P53/IFI16/NF-κB Pathway Regulates Cisplatin-induced Apoptosis In Cervical Cancer Cells

Background:Cervical cancer is the fourth most common female cancer worldwide,with an estimated 570,000 new cases and 311,000 deaths in 2018.Although the human papillomavirus(HPV)vaccine and cervical cancer screening have greatly reduced the incidence of cervical cancer,limited treatment options still lead to a poor prognosis.The standard of care for advanced cervical cancer includes radiation therapy and cisplatin-based chemotherapy.Cisplatin binding to double-stranded DNA(ds DNA)to form cross-linked complexes causing cell cycle arrest,activating the oncogene p53 and inducing mitochondrial dysfunction are considered to be the main mechanisms of cisplatin’s antitumor effects.As a first-line chemotherapeutic agent in the treatment of cervical cancer,tumor cells exhibit a response resistant to the action of cisplatin during the use of cisplatin,which eventually leads to the development of acquired drug resistance and severely limits the use of cisplatin in clinical treatment.NF-κB signaling pathway plays a key role in mediating cell proliferation and inflammatory response.Clinically,the intensity of NF-κB signaling pathway-related protein expression was found to be positively correlated with increased in situ grading of cervical cancer.It is now believed that NF-κB can induce the expression of various cytokines and chemokines,stimulate the transcription of proliferation-regulated genes,maintain mitochondrial homeostasis,and control mitochondrial metabolic reprogramming to promote tumorigenesis,suggesting that NF-κB signaling pathway activation may be associated with antagonizing the effect of cisplatin-induced apoptosis in cervical cancer cells.In addition,there are experimental results suggesting that cisplatin-induced activation of NF-κB pathway,in addition to causing tumor cell cell proliferation,may also mediate innate immune responses involved in tumor cell immune response by promoting transcription of cytokines such asβ-interferon-β(IFN-β)and interleukin-6(IL-6)through IFI16/STING/NF-κB pathway regulation,and it is believed that activation of NF-κB pathway in different ways may produce different results.Therefore,the role of NF-κB pathway in the antitumor effects of cisplatin and the activation mechanism need to be further investigated.Interferon gamma inducible protein 16(IFI16)is an AIM2-like DNA binding protein.As a DNA receptor,IFI16 also recognizes cytoplasmic DNA in human keratin-forming cells and macrophages and mediates innate immune responses by promoting transcription of cytokines such as IFN-β and IL-6 through the IFI16/STING/NF-κB pathway.However,the role of IFI16 in regulating NF-κB in cell proliferation is unclear.It has been shown that IFI16 acts as an amplifier of DNA damage,promotes p53 phosphorylation at the Ser15 site,and ultimately induces p53-dependent apoptosis.This suggests that using IFI16 as an entry point,by investigating the mechanism of p53 activation on NF-κB could provide clues to elucidate the function of NF-κB in the antitumor effect of cisplatin.Objective:To explore the role of IFI16 in the regulation of p53/NF-κB in cisplatin-induced apoptosis of cervical cancer cells,which provides a new idea for targeting inhibition of NF-κB to increase the cisplatin sensitivity of cervical cancer cells.Methods:Ⅰ.Effects of cisplatin damage to DNA-activated p53 on apoptosis and mitochondrial function1.Human cervical cancer cells He La were treated with different concentrations of cisplatin for 24 h.Cell activity was detected by MTT;apoptosis rate was detected by flow cytometry and apoptosis-related protein expression was detected by Western Blot;DNA damage markers were detected by Western Blot after cisplatin(4 μg/m L)treatment for different times(1h,3h,6h,12h)protein histone γH2A.X protein expression levels.2.After stimulating human cervical cancer cells He La with cisplatin(4μg/m L)for different time periods(3h,6h,12h),p53 protein expression changes were detected using Western Blot to determine the timing of cisplatin stimulation in subsequent experiments;after stimulating He La cells with cisplatin(4μg/m L)for 12 h,cell nuclear proteins and cell plasma proteins were isolated and extracted.Western Blot assay was used to detect the protein localization and expression level of p53;q PCR assay was used to detect the expression level of p21 m RNA,a gene downstream of p53.3.Stimulation of human cervical cancer cells He La for 12 h using cisplatin(4μg/m L)and flow cytometry to detect the mitochondrial membrane potential as well as intracellular ROS and to observe changes in mitochondrial function.Ⅱ.Confirmation of the correlation between p53 and IFI16/NF-κB pathway1.Human cervical cancer cells were stimulated with cisplatin(4μg/m L)for 12 h,and nuclear and plasma proteins were isolated and extracted.IL-6 and cyclin D1 m RNA expression level by q PCR;to evaluate whether NF-κB pathway is activated.2.Construct Pmp53 plasmid(overexpressing p53),transfect human cervical cancer cells He La for 24 h,or stimulate with cisplatin(4μg/m L)for 12 h,isolate and extract cell nuclear proteins and cell plasma proteins,and detect the protein expression and subcellular localization of IFI16 and p65 using Western Blot method.