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Gene expression analysis regulated by Dermcidin protein in G-361 malignant melanoma cell line by DNA-microarray

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Nancy Marcela perez Sosa
Total Authors: 1
Document type: Master's Dissertation
Press: São Paulo.
Institution: Universidade de São Paulo (USP). Faculdade de Medicina (FM/SBD)
Defense date:
Examining board members:
Jose Ernesto Belizario; Aline Cadurin Custódio; Andreia Hanada Otake
Advisor: Jose Ernesto Belizario

Dermcidin (DCD) is a human gene mapped to chromosome 12q13 region, only identified in primates and humans, and normally expressed in the eccrine glands of skin and brain. Several studies have confirmed that DCD-derived peptides contribute to innate and immune surveillance and in the oncogenic processes of breast, prostate and skin cancers, as revealed by its role as a growth factor and cell survival. We have further explored DCD function and its tumorigenic potential on skin melanocytes by specifically knocking down its expression in G-361 malignant melanoma cells via expressing constitutively short hairpin RNA against DCD mRNA. Biological and biochemical assays showed that the \"knockdown\" in the expression of DCD in G-361-pLKO control clone and a G-361-IBC-I clone expressing constitutively short hairpin RNA against DCD mRNA decreased significantly the in vitro growth in cell culture and tumor formation in nude mice. Similar results were obtained treating nude mice bearing G-361 melanoma xenografts with rabbit polyclonal antibodies against DCD protein. Here, we present a DNA microarray-based study that identified the genes that are up- and down-regulated in a G-361-pLKO control clone and a G-361-IBC-I clone expressing constitutively short hairpin RNA against DCD mRNA. A total of 372 genes differentially expressed were identified; being 162 genes up-regulated and 212 genes down-regulated. Bioinformatic studies showed that DCD gene silencing modulates canonical pathways and signaling networks mediated APRIL/BAFF receptors and ligands and NF-kB signaling pathway as well as chromatin remodeling mediated by histone family. The mRNA expression levels of 9 genes of interest were validated by RT-qPCR assays. Next we analyzed the proteins present in nuclear extracts from G-361- pLKO and G-361-IBC-I clones by mass spectrometry. We identified 74 proteins in the G-361-pLKO clone and 31 proteins in the G-361-IBC-I. A group of 21 proteins was identified in both sublineages. Bioinformatics analyses by STRING (Search Tool for the Retrieval of Interacting Genes/Proteins) platform showed that a small portion of the proteins identified only in G-361- pLKO cells was predicted to interact directly with DCD. The network formed by these proteins is centered in the p53 protein, a key regulator of survival and cell death program in response to DNA damage and oxidative stress. On the other hand, this network was completed abrogated using the nuclear protein from G-361-IBC-I because of absence of DCD protein. Since most of the proteins identified in nuclear extracts are of the histone family, it is likely that they are acting in the chromatin-remodeling complexes which are important to remodel nucleosomes of the G-361-IBC-I cells. Our results allowed us to suggest that future studies on the expression of histones and their posttranslational modifications may help to unravel the possible role of DCD in the epigenetic regulation of melanoma and other cancers (AU)

FAPESP's process: 12/02497-7 - Expression analysis of genes regulated by protein Dermicidina in malignant melanoma cells G-361 by DNA microarray
Grantee:Nancy Marcela Pérez Sosa
Support Opportunities: Scholarships in Brazil - Master