MATERIALS AND METHODS: miR-205 expression was investigated in 48 cases of inflammatory, benign and malignant tumor tissue array of the neck, oronasopharynx, larynx and salivary glands by Locked Nucleic Acid in situ hybridization (LNA-ISH) technology.
RESULTS: miR-205 expression was significantly differentially expressed across all of the inflammatory, benign and malignant tumor tissues of the neck. A significant increase in miR-205 staining intensity (p<0.05) was observed from inflammation to benign and malignant tumors in head and neck tissue array, suggesting that miR-205 could be a biomarker to differentiate between cancer and non-cancer tissues.
CONCLUSIONS: LNA-ISH revealed that miR-205 exhibited significant differential cytoplasmic and nuclear staining among inflammation, benign and malignant tumors of head and neck. miR-205 was not only exclusively expressed in squamous epithelial malignancy. This study offers information and a basis for a comprehensive study of the role of miR-205 that may be useful as a biomarker and/or therapeutic target in head and neck tumors.
OBJECTIVES: To screen hypermethylated genes with a microarray approach and to validate selected hypermethylated genes with the methylation-specific polymerase chain reaction (MSPCR).
MATERIALS AND METHODS: Genome-wide analysis of normal oral mucosa and OSCC tissues was conducted using the Illumina methylation microarray. The specified differential genes were selected and hypermethylation status was further verified with an independent cohort sample of OSCC samples. Candidate genes were screened using microarray assay and run by MSPCR analysis.
RESULTS: TP73, PIK3R5, and CELSR3 demonstrated high percentages of differential hypermethylation status.
CONCLUSIONS: Our microarray screening and MSPCR approaches revealed that the signature candidates of differentially hypermethylated genes may possibly become potential biomarkers which would be useful for diagnostic, prognostic and therapeutic targets of OSCC in the near future.
MATERIALS AND METHODS: BCR-ABL positive CML cells resistant to imatinib (K562-R) were developed by overexposure of K562 cell lines to the drug. Cytotoxicity was determined by MTS assays and IC50 values calculated. Apoptosis assays were performed using annexin V-FITC binding assays and analyzed by flow cytometry. Methylation profiles were investigated using methylation specific PCR and sequencing analysis of SOCS-1 and SOCS-3 genes. Gene expression was assessed by quantitative real-time PCR, and protein expression and phosphorylation of STAT1, 2 and 3 were examined by Western blotting.
RESULTS: The IC50 for imatinib on K562 was 362 nM compared to 3,952 nM for K562-R (p=0.001). Percentage of apoptotic cells in K562 increased upto 50% by increasing the concentration of imatinib, in contrast to only 20% in K562-R (p<0.001). A change from non-methylation of the SOCS-3 gene in K562 to complete methylation in K562-R was observed. Gene expression revealed down- regulation of both SOCS-1 and SOCS-3 genes in resistant cells. STAT3 was phosphorylated in K562-R but not K562.
CONCLUSIONS: Development of cells resistant to imatinib is feasible by overexposure of the drug to the cells. Activation of STAT3 protein leads to uncontrolled cell proliferation in imatinib resistant BCR-ABL due to DNA methylation of the SOCS-3 gene. Thus SOCS-3 provides a suitable candidate for mechanisms underlying the development of imatinib resistant in CML patients.
MATERIALS AND METHODS: ER+ MCF7 and ER- MDA-MB-231 cell lines were subjected to two-dimensional electrophoresis (2-DE) and spots of interest were identified by matrix-assisted laser desorption/ionization time of- flight/time- of-flight (MALDI-TOF/TOF) mass spectrometry (MS) analysis after downregulation of RhoGDIα using short interfering RNA (siRNA) and upregulated using GFP-tagged ORF clone of RhoGDIα.
