Project description:Rifampicin and efavirenz are commonly used medicines to treat tuberculosis (rifampicin) and HIV/AIDS (efavirenz). Rifampicin and efavirenz are metabolized in the liver and exposure to these medicines can alter microRNA expression in hepatocytes. Changes in microRNA expression may affect metabolism of these medicines and potentially influence how patient's respond to therapy. It is important to understand changes in microRNA expression in a hepatic cell-based model. Differentiated HepaRG cells were used because mRNA expression and induction of drug metabolizing enzymes are comparable to that of primary human hepatocytes yet differentiated HepaRG cells have an extended lifespan. Differentiated HepaRG cells were obtained from Merck Millipore and cultured as an adherent cell line at a density of 315000 cells/well using collagen I coated 24-well plates. Clinically relevant concentrations of rifampicin and efavirenz were selected as 6.4uM efavirenz (dissolved in 100% dimethyl sulfoxide) and 24.4uM rifampicin (dissolved in 100% dimethyl sulfoxide), while 0.02% dimethyl sulfoxide was used as control to minimize toxicity. HepaRG serum-free induction medium was used to dilute efavirenz and rifampicin. On day 7 since thawing differentiated HepaRG cells, these cells were treated with 6.4uM efavirenz or 24.4uM rifampicin or 0.02% dimethyl sulfoxide for a period of 24 hours. Three biological replicates were available for each treatment condition. Total RNA was isolated from the cells, after treatment, using the Quick-RNATM MiniPrep Kit from Zymo Research Corporation. MicroRNA expression profiling was performed using the TaqMan® OpenArray® Human MicroRNA Panel and QuantStudioTM 12K Flex system. The R/Bioconductor package “Automated Analysis of High-Throughput qPCR Data” were used for data analyses. CT values were used for differential expression analysis and microRNAs with a CT value > 35 was considered undetected. MicroRNAs undetected in any of the replicate samples or microRNAs with AmpScore < 1.24 or CqConf < 0.8 were excluded. Quantile normalization was followed by limma analyses to identify differentially expressed microRNAs for efavirenz versus dimethyl sulfoxide and rifampicin versus dimethyl sulfoxide.

Project description:MicroRNAs (miRNAs) are abundant in the circulation and play a central role in diverse biological processes; they may be useful for early diagnosis of hepatocellular carcinoma (HCC). We conducted a two-phase, case-control study (20 pairs for the discovery set and 49 pairs for the validation set) to test the hypothesis that genome-wide dysregulation of circulating miRNAs differentiate HCC cases from controls. Taqman low density arrays were used to examine genome-wide miRNA expression for the discovery set, and quantitative RT-PCR was used to validate candidate miRNAs for both discovery and validation sets. Sixty-six miRNAs were found to be significantly over-expressed in plasma of HCC cases compared to controls after adjusting for false discovery rate (p<0.05). A volcano plot indicated that 7 miRNAs had greater than 2-fold case-control differences with p<0.01. Four significant miRNAs (miR-150, miR-30c, miR-483-5p and miR-520b) detectable in all samples with varied expression levels were further validated in a validation set. MiR-483-5p was statistically significantly over-expressed in HCC cases compared with controls (3.20 vs. 0.82, p<0.0001). HCC risk factors and clinic-pathological characteristics did not influence miR-483-5p expression. The combination of plasma miR-483-5p level and HCV status can significantly differentiate HCC cases from controls with an AUC of 0.908 (p<0.0001). The sensitivity and specificity were, respectively, 75.5% and 89.8%. These preliminary results suggest the importance of dysregulated circulating miR-483-5p as a potential HCC biomarker. Confirmation of aberrant expression of miR-483-5p in a large prospective HCC study will provide support for its application to HCC detection. A hospital-based HCC case-control study including 20 HCC patients and 20 controls is conducted in Columbia University Medical Center (CUMC), which is approved by the Institutional Review Board. Cases were newly diagnosed HCC patients who were treated in the Hepatobiliary Oncology Clinics, CUMC, and examined pathologically. Controls were recruited from volunteers through the Research Recruitment and Minority Outreach (RRMO) core of Herbert Irving Comprehensive Cancer Center (HICCC). Flyers were placed at strategic locations around CUMC where hospital visitors and employees frequent or were handed out at inreach events at the hospital or at outreach events in the community. Interested participants were directed to contact the trained recruitment staff from the RRMO and given further information about participation. Interested participants were excluded from the control group if diagnosed for any kind of cancer or liver disease. Eligible controls were asked to fill out the same demographic and epidemiological questionnaire as HCC cases. In the current study, controls were matched with HCC cases on age (M-BM-15 yrs), gender (male/female) and ethnicities (Caucasian/Hispanic/African-American/Asian).

