Ming Guan

Identification of miR-130a, miR-27b and miR-210 as serum biomarkers for atherosclerosis obliterans.

BACKGROUNDnArteriosclerosis obliterans (ASO) is a kind of peripheral arterial disease. Most patients with ASO have no apparent clinical symptoms early on, but a diagnosis at the early stage is essential to prevent progression. Unfortunately, the specific and sensitive markers for ASO are still lacking currently. In this study, using both tissue samples and blood samples obtained from ASO patients, we aim to find a cluster of miRNAs that can be used as new risk-markers for the diagnosis of ASO in the earlier stages.nnnMETHODSnWe enrolled 104 patients diagnosed with ASO and 105 age-matched controls. We examined the expression levels of a series of miRNAs in both intima samples and serum samples from these patients.nnnRESULTSnLevels of miR-21, miR-130a, miR-27b, let-7f and miR-210 significantly increased, while levels of miR-221 and miR-222 significantly decreased in the sclerotic samples compared with normal samples. Significant increase of miR-130a, miR-27b and miR-210 expression was observed in the serum samples of ASO patients. Moreover, the expression of miR-130a and miR-27b in sera of ASO patients was positively correlated with Fontaine stages.nnnCONCLUSIONSnThe serum levels of miR-130a, miR-27b and miR-210 may serve as potential biomarkers for early stage ASO.

Copy number variations of interleukin-17F, interleukin-21, and interleukin-22 are associated with systemic lupus erythematosus.

OBJECTIVEnSystemic lupus erythematosus (SLE) represents the classic prototype of systemic autoimmune disease. The identification of the Th17 cell subset has provided new understanding regarding the underlying mechanisms of autoimmunity. Copy number variations (CNVS) have been discovered to have phenotypic consequences and are associated with various types of diseases. We undertook this study to explore a possible association between CNVS of Th17 cell-related genes and the risk of SLE.nnnMETHODSnWe extracted genomic DNA and RNA from 938 SLE patients and 1,017 healthy controls. We examined CNVS of Th17 cell-related genes, including retinoic acid receptor-related orphan nuclear receptor γt, STAT-3, interleukin-6 (IL-6), transforming growth factor β, tumor necrosis factor α, IL-17A, IL-17F, IL-21, IL-22, IL-23A, CCL20, and CCR6, and levels of messenger RNA (mRNA) for IL-17F, IL-21, and IL-22.nnnRESULTSnGenotype and allele frequencies for copy number amplifications of IL-17F, IL-21, and IL-22 were found to be significantly higher in SLE patients than in healthy controls. CNVS of IL-17F, IL-21, and IL-22 had no synergistic contribution to SLE. The mRNA expression of IL-17F, IL-21, and IL-22 in the samples with >2 copies of DNA was significantly higher than that in those with 2 copies of DNA.nnnCONCLUSIONnOur findings indicate that CNVS of IL-17F, IL-21, and IL-22 are associated with the risk of SLE.

Copy number variations of the human histamine H4 receptor gene are associated with systemic lupus erythematosus.

Backgroundu2002 Systemic lupus erythematosus (SLE) is a complex genetic disease; the histamine H4 receptor (HRH4) has been shown to be related to different kinds of autoimmune disorders; and copy number variations (CNVs) have been found to be associated with various types of diseases.

High-resolution melting analysis of ADAMTS18 methylation levels in gastric, colorectal and pancreatic cancers

A disintegrin and metalloprotease with trombospondin motifs (ADAMTS) is a family of proteins characterized by the presence of a metalloproteinase domain linked to a variety of specialized ancillary domains. ADAMTS18 is a putative tumor suppressive gene related to nasopharyngeal carcinoma. We used high-resolution melting (HRM) analysis to detect the methylation levels of ADAMTS18 gene in 100 gastric cancers, 100 colorectal cancers, 70 pancreatic cancers, and equal number of adjacent normal tissues. The frequency of ADAMTS18 methylation in all three types of cancers was significantly higher than that in normal tissues. Expression levels of ADAMTS18 were inversely correlated with methylation levels. No significant association was found between ADAMTS18 methylation status and TNM staging in the cancers. In summary, epigenetic regulation of ADAMTS18 was associated with carcinogenesis. The application of HRM analysis is a fast and high-throughput way to investigate the epigenetic status of ADAMTS18.

