E5 human papillomavirus type 16 oncoprotein modify ST3GAL3 and ST6GAL1 gene expression

Human papillomavirus (HPV) is the etiological agent of cervical cancer. Three viral proteins, E5, E6 and E7 have been implicated in cell transformation. Increased expression of sialic acid and sialylated antigens have been reported during cervix transformation, these results agree with the increased mRNA levels of the sialyltransferases genes ST6GAL1 and ST3GAL3 reported in premalignant and malignant tissue of the cervix. E6 and E7 HPV oncoproteins modify the expression of some glycogenes. The role of E5 HPV oncoprotein in the glycogene expression changes in premalignant and malignant cervical tissue has not been reported. The objective of this work was to identify glycogenes that modify their expression by E5 HPV oncoprotein in HaCaT cell line. A gene expression microarray was performed on HaCaT cells that stably expressed the HPV16 E5 oncogene. Analysis revealed alteration in some glycogenes including upregulation of ST6GAL1 and ST3GAL3. The increased mRNA levels of both genes were confirmed by qRT-PCR. In addition, an in-silico analysis was performed to identify glycosylation networks altered in presence of E5 oncoprotein. The analysis showed that E5 could modify the sialic acid expression, keratan sulfate synthesis, N-glycosylation and biosynthesis of glycosaminoglycans. This is the first report of the role of HPV16 E5 oncoprotein on glycogenes expression changes. Moreover, our results suggest that the increase of the sialyltransferases genes reported in premalignant and malignant cervical tissue, could be the result of the expression of E5 oncoprotein. These results provide information of the possible role of HPV infection on the sialylation changes in the cervical epithelium identified in premalignant lesions and cancer. Two-condition experiment, HaCat cell groups (HaCat-pMSG, and HaCat-E5) One replicate array. 10 µg of total RNA were used for cDNA synthesis incorporating dUTP-Alexa555 (for HaCat-pMSG) or dUTP-Alexa647 (for HaCat-E5) employing the First-Strand cDNA labeling kit (Invitrogen). Incorporation of fluorophore was analyzed by using the absorbance at 555 nm for Alexa555 and 650 nm for Alexa647. Equal quantities of labeled cDNA were hybridized using hybridization solution UniHyb (TeleChem International INC). The arrays were incubated for 14 h at 42°C, and then washed tree times with 1X SCC, 0.05 % SDS at room temperature. Please note that [1] the experiment was performed technically as dual channel (eg, Cy3 and Cy5-labeled samples hybridized to the same array) but the results was processed as though they are single channel (Cy3 and Cy5 signals are calculated; Cy3/Cy5 ratios are not calculated) and that [2] In the microarray assay, each gene was evaluated as duplicate for the same condition, generating two values corresponding to each spot.