Adrenocortical carcinoma (ACC) is an aggressive tumor showing frequent metastatic spread and poor survival. Although recent genome-wide studies of ACC have contributed to our understanding of the disease, major challenges remain for both diagnostic and prognostic assessments. The aim of this study was to identify specific microRNAs (miRNAs) associated with malignancy and survival of ACC patients. miRNA expression profiles were determined in a series of ACC, adenoma, and normal cortices using microarray. A subset of miRNAs showed distinct expression patterns in the ACC compared with adrenal cortices and adenomas. Among others, miR-483-3p, miR-483-5p, miR-210, and miR-21 were found overexpressed, while miR-195, miR-497, and miR-1974 were underexpressed in ACC. Inhibition of miR-483-3p or miR-483-5p and overexpression of miR-195 or miR-497 reduced cell proliferation in human NCI-H295R ACC cells. In addition, downregulation of miR-483-3p, but not miR-483-5p, and increased expression of miR-195 or miR-497 led to significant induction of cell death. Protein expression of p53 upregulated modulator of apoptosis (PUMA), a potential target of miR-483-3p, was significantly decreased in ACC, and inversely correlated with miR-483-3p expression. In addition, high expression of miR-503, miR-1202, and miR-1275 were found significantly associated with shorter overall survival among patients with ACC (P values: 0.006, 0.005, and 0.042 respectively). In summary, we identified additional miRNAs associated with ACC, elucidated the functional role of four miRNAs in the pathogenesis of ACC cells, demonstrated the potential involvement of the pro-apoptotic factor PUMA (a miR-483-3p target) in adrenocortical tumors, and found novel miRNAs associated with survival in ACC.
The role of polymorphic CYP2B6 in cyclophosphamide (CPA) bioactivation was investigated in human liver microsomes. A total of 67 human liver specimens were first genotyped with respect to the CYP2B6*5 and CYP2B6*6 variant alleles. CYP2B6 apoprotein levels in 55 liver microsomal preparations were assessed by immunoblotting. 4-Hydroxy-CPA and hydroxy-bupropion were quantified by using HPLC and LC-MS, respectively. 7-Ethoxy-4-trifluoromethyl coumarin O-deethylase activity was measured fluorometrically. The frequencies of CYP2B6*5 and CYP2B6*6 mutant alleles were 9.0 and 16.4%, respectively. CYP2B6 protein expression was detected in 80% of the samples, with a large variation (0.003-2.234, arbitrary units). There was a high correlation between CYP2B6 apoprotein content and CPA 4-hydroxylation (n ¼ 55, r ¼ 0.81, Po0.0001). When based on the CYP2B6 apoprotein levels, the *6 carriers had significantly higher CPA 4-hydroxylation (Po0.05). CPA 4-hydroxylation also correlated significantly with other CYP2B6-specific reactions (n ¼ 20, Po0.0001). V max and K m for CPA 4-hydroxylation in recombinant CYP2B6 enzyme were 338 nmol/min/nmol enzyme and 1.4 mM, respectively. CYP2B6 showed much higher in vitro intrinsic clearance than previously observed in recombinant CYP2C19 and CYP2C9 variants in yeast expression system. Our results demonstrate that the polymorphic CYP2B6 is a major enzyme in the bioactivation of CPA. Moreover, we identified a strong impact of CYP2B6*6 on CPA 4-hydroxylation.
Gastrointestinal stromal tumors (GISTs), which arise from the interstitial cells of Cajal, are the most common nonepithelial tumors of the gastrointestinal tract. It is now well known that imatinib, a new molecularly targeted tyrosine kinase receptor blocker, results in a dramatic response and markedly improved long-term survival in patients with GISTs. The increasing recognition of GISTs and the prolonged survival have made imaging increasingly important not only for diagnosis but also for monitoring the effects of treatment and detecting tumor progression. Computed tomography (CT) is the imaging modality of choice for these purposes. The imaging findings at initial presentation, during treatment, and at tumor progression were studied in 113 patients with primary and advanced GISTs before and up to 37 months after imatinib treatment. GISTs occur anywhere along the gastrointestinal tract, most commonly in the stomach and small bowel. At contrast material-enhanced CT, localized primary GISTs are typically exophytic, large, hypervascular masses. When the tumors respond to treatment, the changes in tumor size may initially vary; however, GISTs typically become homogeneous and hypoattenuating, with disappearance of enhancing tumor nodules and tumor vessels in the early posttreatment period. Development of a nodule within the treated tumor is unique to GISTs and indicates recurrence regardless of changes in tumor size.
MicroRNAs (miRNAs) are short non-coding RNA regulators that control gene expression mainly through post-transcriptional silencing. We previously identified miR-205 in a signature for human cervical cancer using a deep sequencing approach. In this study, we confirmed that miR-205 expression was frequently higher in human cervical cancer than their matched normal tissue samples. Functionally, we demonstrate that miR-205 promotes cell proliferation and migration in human cervical cancer cells. To further understand the biological roles of miR-205, we performed in vivo crosslinking and Argonaute 2 immunoprecipitation of miRNA ribonucleoprotein complexes followed by microarray analysis (CLIP-Chip) to identify its potential mRNA targets. Applying CLIP-Chip on gain- and loss-of-function experiments, we identified a set of transcripts as potential targets of miR-205. Several targets are functionally involved in cellular proliferation and migration. Two of them, CYR61 and CTGF, were further validated by Western blot analysis and quantification of mRNA enrichment in the Ago2 immunoprecipitates using qRT-PCR. Furthermore, both CYR61 and CTGF were downregulated in cervical cancer tissues. In summary, our findings reveal novel functional roles and targets of miR-205 in human cervical cancer, which may provide new insights about its role in cervical carcinogenesis and its potential value for clinical diagnosis.
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