0-fold compared with normal IEC-6 cells. Of these, nine genes were up-regulated and 2 were down-regulated (Table 4). The category of altered genes included apoptosis and cell senescence (Cflar, Bax), cell cycle control and DNA damage repair (Mdm2, Ccne1), angiogenesis (Ifna1, Egfr), adhesion (Itgav, Cdh1)
and signal transduction (Fos, Myc, Rasa1). Table 4 Differentially expressed genes related to cell transformation GeneBank no Symbol Description Ratio NM_057138 Cflar CASP8 and FADD-like apoptosis regulator, 2.06 NM_017059 Bax Bcl2-associated X protein, 2.23 XM_235169 Mdm2 Transformed GDC-0449 mouse 3T3 cell double minute 2, 2.73 XM_574426 Ccne1 Cyclin E 2.17 NM_001014786 Ifna1 Interferon-alpha 1 7.38 NM_031507 Egfr Epidermal growth factor receptor 2.50 NM_022197 Fos FBJ murine osteosarcoma viral oncogene homolog 6.50 NM_012603 Myc Myelocytomatosis viral oncogene homolog (avian) 3.43 XM_230950 Itgav_predicted Integrin alpha V (predicted) 3.22 NM_031334 Cdh1 this website Cadherin 1 0.07 NM_013135 Rasa1 RAS p21 protein activator 1 0.37 Verification of differential expression genes by real-time PCR To confirm and validate the results obtained from microarray, we analyzed the expression of selected differentially
expressed genes CH5183284 by real-time qPCR. Six genes were selected from the up-regulated and downregulated genes because of its ratio and putative gene functions. The ratios representing gene expression changes were log2-transformed in the histograms for these genes. The validation experiments showed expression patterns of other genes comparable to the microarray data (Fig. 2). This implies that the data obtained from microarray analysis were reliable. Figure 2 Comparison of data obtained by real-time PCR and microarray analysis in transformed and normal IEC-6 cells. Using the housekeeping GPADH gene as a reference gene, five selected genes were assessed
for expression at the mRNA level by Real-time PCR. The ratio, representing the relative value of the gene expression level, was 5-Fluoracil research buy expressed as a logarithm (log2). Corresponding values obtained by microarray analysis were presented for comparison. Changes of miRNAs expression To determine the alteration of miRNA expression in transformed IEC-6 cells, total RNA samples from normal and transformed IEC-6 cells were isolated and hybridized to miRNA microarrays, comprising LNA-modified probes for all rat miRNAs in release 9.2 of the miRBase microRNA Registry. Expression profiling showed that a large set of miRNAs was expressed in IEC-6 cells. In agreement with other reports, several miRNAs, including miR-320, miR-494, miR-503, and members of the let-7 family, were highly expressed in IEC-6 cells, giving strong hybridization signals on the miRNA arrays. The top 5 miRNAs, which were highly expressed in IEC-6 cells, were miR-320, miR-494, miR-503, miR-185 and miR-206. Among them, the expression of miR-185 was altered in transformed IEC-6 cells.