A novel normalization method for effective removal of systematic variation in microarray data

Normalization of cDNA and oligonucleotide microarray data has become a standard procedure to offset non-biological differences between two samples for accurate identification of differentially expressed genes. Although there are many normalization techniques available, their ability to accurately remove systematic variation has not been sufficiently evaluated. In this study, we performed experimental validation of various normalization methods in order to assess their ability to accurately offset non-biological differences (systematic variation). The limitations of many existing normalization methods become apparent when there are unbalanced shifts in transcript levels. To overcome this limitation, we have proposed a novel normalization method that uses a matching algorithm for the distribution peaks of the expression log ratio. The robustness and effectiveness of this method was evaluated using both experimental and simulated data

Investigating the influence of Epstein-Barr virus on the p53 pathway in nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NPC) is a malignancy arising from the epithelial lining of nasopharynx. It is one of the most common malignancies in the southern area of China and South-East Asia. NPC is frequently reported in Sarawak, Malaysia especially in males and is endemic to certain ethnic groups, including the Bidayuh and Chinese. The development of NPC is associated with multiple factors, with one common mechanism involving persistent infection with Epstein-Barr virus (EBV). This study will aim to gain a clearer understanding regarding the mechanism of EBV influencing p53 pathway to facilitate NPC development. Mutations in p53 mutation have been associated with oncogenesis of several human malignancies, however such mutations are relatively rare in NPC and the mechanisms through which p53 in inactivated in this malignancy remain poorly understood. In this study, we study the activity of p53 in immortalised nasopharyngeal epithelial cells using nutlin-3, an MDM2 antagonist. The impact of EBV infection on the protein expression of p53 and its ability to stimulate its transcriptional target genes is studied. The proteins levels of p53 and its transcriptional activation of target genes was significantly higher in EBV-infected cells, indicating that EBV infection alone is not sufficient to attenuate the p53 pathway in a non-malignant nasopharyngeal cell line. These observations will provide the fundamental steps towards the understanding of p53 and EBV viral proteins interaction that causes NPC

systematic variation

novel normalization method for effective removal o

Applied numerical analysis & computational mathematics : ANACM

Annexin 1 (ANXA1) is an endogenous anti-inflammatory protein implicated in cancer. ANXA1 was previously shown to be regulated by hsa-miR-196a. However, whether ANXA1 itself regulates microRNA (miR) expression is unknown. Therefore, we investigated the regulation of miR by ANXA1 in MCF7 breast cancer cells. MCF7-EV (Empty vector) and MCF7-V5 (ANXA1-V5 expressing cells) were subjected to a miR microarray. Microarray analysis revealed a number of miRNAs which were dysregulated in MCF7-V5 cells. 2 novel miRNAs (miR562 and miR26b*) were validated, cloned and functionally characterized. As ANXA1 constitutively activates NF-κB activity to modulate breast cancer metastasis, we found that miR26b* and miR562 directly targeted the canonical NF-κB pathway by targeting the 3' UTR and inhibiting expression of Rel A (p65) and NF-κB1 (p105) respectively. MiR562 inhibited wound healing, which was reversed when ANXA1 was overexpressed. Overexpression of either miR562 or miR26b* in MCF-7 cells enhanced endothelial tube formation when cocultured with human umbilical cord endothelial cells while conversely, treatment of MCF7 cells with either anti-miR562 or anti-miR26b* inhibited endothelial tube formation after co-culture. Further analysis of miR562 revealed that miR562-transfected cell conditioned media enhances endothelial cell tube formation, indicating that miR562 increased angiogenic secreted factors from MCF-7 breast tumor cells. TNFα was increased upon overexpression of miR562, which was reversed when ANXA1 was co-transfected In conclusion, this data suggests that ANXA1-regulated miR26b* and miR562 may play a role in wound healing and tumor-induced endothelial cell tube formation by targeting NF-κB expression and point towards a potential therapeutic target for breast cancer

miR26b* and miR562 overexpression in MCF7 cells enhances endothelial cell angiogenesis while silencing miR26b* and miR562 inhibits angiogenesis.

<p>MCF7 cells were transfected with empty vector (EV), miR26b* or miR562 and a co-culture using transwells was performed with HUVEC in matrigel. (A,C) Average number of tubes formed per field of view and (B,D) average tube length was analyzed. Similar co-culture experiments were performed with MCF7 cells silenced with control, anti-miR26b* and anti-miR562. (D,G) Average number of tubes formed per field of view and (F,H) average tube length was analyzed. * p<0.05 ** p<0.01 vs control cells.</p

Expression of miR26b* and miR562 in breast cancer cells.

<p>(A–C) RNA from MCF10A breast epithelial cells, MCF7 and MDA-MB231 breast cancer cells were isolated and expression levels of ANXA1, miR26b* and miR562 were measured. (D–E) MCF7 cells were stably transfected with a ANXA1 overexpression vector and levels of ANXA1, miR26b* and miR562 were assessed. * p<0.05 ** p<0.01 vs MCF-10A cells or EV. Values are represented as fold change vs MCF-10A cells or EV.</p

Microarray and qPCR validation of microRNA dysregulation in MCF-7 cells overexpressing ANXA1.

<p>(A) Heat map showing pattern of dysregulation observed in miRs during miR microarray analysis when ANXA1 was over-expressed in MCF7 cells. Green bars represent down-regulation of miR expression and red bars represent up-regulation of miR expression. The intensity corresponds to the degree of dysregulation of expression compared to MCF7-EV cells. (B) Correlations between 3 microarray runs and 3 qPCR validations of 12 miRs chosen. p values shown are vs MCF7-EV control cells. (C) Chromosome location of miR26b* and miR562. (E) miR26b* and (F) miR562 expression in breast cancer cell lines. * p<0.05 ** p<0.01 vs MCF10A cells</p

Primer sequences used for quantitative PCR.

<p>Primer sequences used for quantitative PCR.</p