EGFR Regulates The Radiosensitivity Of Cervical Cancer Cell By Affecting The Nuclear Distribution Of NRF2

Objective:EGFR is a multifunctional glycoprotein widely distributed on the cell membrane of various human tissues.It can regulate cell behavior by activating downstream signaling pathways through phosphorylation cascade reactions,and its role in tumor prevention and treatment has been a focus of research.However,there are fewer studies on EGFR affecting radiation sensitivity of cervical cancer cells and the mechanism remains unclear.Recently,it has been shown that EGFR can function through NRF2 signaling pathway.But how EGFR increases the radiation sensitivity of cervical cancer cells through NRF2 has not been elucidated.This study investigated the effect of EGFR on radiation sensitivity of cervical cancer cells and DNA damage response mechanism of EGFR in cervical cancer cells subjected to ionizing radiation damage.Methods:Small interfering RNA(siRNA)was used to knock down EGFR and NRF2 in HeLa cellsο After γ-rays exposure,the cell survival and proliferation abilities of HeLa cells were observed by CCK-8 assay;comet assay and y-H2AX immunofluorescence assay were performed to detect the effect of EGFR and NRF2 on DNA damage and repair ability of cervical cancer cells;The irradiated p-ATR(Ser1989),p-CHK1(Ser345),HO-1 proteins were detected by Western blot assay;The effect of EGFR on cervical cancer cell cycle was detected by flow cytometry.EGFR,NRF2,HO-1 mRNA levels were measured by RT qPCR.Detection of p-GSK3β by Western blotting(Ser9)to explore the effect of EGFR downstream signaling pathway on NRF2 nucleation;Effect of EGFR on NRF2 degradation using siRNA knockdown of KEAP1 or MG 132 inhibition of proteasome treatment of cervical cancer cells.Result:1.Knocking down EGFR and NRF2 inhibited the cell survival and proliferative ability of cervical cancer HeLa cells;2.Knocking down EGFR and NRF2 weakened the DNA damage repair ability of cervical cancer HeLa cells;3.Knocking down EGFR and NRF2 hindered the G2/M phase cell cycle arrest of cervical cancer HeLa cells after radiation;4.Knocking down EGFR and NRF2 inhibited ATR/CHK1 mediated DNA damage response signals and antioxidant protein HO-1 expression;5.Knocking down EGFR suppressed radiation induced elevation of NRF2 nuclear localization6.EGFR did not promote NRF2 out of nucleus through p-GSK-3 β;7.EGFR regulates the nuclear distribution of NRF2 through a ubiquitin proteasome degradation pathway independent of KEAP1 mediation;Conclusions:In cervical cancer HeLa cells,EGFR regulated NRF2 nuclear entry in a KEAP1-independent manner,downregulated the expression of antioxidant proteins,and reduced radiation-induced cell cycle arrest through the ATR-CHK1 signaling pathway,which decreased the antioxidant capacity and DNA damage repair capacity of cervical cancer cells,thereby increasing the radiosensitivity of cervical cancer HeLa cells.