Project description:Mantle cell lymphoma (MCL) is an aggressive B-cell non-HodgkinM-bM-^@M-^Ys lymphoma (NHL). In cancers, tumour suppressive microRNAs may be silenced by DNA hypermethylation. By microRNA profiling, miR-155-3p was significantly upregulated upon demethylation treatment of MCL cell lines with 5-aza-2M-bM-^@M-^Y-deoxycytidine (5-azadC). Methylation-specific PCR, verified by pyrosequencing, showed complete methylation of miR-155-3p in one MCL cell line (REC-1). 5-azadC treatment of REC-1 led to demethylation and re-expression of miR-155-3p. Over-expression of miR-155-3p led to increased sub-G1 apoptotic cells and reduced cellular viability, demonstrating its tumour suppressive properties. By luciferase assay, lymphotoxin-beta (LT-M-NM-2) was validated as a miR-155-3p target. In 31 primary MCL, miR-155-3p was found hypermethylated in 6(19%) cases. To test if methylation of miR-155-3p was MCL-specific, miR-155-3p methylation was tested in an additional 191 B-cell, T-cell and NK-cell NHLs, yielding miR-155-3p methylation in 66(34.6%) including 36(27%) non-MCL B-cell, 24(53%) T-cell and 6(46%) of NK-cell lymphoma. Moreover, in 72 primary NHL samples with RNA, miR-155-3p methylation correlated with miR-155-3p downregulation (p=0.030), and LT-M-NM-2 upregulation (p=0.004). Collectively, miR-155-3p is tumour suppressive microRNA hypermethylated in MCL and other NHL subtypes. As miR-155-3p targets LT-M-NM-2, which is an upstream activator of the non-canonical NF-kB signalling, miR-155-3p methylation is potentially important in lymphomagenesis Total RNA isolated from MINO and JEKO-1 before and after 5-azadC treatment were converted into cDNA by MegaplexTM RT Primers and TaqManM-BM-. MicroRNA Reverse Transcription Kit. cDNA was pre-amplified using MegaplexTM PreAmp Primer and loaded onto 384-well format TaqmanM-BM-. human microRNA array A V2.0 & B V3.0. Real-time PCR was performed on 7900HT Real-Time PCR system and raw data were analyzed normalizing to mean of three endogenous controls (U6snRNA, RNU44 and RNU48). Relative microRNA levels were determined by M-NM-^TM-NM-^TCt using endogenous controls and untreated controls using SDS 2.4 and RQ manager 1.2. All experimental procedures and analyses were performed according to manufacturerM-bM-^@M-^Ys instruction, using reagents, system and softwares acquired from Applied Biosystems (Foster City, USA).

Project description:Most (70%) epithelial ovarian cancers (EOCs) are diagnosed late. Non-invasive biomarkers that facilitate disease detection and predict outcome are needed. The microRNAs (miRNAs) represent a new class of biomarkers. This study was to identify and validate plasma miRNAs as biomarkers in EOC. We evaluated plasma samples of 360 EOC patients and 200 healthy controls from two institutions. All samples were grouped into screening, training and validation sets. We scanned the circulating plasma miRNAs by TaqMan low-density array in the screening set and identified/validated miRNA markers by real-time polymerase chain reaction assay in the training set. Receiver operating characteristic and logistic regression analyses established the diagnostic miRNA panel, which were confirmed in the validationsets. We found higher plasma miR-205 and lower let-7f expression in cases than in controls. MiR-205 and let-7f together provided high diagnostic accuracy for EOC, especially in patients with stage I disease. The combination of these two miRNAs and carbohydrate antigen-125 (CA-125) further improved the accuracy of detection. MiR-483-5p expression was elevated in stages III and IV compared with in stages I and II, which was consistent with its expression pattern in tumor tissues. Furthermore, lower levels of let-7f were predictive of poor prognosis in EOC patients. Our findings indicate that plasma miR-205 and let-7f are biomarkers for ovarian cancer detection that complement CA-125; let-7f may be predictive of ovarian cancer prognosis. We designed a multi-stage, retrospective, nested case-control study to determine whether serum miRNA profiles can be used to predict EOC development. All samples were grouped into screening, training, and validation sets. A total of 560 plasma samples (360 cases and 200 controls) were obtained from Tianjin Medical University Cancer Institute and Hospital (TCIH) and Cancer Center of Nanjing Medical University. The cases and controls were well matched for age. The International Federation of Gynaecology and Obstetrics (FIGO) staging system was used to stage cases. All patients and controls were genetically unrelated, ethnic Han Chinese women who were permanent residents of the urban area of Tianjin and Nanjing. Controls with cardiovascular, respiratory, digestive, urinary, reproductive, and endocrine diseases were excluded. The study protocols were approved by the Tianjin and Nanjing Center review committees. In screening set, to detect generalizable miRNA signatures, we pooled serum samples of 10 early-stage cases (stage I), 10 late-stage cases (stage IIIc-IV), and 10 healthy controls, respectively, and analyzed these three pool samples using a TaqMan low-density array (TLDA set 2.0, Applied Biosystems) chip screening in the discovery stage. In training set, the miRNAs identified in the screening set were validated using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) on individual plasma samples of 76 EOC patients and 30 normal controls. In validation set, the validation set comprised a two-phase process. Promising associations from the screening and training set were evaluated in the validation phase I set, comprising 134 cases and 70 controls from Tianjin Center. The validation phase II set included samples of 77 EOC cases and 50 normal controls were from Tianjin Center and 73 EOC cases and 50 normal controls were from Nanjing Center.

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MiRNA expression profile from ANGPTL1 overexpression lung cancer cell

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