High-resolution melting analysis of ADAMTS9 methylation levels in gastric, colorectal, and pancreatic cancers

ADAMTS (a disintegrin and metalloprotease with thrombospondin motifs) is a family of proteins characterized by the presence of a metalloproteinase domain linked to a variety of specialized ancillary domains. The ADAMTS9 gene (ADAM metallopeptidase with thrombospondin type 1 motif, 9); has been characterized as a novel tumor suppressor gene in and epigenetically silenced in association with lymph node metastases in nasopharyngeal carcinoma. High-resolution melting (HRM) analysis has been used as a tool for analysis of promoter methylation. Here, we report HRM analysis used to detect the methylation levels of ADAMTS9 gene in 100 gastric cancers, 100 colorectal cancers, 70 pancreatic cancers, and an equal number of adjacent normal tissues. The frequency of ADAMTS9 methylation in all three types of cancers was significantly higher than in normal tissues. Consistent with previous reports, expression levels of ADAMTS9 were inversely correlated with methylation levels. There was no significant association between ADAMTS9 methylation status and tumor-node-metastasis staging in all three types of cancers. In summary, application of HRM analysis to large numbers of clinical samples is a rapid and high-throughput way to investigate the epigenetic status of ADAMTS9. The present study is novel in evaluating the prevalence of ADAMTS9 methylation based on a large number of tumor samples and showing that epigenetic regulation of ADAMTS9 was associated with carcinogenesis.

Copy Number Variations of EphA3 Are Associated With Multiple Types of Hematologic Malignancies

BACKGROUNDnEphA3 is a component of the Eph receptor family, the largest subgroup of the receptor tyrosine kinase (RTK) family. A recent array-based study implicated the presence of copy-number variations (CNVs) of EphA3 in the genomes of acute myelogenous leukemia. CNVs are present in the general population at varying degrees, and have been found to associate with various types of diseases including hematologic malignancies. However, most of the current studies focused on the genome-wide screening of CNVs, and the functional impact of such regions needs to be extensively investigated in large number of clinical samples.nnnPATIENTS AND METHODSnIn our study, we collected 617 bone marrow samples from multiple types of hematologic malignancies as well as healthy controls. DNA copy numbers and mRNA levels of EphA3 in these samples were examined.nnnRESULTSnWe found significant association between the CNVs of EphA3 and these hematologic malignancies including acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), multiple myeloma (MM), and myelodysplastic syndrome (MDS). We also observed a positive correlation between the relative mRNA level and gene dosage of EphA3.nnnCONCLUSIONnThe CNVs of EphA3 were associated with multiple types of hematologic malignancies including ALL, AML, CLL, CML, MM, and MDS.

MDA-9 and GRP78 as potential diagnostic biomarkers for early detection of melanoma metastasis

Metastatic melanoma, the primary cause of skin cancer-related death, warrants new diagnostic and therapeutic approaches that target the regulatory machinery at molecular level. The heterogeneity and complexity of melanoma result in the difficulty to find biomarkers and targets for early detection and treatment. Here, we investigated metastasis-associated proteins by comparing the proteomic profiles of primary cutaneous melanomas to their matched lymph node metastases, which minimizes heterogeneity among samples from different patients. Results of two-dimensional gel electrophoresis (2-DE) followed by proteomic analysis revealed eight differentially expressed proteins. Among them, seven proteins (α-enolase, cofilin-1, LDH, m-β-actin, Nm23, GRP78, and MDA-9) showed increased and one (annexin A2) showed decreased expression in metastatic lymph node tissues than in primary melanomas. MDA-9 and GRP78 were the most highly expressed proteins in lymph node metastases, which was validated by immunohistochemical staining. Moreover, exosomes from serum samples of metastatic melanoma patients contained higher levels of MDA-9 and GRP78 than those of patients without metastases, indicating the potential of MDA-9 and GRP78 to be biomarkers for early detection of metastasis. Further, small interfering RNA (siRNA)-mediated knockdown confirmed a functional role for MDA-9 and GRP78 to promote cell invasion in the A375 cells. Finally, we showed that GRP78 co-localized with MDA-9 in 293T cells. Taken together, our findings support MDA-9, co-expressed with GRP78, as a melanoma protein associated with lymph node metastasis. Investigating how MDA-9 and GRP78 interact to contribute to melanoma metastasis and disease progression could reveal new potential avenues of targeted therapy and/or useful biomarkers for diagnosis and prognosis.

Semi-quantitative detection of GADD45-gamma methylation levels in gastric, colorectal and pancreatic cancers using methylation-sensitive high-resolution melting analysis.

