178 publications from this institution
Quantification of gene expression by real-time RT-PCR has become the gold standard to which all other quantification methods are compared. Two very simple precautions, normalizing the cDNA to contain equal amounts of RNA and validation of the internal control gene, combined with RNA of sufficient quality, will almost certainly guarantee high quality data time after time. Described here is a protocol to normalize the RNA content of multiple cDNA reactions using the NanoDrop ® ND-1000 Spectrophotometer.
<div>Abstract<p>Patients with advanced hepatocellular carcinoma (HCC) face a dismal prognosis because of a lack of any effective therapies. To address this situation, we conducted a preclinical investigation of the therapeutic efficacy of oligonucleotides directed against the oncogenic microRNA miR-221, which has been implicated in HCC. Of 9 chemistries evaluated, we determined that a 2′-<i>O</i>-methyl phosphorothioate-modified anti-miR-221 oligonucleotide was most effective at reducing proliferation <i>in vitro</i>. A cholesterol-modified isoform of anti-miR-221 (chol-anti-miR-221) exhibited improved pharmacokinetics and liver tissue distribution compared with unmodified oligonucleotide. Chol-anti-miR-221 significantly reduced miR-221 levels in liver within a week of intravenous administration and <i>in situ</i> hybridization studies confirmed accumulation of the oligonucleotide in tumor cells <i>in vivo</i>. Within the same period, chol-anti-miR-221 reduced tumor cell proliferation and increased markers of apoptosis and cell-cycle arrest, elevating the tumor doubling time and increasing mouse survival. Taken together, our findings offer a preclinical proof of efficacy for chol-anti-miR-221 in a valid orthotopic mouse model of HCC, suggesting that this targeted agent could benefit treatment for patients with advanced HCC. <i>Cancer Res; 71(24); 7608–16. ©2011 AACR</i>.</p></div>
Abstract Circulating extracellular vesicles (EVs) have gained significant attention for discovering tumor biomarkers. However, isolating EVs with well-defined homogeneous populations from complex biological samples is challenging. Different isolation methods have been found to derive different EV populations carrying different molecular contents, which confounds current investigations and hinders subsequent clinical translation. Therefore, standardizing and building a rigorous assessment of isolated EV quality associated with downstream molecular analysis is essential. To address this need, we introduce a statistical algorithm (ExoQuality Index, EQI) by integrating multiple EV characterizations (size, particle concentration, zeta potential, total protein, and RNA), enabling direct EV quality assessment and comparisons between different isolation methods. We also introduced a novel capture-release isolation approach using a pH-responsive peptide conjugated with NanoPom magnetic beads (ExCy) for simple, fast, and homogeneous EV isolation from various biological fluids. Bioinformatic analysis of next-generation sequencing (NGS) data of EV total RNAs from pancreatic cancer patient plasma samples using our novel EV isolation approach and quality index strategy illuminates how this approach improves the identification of tumor associated molecular markers. Results showed higher human mRNA coverage compared to existing isolation approaches in terms of both pancreatic cancer pathways and EV cellular component pathways using gProfiler pathway analysis. This study provides a valuable resource for researchers, establishing a workflow to prepare and analyze EV samples carefully and contributing to the advancement of reliable and rigorous EV quality assessment and clinical translation.
