Hyper-methylated miR-203 dysregulates ABL1 and contributes to the nickel-induced tumorigenesis.

Nickel compounds have been found to be carcinogenic based upon epidemiological, animal and cell culture studies. Previous studies suggest that epigenetic mechanisms play a role in Nickel-induced carcinogenesis such as DNA methylation and histone modification. In this study, we investigated the role of microRNAs (miRNAs) in nickel-induced carcinogenesis. The expression of several miRNAs which may function as tumor suppressor genes revealed a strong downregulation of miR-203 in Ni3S2-transformed 16HBE cells (NSTCs). Meanwhile, we observed hypermethylation of CpGs in miR-203 promoter and first exon area, and proved that the hyper-methylated miR-203 was involved in the Nickel-induced tumorigenesis. Moreover, we identified that miR-203 may suppress the tumorigenesis at least in part through negatively regulating its target gene ABL1. Our findings indicate that DNA methylation-associated silencing of tumor suppressor miRNAs contributes to the development of Nickel-induced cancer.

Effects of miR-541 on neurite outgrowth during neuronal differentiation.

MicroRNA (miRNAs) are short non-coding RNA molecules that downregulate gene expression at post-transcriptional level. miRNAs are post-transcriptional regulators of gene expression important for neuron development and function. This report demonstrated that a putative and chemically synthesized miRNA rno-mir-541 played an important role in the neuron development. Differentiation of PC12 cells with nerve growth factor (NGF) is associated with neurite outgrowth, a process that involves upregulation of Synapsin I. We predicted, detected and assessed the expression levels of a number of possible miRNAs for synapsin I in rats and our outcomes showed that rno-mir-541 was associated with rat synapsin I expression. miR-541, a brain specific miRNA, plays an important role in repressing neurite extension in cultured PC12 neurons. The neurites of PC12 cells was shortened drasticly as a result of the overexpression of rno-mir-541. In contrast, the neurites of PC12 cell developed well after the knockdown of rno-mir-541 by RNA interference. Our study showed that rno-mir-541 played an important role in neuron-cell proliferation and neurite outgrowth through suppressing the expression of its target gene synapsin I. Furthermore, the introduction of NGF causes downregulation of miR-541, de-repression of its target, Synapsin-I and allows for neuritogenesis. Thus, miR-541 functions in neuronal precursors as an endogenous conditional component between NGF and Synapsin-I.

Characterization of de novo synthesized proteins released from human colorectal tumour explants.

Tumours release many proteins into their microenvironment. These proteins may enter the blood stream and have value as cancer biomarkers. We examined the range of proteins released by colorectal cancer (CRC) liver metastasis (LM) specimens and normal colon mucosa during 16 h culture as explants in the presence of [35S]-methionine. Proteins released into the conditioned media were isolated and separated by 2-DE and detected by CBB stain and de novo synthesized proteins by autoradiography. The majority of proteins released by CRC LM explants in short-term culture were plasma proteins from tumour interstitial fluid and tissue breakdown products including mitochondrial and nuclear proteins from pre-existing necrotic cells within the tumours. De novo synthesized proteins were present at a lower abundance and included a high proportion of cytoplasmic proteins in addition to classically secreted proteins. Many cytoplasmic proteins were also present in the autoradiograph secretomes of four CRC cell lines examined, despite high cell viability (>97%), suggestive of an alternative release mechanism. The secretome profiles varied significantly between different patients, and also between different cell lines, despite low intra-experimental variation. Quantitative analysis of the autoradiograph secretome profiles prepared from tumour and normal colon mucosa tissues revealed 32 protein spots that were differentially abundant between the normal and cancer tissue secretome, including desmocollin-2 and fibrinogen gamma chain, which were upregulated and downregulated in the CRC LM secretomes, respectively. Further characterization of de novo synthesized proteins released from human tumours may help to discover a novel set of serological markers for CRC.

Compatibility of toluidine blue with laser microdissection and saturation labeling DIGE.

Tissue fixation and staining protocols for laser microdissection are frequently not fully compatible with subsequent proteomic analysis. We compared the effect of three common histological stains (toluidine blue (TB), hemotoxylin, and hematoxylin and eosin (HE)) on tissue visualization, protein recovery, the saturation labeling reaction, and 2-D electrophoresis. TB provided the best visualization of colorectal tumor tissue during laser microdissection (LMD) and had a comparable effect on protein recovery and the saturation labeling reaction with hematoxylin, provided a modified 2-D clean-up protocol was used. Eosin inhibited both protein recovery and the saturation labeling reaction.

Metastatic susceptibility locus, an 8p hot-spot for tumour progression disrupted in colorectal liver metastases: 13 candidate genes examined at the DNA, mRNA and protein level.

BACKGROUND: Mortality from colorectal cancer is mainly due to metastatic liver disease. Improved understanding of the molecular events underlying metastasis is crucial for the development of new methods for early detection and treatment of colorectal cancer. Loss of chromosome 8p is frequently seen in colorectal cancer and implicated in later stage disease and metastasis, although a single metastasis suppressor gene has yet to be identified. We therefore examined 8p for genes involved in colorectal cancer progression. METHODS: Loss of heterozygosity analyses were used to map genetic loss in colorectal liver metastases. Candidate genes in the region of loss were investigated in clinical samples from 44 patients, including 6 with matched colon normal, colon tumour and liver metastasis. We investigated gene disruption at the level of DNA, mRNA and protein using a combination of mutation, semi-quantitative real-time PCR, western blotting and immunohistochemical analyses. RESULTS: We mapped a 2 Mb region of 8p21-22 with loss of heterozygosity in 73% of samples; 8/11 liver metastasis samples had loss which was not present in the corresponding matched primary colon tumour. 13 candidate genes were identified for further analysis. Both up and down-regulation of 8p21-22 gene expression was associated with metastasis. ADAMDEC1 mRNA and protein expression decreased during both tumourigenesis and tumour progression. Increased STC1 and LOXL2 mRNA expression occurred during tumourigenesis. Liver metastases with low DcR1/TNFRSF10C mRNA expression were more likely to present with extrahepatic metastases (p = 0.005). A novel germline truncating mutation of DR5/TNFRSF10B was identified, and DR4/TNFRSF10A SNP rs4872077 was associated with the development of liver metastases (p = 0.02). CONCLUSION: Our data confirm that genes on 8p21-22 are dysregulated during colorectal cancer progression. Interestingly, however, instead of harbouring a single candidate colorectal metastasis suppressor 8p21-22 appears to be a hot-spot for tumour progression, encoding at least 13 genes with a putative role in carcinoma development. Thus, we propose that this region of 8p comprises a metastatic susceptibility locus involved in tumour progression whose disruption increases metastatic potential.