Integration of gene and miRNA expression profiles in clear cell renal carcinoma cell lines and relationship with VHL gene status

Clear cell renal cell carcinoma (ccRCC) is the predominant form of kidney cancer, representing 75–80% of primary malignancies of the kidney. The status of the von Hippel-Lindau (VHL) tumor suppressor gene, an important regulator of the hypoxia pathway via the hypoxia-inducible factors (HIFs), has been correlated to RCC pathogenesis. VHL biallelic inactivation occurs in 80% of sporadic ccRCCs and in all inherited cases, while the remaining 20% harbours wild type gene, thus this molecular heterogeneity needs further investigations. The role of microRNAs (miRNAs) in cancer development and progression is expanding, since a number of evidences suggested that miRNA expression is implicated in tumorigenesis, and miRNAs might function as tumour suppressors and oncogenes in a contest-dependent way. We used Caki-2 and A498 cell lines as in vitro model of ccRCC pathology, and HK-2 (normal proximal tubular epithelial cell line) as reference sample. We characterized the VHL status by direct sequencing and the HIF status by western blot. Affymetrix microarray platforms were applied to assess miRNA profiles (onto GeneChip® miRNA Array, comprising 847 human mature miRNAs) and gene expression profiles (onto GeneChip® Human Gene 1.0 ST Array, including 19,793 annotated genes). Analysis of differentially expressed miRNAs (DEMs) outlined specific miRNAs in both Caki-2 and A498 that have been found related to ccRCC (e.g. miR-155, miR-21 and miR-221), and in addition some DEMs found only in A498 (VHL-/-) that are involved in hypoxia pathway (e.g. miR-210). Functional enrichment analysis showed that some modulated gene (DEG) have a known role in hypoxia and p53 signalling pathways. Additionally, we performed an integrated analysis to combine gene and miRNA expression profiles, under the assumption that, since miRNAs tend to down-regulate their targets, expression profiles of miRNAs and real targets are expected to be anti-correlated. This integrated analysis exploits miRNAs and target expression information in order to identify most probable functional regulatory interactions occurring in the ccRCC cells, and to reconstruct and study the corresponding post-transcriptional regulatory network. The further integration of these results with DEGs and DEMs will facilitate the elucidation of regulatory circuits important for tumorigenesis and biological processes under relevant post-transcriptional regulation in ccRCC and the interpretation of these results on the basis of VHL status.