Abstract Breast cancer is the most commonly diagnosed cancer and the second leading cause of death among women. Triple Negative Breast Cancer (TNBC) represents 10-20% of breast cancer patients. It is considered the most aggressive and challenging type of breast cancer due to the lack of effective targeted therapies. Metastatic TNBC commonly migrates to the lung, brain and liver. Chemotherapy is the only option for these patients and it is hard to predict the success of chemotherapy. It is crucial to investigate the molecular changes leading to TNBC and develop new therapies targeting the molecular changes that cause this disease. microRNA (miRNA) has been implicated in many cancers by deregulating the expression of genes having key roles in cancer initiation and/or progression. We measured the mature levels of miR-205 in five breast cell lines: MCF10A non-tumorigenic normal like epithelial cell line, MCF7 cancer cell line expressing both estrogen and progesterone receptors (ER+/PR+); MDA-MB-436, MDA-MB-231 and BT549 TNBC cell lines (ER-/PR-/HER2-). We also assayed miR-205 in 33 human breast tissues (benign, tumor and metastatic). miR-205 levels were significantly down regulated in breast cancer cell lines compared to normal-like breast epithelial cells and in tumor and metastatic tissues compared to benign. We identified two miR-205 predicted binding sites in the 3’ untranslated region of the high mobility group gene, HMGB3. Both Dual-luciferase reporter assay and Western blotting confirmed that miR-205 regulates HMGB3. To explore the function of miR-205 and HMGB3 in breast cancer, WST-1 proliferation and Matrigel invasion assays were done using the TNBC cell lines MDA-MB-231 and BT549 transiently transfected with precursor miR-205 oligonucleotide (pre-miR-205 oligo) or HMGB3 small interfering RNA (siRNA). Both treatments reduced the proliferation and invasion of the cancer cells, demonstrating that knockdown of HMGB3 with miRNA or siRNA is effective in reducing the malignant phenotype of this protein. The messenger RNA (mRNA) levels of HMGB3 significantly increased in the tumor and metastatic groups in a stage dependent manner suggesting a tumor initiating and progression role. Western blotting of HMGB3 protein from 5 matched pairs of breast tissue showed that HMGB3 protein was not detected in benign tissues but was present in tumor. In conclusion, regulation of HMGB3 by miR-205 reduced both proliferation and invasion of breast cancer cells. Since the down regulation of miR-205 in patients’ tumor specimens correlate with the high HMGB3 protein levels in tumor, our findings suggest that reintroducing miR-205 and targeting HMGB3 are potential therapies for TNBC. Citation Format: Ola Elgamal, Jong-Kook Park, Thomas D. Schmittgen. miR-205 functions as a tumor suppressor in breast cancer by targeting HMGB3. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4204. doi:10.1158/1538-7445.AM2013-4204
Our previous study demonstrated that the anticancer agent cis-diamminedichloroplatinum (II) (cis-DDP) inhibited the self-splicing activity of the Tetrahymena rRNA. The present study investigated the effects of cis-DDP on pre-mRNA splicing using a HeLa cell nuclear extract. A 2-h exposure of cis-DDP inhibited the splicing of the human B-globin pre-mRNA in a concentration-dependent manner. The concentration required for 50% inhibition of splicing (IC50) was 51 microM. Complete inhibition of spliceosome assembly occurred when the extracts were incubated with 150 microM cis-DDP. The inhibition of splicing by cis-DDP occurred at early events during spliceosome formation and to a greater extent if the extract was pre-incubated with cis-DDP in the absence of pre-mRNA. Splicing was inhibited when both pre-mRNA and cis-DDP were added simultaneously to the reaction mixture but not when cis-DDP was added 30 min after splicing was initiated with pre-mRNA. Clinically useful platinum analogs (ormaplatin, carboplatin, cis-tetraplatin and iproplatin) as well as the clinically ineffective Pt(dien)C1+, compound were tested for their ability to inhibit pre-mRNA splicing. The Pt(dien)C1+ compound, which acts in a monofunctional manner only, failed to inhibit splicing. A varying degree of splicing inhibition was observed for the other platinum analogs studied; the inhibitory activity decreased in the following order: ormaplatin > cis-tetraplatin > cis-DDP > iproplatin > carboplatin. We describe a novel mechanism that may be involved in the activity and/or toxicity of platinum agents.
The purpose of this study was to establish experimental conditions to produce apoptosis by the fluorinated pyrimidine 5-fluorouridine and to examine the changes in gene expression that occurred during cell death. HCT-116 colorectal carcinoma cells were exposed to 10 microM 5-fluorouridine alone or in the presence of 1 mM uridine, 30 microM thymidine or both uridine and thymidine. A time-dependent increase in the percentage of apoptotic cells and a decrease in the percentage of viable cells were observed when the cells were treated with 5-fluorouridine in the absence of uridine (p < 0.001) but not in the presence of uridine. cDNA microarray analysis was used to study the expression of 1,200 different genes during apoptosis by 5-flurouridine. The expression of 33 genes was upregulated by 5-fold or greater at 16 and 24 h of 5-fluorouridine exposure. The largest cluster of upregulated genes included a group of genes classified as growth factors, cytokines and chemokines (e.g. interleukin-3, interleukin-4, B-cell growth factor 1 and stem cell growth factor). The expression of MIC-1 increased up to 100-fold during 5-flurouridine exposure. One hundred and twenty-four genes were downregulated by 5-fold or greater following exposure to 5-fluorouridine. The downregulated genes were distributed throughout the six different classifications on the array. Our data demonstrate a diverse pattern of gene expression during the fluorouridine-induced apoptosis and suggest that mechanisms besides a global inhibition of RNA synthesis/ processing contribute to the RNA-directed cytotoxicity of fluoropyrimidines.
