Evaluation of siRNA function on Human papillomavirus E5 and epidermal growth factor receptor gene expression in CaSki cells

Abstract

Cervical cancer is a growing global disease in women. This cancer can have different causes, but infection with some types of HPV is the most important cause. HPV E5 protein could be considered an oncogene acting in the early stage of oncogenesis. There are several studies demonstrate intracellular binding targets for E5 including some members of the EGF receptor (EGFR) family. Since HPV oncogenes are expressed only in cells infected with the virus, and only such cells are able to enter the cancer pathway, instead of chemotherapy drugs, siRNAs can be used to target HPV oncogenes. The main purpose of this study is the evaluation of siRNA function on Human papillomavirus E5 and epidermal growth factor receptor gene expression in CaSki cells. Material and methods: The Caski cell containing HPV-16 integrated DNA and E5 transcripts was obtained from the Iranian Biological Resource Center (IBRC). Moreover, the siRNA used in this study was obtained from Bioneer Co. Cytotoxic effects of E5-siRNA on CaSki cells were determined using the MTT assay. The following qRT-PCR was used to determine the effect of this transfection on the HPV-16 E5 mRNA expression. Flowcytometric studies were also used to determine the rate of induction of apoptosis using Annexin V-FITC/PI assay. Moreover, Cell cycle arrest was determined by flow cytometry. Finally, qRT-PCR was used to study the effect of E5 silencing on the expression levels of other HPV oncoproteins, apoptosis-related genes, and the initiator of the EGFR signaling pathway. Results: The expression of E5 mRNA was reduced after using E5 siRNA in CaSki cervical cancer cells. The transfection of E5 siRNA caused cell cycle arrest in the sub-G1 phase. Besides, the use of E5 siRNA decreased the expression of PTGS2 and BCL2, increased BAX, HPV E6, HPV E7 gene expression, and does not have any significant change in the expression of EGFR. Also, the results showed increased induction of apoptosis following the transfection of cancer cells.