RESULTS: The results showed a total of 35 proteins that were either up- or down-regulated in these cells. Here we identifed 9 and 15 proteins differentially expressed with silencing of RhoGDIα in MCF-7 and the MDA-MB-231 cells, respectively. In addition, 10 proteins were differentially expressed in the upregulation of RhoGDIα in MCF7, while only one protein was identified in the upregulation of RhoGDIα in MDA-MB-231. Based on the biological functions of these proteins, the results revealed that proteins involved in cell migration are more strongly altered with RhoGDI-α activity. Although several of these proteins have been previously indicated in tumorigenesis and invasiveness of breast cancer cells, some ohave not been previously reported to be involved in breast cancer migration. Hence, these proteins may serve as useful candidate biomarkers for tumorigenesis and invasiveness of breast cancer cells.
CONCLUSIONS: Future studies are needed to determine the mechanisms by which these proteins regulate cell migration. The combination of RhoGDIα with other potential biomarkers may be a more promising approach in the inhibition of breast cancer cell migration.
OBJECTIVE: This study aimed to evaluate the anticancer effects of Strobilanthes crispus juice on hepatocellular carcinoma cells.
MATERIALS AND METHODS: MTT assays, flow cytometry, comet assays and the reverse transcription- polymerase chain reaction (RT-PCR) were used to determine the effects of juice on DNA damage and cancer cell numbers.
RESULTS: This juice induced apoptosis after exposure of the HepG2 cell line for 72 h. High percentages of apoptotic cell death and DNA damage were seen at the juice concentrations above 0.1%. It was found that the juice was not toxic for normal cells. In addition, juice exposure increased the expression level of c-myc gene and reduced the expression level of c-fos and c-erbB2 genes in HepG2 cells. The cytotoxic effects of juice on abnormal cells were in dose dependent.
CONCLUSIONS: It was concluded that the Strobilanthes crispus juice may have chemopreventive effects on hepatocellular carcinoma cells.
OBJECTIVES: The objectives of this study were to investigate the CELSR3 hypermethylation level in oral squamous cell carcinomas (OSCCs) using methylation-sensitive high-resolution melting analysis (MS-HRM) and to correlate CELSR3 methylation with patient demographic and clinicopathological parameters.
MATERIALS AND METHODS: Frozen tissue samples of healthy subjects' normal mucosa and OSCCs were examined with regard to their methylation levels of the CELSR3 gene using MS-HRM.
RESULTS: MS-HRM analysis revealed a high methylation level of CELSR3 in 86% of OSCC cases. Significant correlations were found between CELSR3 quantitative methylation levels with patient ethnicity (P=0.005), age (P=0.024) and pathological stages (P=0.004). A moderate positive correlation between CELSR3 and patient age was also evident (R=0.444, P=0.001).
CONCLUSIONS: CELSR3 promoter hypermethylation may be an important mechanism involved in oral carcinogenesis. It may thus be used as a biomarker in OSCC prognostication.
METHODS: A total of 25 CLL patients and 25 normal individuals were recruited in this study. The methylation status of ADAM12 was determined using Methylation-Specific PCR (MSP); whereas, DNA sequencing method was applied for validation of the MSP results.
RESULTS: Among CLL patients, 12 (48%) were partially methylated and 13 (52%) were unmethylated. Meanwhile, 5 (20%) and 20 (80.6%) of healthy individuals were partially methylated and unmethylated, respectively. There was a statistically significant association between the status of methylation at ADAM12 and the presence of CLL (p=0.037).
CONCLUSION: The aberrant methylation of ADAM12 found in this study using MSP assay may provide new exposure to CLL that may improve the gaps involved in genetic epigenetic study in CLL.
MATERIALS AND METHODS: MicroRNA software predicted that miR21 targets VCL while miR29a targets CX3CL1. Twenty benign prostatic hyperplasia (BPH) and 16 high grade CaP formalinfixed paraffin embedded (FFPE) specimens were analysed. From the bone scan results, high grade CaP samples were further classified into CaP with no BM and CaP with BM. Transient transfection with respective microRNA inhibitors was done in both RWPE1 (normal) and PC3 cell lines. QPCR was performed in all FFPE samples and transfected cell lines to measure VCL and CX3CL1 levels.
RESULTS: QPCR confirmed that VCL messenger RNA (mRNA) was significantly down regulated while CX3CL1 was upregulated in all FFPE specimens. Transient transfection with microRNA inhibitors in PC3 cells followed by qPCR of the targeted genes showed that VCL mRNA was significantly up regulated while CX3CL1 mRNA was significantly downregulated compared to the RWPE1 case.