PurposeGADD45 is a family of proteins involved in DNA damage response and cell growth arrest. GADD45G was identified as an interleukin-2-induced immediate-early gene, and methylation of GADD45G was studied in various tumor cell lines and a few primary tumor samples. High-resolution melting (HRM) analysis has been used as a novel tool for analysis of promoter methylation.MethodsIn our study, we used HRM analysis to detect the methylation levels of GADD45G gene in 100 gastric cancers, 100 colorectal cancers, 70 pancreatic cancers and equal number of adjacent normal tissues.ResultsThe frequency of GADD45G methylation in all three types of cancers was significantly higher than that in normal tissues. Consistent with previous reports, expression levels of GADD45G were inversely correlated with methylation levels. But we did not find significant association between GADD45G methylation status and TNM staging in all three types of cancers.ConclusionsIn summary, application of HRM analysis to large amount of clinical samples proves to be a fast and high-throughput way to investigate the epigenetic status of GADD45G. And this is the first study to evaluate the prevalence of GADD45G methylation based on large amount of tumor samples, showing that epigenetic regulation of GADD45G was associated with carcinogenesis.

Downregulation of EphA5 by promoter methylation in human prostate cancer

BackgroundEphA5 is a member of the Eph/ephrin family and plays a critical role in the regulation of carcinogenesis. A significant reduction of EphA5 transcripts in high-grade prostate cancer tissue was shown using a transcriptomic analysis, compared to the low-grade prostate cancer tissue. As less is known about the mechanism of EphA5 downregulation and the function of EphA5, here we investigated the expression and an epigenetic change of EphA5 in prostate cancer and determined if these findings were correlated with clinicopathologic characteristics of prostate cancer.MethodsSeven prostate cell lines (RWPE-1, LNCap, LNCap-LN3, CWR22rv-1, PC-3, PC-3M-LN4, and DU145), thirty-nine BPH, twenty-two primary prostate carcinomas, twenty-three paired noncancerous and cancerous prostate tissues were examined via qRT-PCR, methylation-specific PCR, bisulfite sequencing, immunohistochemistry and western blotting. The role of EphA5 in prostate cancer cell migration and invasion was examined by wound healing and transwell assay.ResultsDownregulation or loss of EphA5 mRNA or protein expression was detected in 28 of 45 (62.2%) prostate carcinomas, 2 of 39 (5.1%) hyperplasias, and all 6 prostate cancer cell lines. Methylation of the EphA5 promoter region was present in 32 of 45 (71.1%) carcinoma samples, 3 of 39 (7.7%) hyperplasias, and the 6 prostate cancer cell lines. Among 23 paired prostate carcinoma tissues, 16 tumor samples exhibited the hypermethylation of EphA5, and 15 of these 16 specimens (93.8%) shown the downregulation of EphA5 expression than that of their respectively matched noncancerous samples. Immunostaining analysis demonstrated that the EphA5 protein was absent or down-regulated in 10 of 13 (76.9%) available carcinoma samples, and 8 of these 10 samples (80.0%) exhibited hypermethylation. The frequency of EphA5 methylation was higher in cancer patients with an elevated Gleason score or T3-T4 staging. Following the treatment of 6 prostate cancer cell lines with 5-aza-2′-deoxycytidine, the levels of EphA5 mRNA were significantly increased. Prostate cancer cells invasion and migration were significantly suppressed by ectopic expression of EphA5 in vitro.ConclusionOur study provides evidence that EphA5 is a potential target for epigenetic silencing in primary prostate cancer and is a potentially valuable prognosis predictor and thereapeutic marker for prostate cancer.

Downregulation of DLC-1 Gene by Promoter Methylation during Primary Colorectal Cancer Progression

Purpose. DLC-1 is a tumor suppressor gene frequently silenced in human cancers. However, the pathogenicity of DLC-1 epigenetic silencing in the mucosa-adenoma-carcinoma transformation process of colorectal cancer (CRC) has not been studied. Methods. Promoter methylation status of DLC-1 was evaluated in 4 human CRC cell lines, 48 normal mucosa, 57 adenomas, and 80 CRC tissues with methylation-sensitive high-resolution melting analysis (MS-HRMA), while the mRNA expression was examined by qPCR. HRMA was utilized to detect the KRAS codon 12, 13 and BRAF V600Emutations. Results. Partial (1%–10%) and extensive (10%–100%) DLC-1 promoter methylations were observed in 10% and 0% of normal mucosa, 46% and 14% of adenomas, and 60% and 36% of CRCs, respectively. The promoter methylation of DLC-1 was related with the reduction of gene expression and the advanced Dukes stages (Stage C and D). DLC-1 promoter methylation and KRAS mutations are common concurrent pathological alternations. Conclusions. Epigenetic alternation plays a key role in the transcriptional silencing of DLC-1. It is also an independent risk factor related to the carcinogenesis of colorectal tumors and spans over its pathogenesis process. Therefore, DLC-1 promoter methylation quantitation may have a promising significance in the evaluation and management of CRC patients.