BackgroundQualitative alterations or abnormal expression of microRNAs (miRNAs) in colon cancer have mainly been demonstrated in primary tumors. Poorly overlapping sets of oncomiRs, tumor suppressor miRNAs and metastamiRs have been linked with distinct stages in the progression of colorectal cancer. To identify changes in both miRNA and gene expression levels among normal colon mucosa, primary tumor and liver metastasis samples, and to classify miRNAs into functional networks, in this work miRNA and gene expression profiles in 158 samples from 46 patients were analysed.ResultsMost changes in miRNA and gene expression levels had already manifested in the primary tumors while these levels were almost stably maintained in the subsequent primary tumor-to-metastasis transition. In addition, comparing normal tissue, tumor and metastasis, we did not observe general impairment or any rise in miRNA biogenesis. While only few mRNAs were found to be differentially expressed between primary colorectal carcinoma and liver metastases, miRNA expression profiles can classify primary tumors and metastases well, including differential expression of miR-10b, miR-210 and miR-708. Of 82 miRNAs that were modulated during tumor progression, 22 were involved in EMT. qRT-PCR confirmed the down-regulation of miR-150 and miR-10b in both primary tumor and metastasis compared to normal mucosa and of miR-146a in metastases compared to primary tumor. The upregulation of miR-201 in metastasis compared both with normal and primary tumour was also confirmed. A preliminary survival analysis considering differentially expressed miRNAs suggested a possible link between miR-10b expression in metastasis and patient survival. By integrating miRNA and target gene expression data, we identified a combination of interconnected miRNAs, which are organized into sub-networks, including several regulatory relationships with differentially expressed genes. Key regulatory interactions were validated experimentally. Specific mixed circuits involving miRNAs and transcription factors were identified and deserve further investigation. The suppressor activity of miR-182 on ENTPD5 gene was identified for the first time and confirmed in an independent set of samples.ConclusionsUsing a large dataset of CRC miRNA and gene expression profiles, we describe the interplay of miRNA groups in regulating gene expression, which in turn affects modulated pathways that are important for tumor development.
Our data show an association between testiculopathy and alteration of the bone status, despite unvaried androgen and estrogen levels and no other evident cause of vitamin D reduction. Further studies in larger cohorts are needed to confirm our results.
Alternative splicing (AS) is a common mechanism which creates diverse RNA isoforms from a single gene, potentially increasing protein variety. Growing evidence suggests that this mechanism is closely related to cancer progression. In this study, whole transcriptome analysis was performed with GeneChip Human exon 1.0 ST Array from 80 samples comprising 23 normal colon mucosa, 30 primary colorectal cancer and 27 liver metastatic specimens from 46 patients, to identify AS events in colorectal cancer progression. Differentially expressed genes and exons were estimated and AS events were reconstructed by combining exon-level analyses with AltAnalyze algorithms and transcript-level estimations (MMBGX probabilistic method). The number of AS genes in the transition from normal colon mucosa to primary tumor was the most abundant, but fell considerably in the next transition to liver metastasis. 206 genes with probable AS events in colon cancer development and progression were identified, that are involved in processes and pathways relevant to tumor biology, as cell-cell and cell-matrix interactions. Several AS events in VCL, CALD1, B3GNT6 and CTHRC1 genes, differentially expressed during tumor development were validated, at RNA and at protein level. Taken together, these results demonstrate that cancer-specific AS is common in early phases of colorectal cancer natural history.
MiR-182 expression was evaluated by qRT-PCR and in situ hybridization in 20 tubular adenomas, 50 colorectal carcinoma (CRC), and 40 CRC liver metastases. Control samples obtained from patients with irritable bowel syndrome, or tumor-matched normal colon mucosa were analyzed (n=50). MiR-182 expression increased progressively and significantly along with the colorectal carcinogenesis cascade, and in CRC liver metastases. The inverse relation between miR-182 and the expression of its target gene ENTPD5 was investigated by immunohistochemical analysis. We observed that normal colocytes featured a strong ENTPD5 cytoplasmic expression whereas a significantly and progressively lower expression was present along with dedifferentiation of the histologic phenotype. Plasma samples from 51 CRC patients and controls were tested for miR-182 expression. Plasma miR-182 concentrations were significantly higher in CRC patients than in healthy controls or patients with colon polyps at endoscopy. Moreover, miR-182 plasma levels were significantly reduced in post-operative samples after radical hepatic metastasectomy compared to preoperative samples. Our results strengthen the hypothesis of a central role of miR-182 dysregulation in colon mucosa transformation, demonstrate the concomitant progressive down-regulation of ENTPD5 levels during colon carcinogenesis, and indicate the potential of circulating miR-182 as blood based biomarker for screening and monitoring CRC during the follow-up.
For fertilization to occur in mammals, ejaculated spermatozoa must reach the egg which, following ovulation has moved from the ovary into the Fallopian tube. Two active mechanisms of spermatozoa guidance have been shown in mammals: thermotaxis and chemotaxis. The identity of most of human spermatozoa chemoattractants is unknown, and herein we tested if SDF1 (chemokine stromal cell-derived factor-1) and its pathway is involved in spermatozoa chemotaxis. We found that SDF1 is expressed in the oocytes, endometrium and follicular fluid, as well as its specific receptor CXCR4 (chemokine CXC motif receptor 4) is expressed in the head of spermatozoa. By SDF1 gradient experiments, we stated that SDF1 is able to induce hyperactivation in spermatozoa leading to accumulation, to give rise to an increase in intracellular calcium concentration, and to preserve the mitochondrial status and not to induce acrosome reaction. Our findings suggest these phenomena could reflect spermatozoa chemotaxis, and that SDF1 action could represent an important event leading to egg fertilization, even if further studies regarding the link between spermatozoa accumulation and chemotaxis are mandatory. These data suggest that the SDF-1/CXCR4 signalling could be used to manipulate the human fertilization, to improve both the outcome of physiological or assisted reproduction, and to develop new contraceptive methods, by development of SDF1 or CXCR4 antagonist.
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