CONCLUSIONS: The downregulation of VCL in FFPE specimens is most likely regulated by miR21 based on the in vitro evidence but the exact mechanism of how miR21 can regulate VCL is unclear. Upregulated in CaP, CX3CL1 was found not regulated by miR29a. More microRNA screening is required to understand the regulation of this chemokine in CaP with bone metastasis. Understanding miRNAmRNA interactions may provide additional knowledge for individualized study of cancers.
METHODS: In this study, plasma miRNA profiles from eight early-stage breast cancer patients and nine age-matched (± 2 years) healthy controls were characterized by miRNA array-based approach, followed by differential gene expression analysis, Independent T-test and construction of Receiver Operating Characteristic (ROC) curve to determine the capability of the assays to discriminate between breast cancer and the healthy control.
RESULTS: Based on the 372-miRNAs microarray profiling, a set of 40 differential miRNAs was extracted regarding to the fold change value at 2 and above. We further sub grouped 40 miRNAs of breast cancer patients that were significantly expressed at 2-fold change and higher. In this set, we discovered that 24 miRNAs were significantly upregulated and 16 miRNAs were significantly downregulated in breast cancer patients, as compared to the miRNA expression of healthy subjects. ROC curve analysis revealed that seven miRNAs (miR-125b-5p, miR-142-3p, miR-145-5p, miR-193a-5p, miR-27b-3p, miR-22-5p and miR-423-5p) had area under curve (AUC) value > 0.7 (AUC p-value < 0.05). Overlapping findings from differential gene expression analysis, ROC analysis, and Independent T-Test resulted in three miRNAs (miR-27b-3p, miR-22-5p, miR-145-5p). Cohen's effect size for these three miRNAs was large with d value are more than 0.95.
CONCLUSION: miR-27b-3p, miR-22-5p, miR-145-5p could be potential biomarkers to distinguish breast cancer patients from healthy controls. A validation study for these three miRNAs in an external set of samples is ongoing.
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METHODS: BCR-ABL positive K562 CML cells were treated with TQ. Cytotoxicity was determined by Trypan blue exclusion assay. Apoptosis assay was performed by annexin V-FITC/PI staining assay and analyzed by flow cytometry. Transcription levels of BCR ABL, JAK2, STAT3, STAT5A and STAT5B genes were evaluated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Protein levels of JAK2 and STAT5 were determined by Jess Assay analysis.
RESULTS: TQ markedly decreased the cell proliferation and induced apoptosis in K562 cells (P < 0.001) in a concentration dependent manner. TQ caused a significant decrease in the transcriptional levels of BCR ABL, JAK2, STAT3, STAT5A and STAT5B genes (P < 0.001). TQ induced a significant decrease in JAK2 and STAT5 protein levels (P < 0.001).
CONCLUSION: our results indicated that TQ inhibited cell growth of K562 cells via downregulation of BCR ABL/ JAK2/STAT3 and STAT5 signaling and reducing JAK2 and STAT5 protein levels.
METHODOLOGY: A cross-sectional study of 32 archived tissue blocks of TNKcNHLs were immunohistochemically stained with c-Myc. The results were microscopically evaluated and statistically analysed to examine the association between the clinicopathological data with the c-Myc expression.
RESULTS: c-Myc protein expressions were detected in 25/32 (78.1%) cases. The median age was 38-years. Malay ethnicity (92.0%) with 21 males and 11 females. c-Myc expressions were seen in T lymphoblastic lymphoma (20%), ALK-positive ALCL (16%) ,PTCL,NOS (16%), extra nodal NK/T-cell lymphoma, nasal type (12%), extra-nodal involvement (78.1%), elevated serum LDH (83.3%) and high ECOG performance status (82.4%). However, no statistical significant of c-Myc in association with the clinicopathological parameters (p > 0.05).
CONCLUSION: There was no statistically significant association of clinicopathological parameters and histological subtypes of TNKcNHLs contributed by small samples tested. However, the attribution of c-Myc in this disease should be further explored.