IADR Abstract Archives
Role of ARHGAP29 Isoforms Expression in Cancer Therapy-Induced Oral Mucositis
Objectives: Cancer therapy-induced oral mucositis (CTOM) is a dose-limiting side effect caused by chemotherapy and radiation therapy, which may cause painful ulceration of the oral mucosa. We have previously identified a single nucleotide polymorphism (SNP) rs4847278 associated with CTOM severity. This SNP, located in ABCA4 enhancer region, might affect ARHGAP29 expression and impact the maintenance of oral mucosa integrity. ARHGAP29 transcription may be upregulated by IRF6, thereby impacting mucosal healing. Our objective was to determine whether siRNA knockdown of specific ARHGAP29 isoforms impairs wound healing.
Methods: Wound healing assays were performed using the immortalized primary oral keratinocyte cell line, OKF6-TERT2, by introducing a physical scratch. At 24-48hrs, the expression of ARHGAP29 and IRF6 was observed at the wound closure edge by immunofluorescence. siRNA knockdown of ARHGAP29 isoforms A or B was performed in parallel and mRNA harvested after 48hrs (i.e., 24hrs post-scratch). The mRNA expression of two ARHGAP29 isoforms (A, B) was determined by semi-qRT-PCR. Corresponding protein expression was determined 72hrs after siRNA knockdown by Western blot (i.e., 48hrs post-scratch).
Results: OKF6-TERT2 cell line demonstrated 81% closure after 24hrs and 100% after 48hrs post-scratch. Immunocytochemistry revealed increased IRF6 presence at the leading edge of the scratch and was co-expressed with ARHGAP29. siRNA knockdown of ARHGAP29 isoforms A and B reduced total mRNA expression by 42%, while protein expression was reduced by 65% and 25%, respectively. After 48hrs siRNA knockdown of both isoforms A and B (24hrs post-scratch), wound healing was reduced by 12.5%. Knockdown of isoforms A or B alone resulted in a wound healing reduction by 5% and 11.5%, respectively.
Conclusions: IRF6 and ARHGAP29 are involved in keratinocyte migration and are overexpressed in response to physical wounding. Confirmation that SNP rs4847278 may impact ARHGAP29 isoforms expression and therefore promote CTOM awaits introduction of this polymorphism in different oral epithelial cell lines.
Methods: Wound healing assays were performed using the immortalized primary oral keratinocyte cell line, OKF6-TERT2, by introducing a physical scratch. At 24-48hrs, the expression of ARHGAP29 and IRF6 was observed at the wound closure edge by immunofluorescence. siRNA knockdown of ARHGAP29 isoforms A or B was performed in parallel and mRNA harvested after 48hrs (i.e., 24hrs post-scratch). The mRNA expression of two ARHGAP29 isoforms (A, B) was determined by semi-qRT-PCR. Corresponding protein expression was determined 72hrs after siRNA knockdown by Western blot (i.e., 48hrs post-scratch).
Results: OKF6-TERT2 cell line demonstrated 81% closure after 24hrs and 100% after 48hrs post-scratch. Immunocytochemistry revealed increased IRF6 presence at the leading edge of the scratch and was co-expressed with ARHGAP29. siRNA knockdown of ARHGAP29 isoforms A and B reduced total mRNA expression by 42%, while protein expression was reduced by 65% and 25%, respectively. After 48hrs siRNA knockdown of both isoforms A and B (24hrs post-scratch), wound healing was reduced by 12.5%. Knockdown of isoforms A or B alone resulted in a wound healing reduction by 5% and 11.5%, respectively.
Conclusions: IRF6 and ARHGAP29 are involved in keratinocyte migration and are overexpressed in response to physical wounding. Confirmation that SNP rs4847278 may impact ARHGAP29 isoforms expression and therefore promote CTOM awaits introduction of this polymorphism in different oral epithelial cell lines.