 % confidence interval (CI) 1.0-3.26; P = 0.05], low expression of miR-16 (HR 2.26; 95 % CI 1.51-3.40; P < 0.001) and miR-451 (HR 2.01; 95 % CI 1.36-2.96; P < 0.001) predicted shorter OS, while tumor status (HR 1.59; 95 % CI 0.73-3.48 P = 0.242), lymph node metastasis (HR 1.41; 95 % CI 0.71-2.82; P = 0.326) and tumor size (HR 1.53; 95 % CI 0.92-2.55; P = 0.099) were not. Moreover, patients with both miR-16 and miR-451 high expression have better OS than those with two miRNAs unchanged or low expression in GC tissues. Patients with both miR-16 and miR-451 high have better OS than patients with single miR-451 high expression. CONCLUSIONS: High expression of miR-16 and miR-451 was associated with longer OS in GC patients. Especially patients with miR-16 and miR-451 double high expression will predict better OS. MiR-16 and miR-451 may be used as novel makers to evaluate prognosis and provide a new treatment target in GC.