Ⅲ.Exploring the mechanism of NF-κB pathway activation by IFI16 to regulate cisplatin-induced apoptosis in cervical cancer cells1.Construct si RNA-IFI16(knockdown IFI16)was transfected with human cervical cancer cells He La for 24 h,followed by stimulation with cisplatin(4μg/m L)for 12 h,MTT colorimetric assay for cell activity,Western Blot assay for apoptosis-related protein expression,and flow cytometry for apoptosis rate;flow cytometry for mitochondrial membrane potential as well as intracellular ROS and observation of changes in mitochondrial function.2.Mouse cervical cancer cell U14 subcutaneous tumor model was constructed,and 15μg negative non-target si RNA or a mixture of 2’-o-Me modified si IFI16 1/2(dissolved in 50μL PBS)and 30μL RNAFit transfection agents were injected at multiple points in the tumor every other day.Intraperitoneal injection of cisplatin(3mg/kg)began the next day.The status of mice was observed every day,and the weight and tumor size of mice were measured.After 1 week,the mice were sacrificed,and the subcutaneous tumor was weighed and photographed.3.Construct si RNA-IFI16(knockdown IFI16)was transfected with human cervical cancer cells He La for 24 h,followed by stimulation with cisplatin(4μg/m L)for 12 h,isolation and extraction of cell nuclear proteins and cell plasma proteins,application of Western Blot and immunofluorescence to detect the protein expression of p65 in the nucleus;application of q PCR to detect NF-κB signaling pathway target gene IL-6 and cyclin D1 m RNA expression level by q PCR.4.si RNA-IFI16(knockdown IFI16)was constructed and transfected with human cervical cancer cells He La for 24 h,then stimulated with cisplatin(4μg/m L)for 12 h,and the nuclear and plasma proteins were isolated and extracted,and the protein localization and expression levels of IFI16 and p53 were detected by Western Blot,and the downstream gene p21 was detected by q PCR.m RNA expression levels,as a way to evaluate whether p53 functions as a transcription factor.5.Construct si RNA-IFI16(knockdown IFI16)to transfect human cervical cancer cells He La for 24 h,followed by stimulation with cisplatin(4μg/m L)for 12 h,and apply Western Blot to detect the protein expression level of P-STING and STING,and q PCR to detect the m RNA expression level of IFN-β,the target gene of STING pathway.to evaluate the activation level of STING signaling.6.STING inhibitor H-151(1μM)was pretreated for 2h,and then He La cells were stimulated with cisplatin(4μg/m L).q PCR was used to detect the m RNA expression level of IFN-β,the target gene of STING pathway;cell nuclear proteins and cell plasma proteins were isolated and extracted,and cell activity was detected by MTT colorimetric assay,and apoptosis-related protein was detected by Western Blot The protein expression of p65 in the nucleus was detected by Western Blot.Results:Ⅰ.Cisplatin increases p53 expression through DNA damage and induces apoptosis1.The IC50 of cisplatin treatment for human cervical cancer cells He La to cisplatin was about 4 μg/m L at 24 h.The cell activity decreased,the apoptosis rate increased,and the expression level of Cleaved-caspase3 protein increased;the expression level of γH2A.X protein increased significantly at 12 h of cisplatin treatment.2.12 h of cisplatin treatment,the p53 protein expression level began to increase significantly,and there was obvious nucleation,and the p53 downstream gene p21 m RNA expression level increased.3.An increase in intracellular ROS content and a decrease in mitochondrial membrane potential in human cervical cancer cells He La cells treated with cisplatin for 12 h.Ⅱ.p53 induces increased IFI16 expression and activates NF-κB pathway1.p65 protein expression level in the nucleus of human cervical cancer cells He La cells was significantly increased by cisplatin treatment for 12 h,and IL-6 and cyclin D1 m RNA expression level was significantly higher than that of the control group;IFI16 protein expression level was significantly increased in whole cells and nucleus.2.Overexpression of p53,significantly higher p65 protein expression levels in the nuclei of human cervical cancer cells He La,significantly higher IL-6 and cyclin D1 m RNA expression levels than in the control group;significantly higher IFI16 protein expression levels in whole cells and nuclei.Ⅲ.Mechanism of NF-κB pathway activation by IFI16 against cisplatin-induced apoptosis in cervical cancer cells1.Knockdown of IFI16 followed by cisplatin stimulation for 12 h resulted in decreased cell activity,increased apoptosis levels,increased Cleaved-caspase3 protein expression levels,increased intracellular ROS content and decreased mitochondrial membrane potential compared to the group with cisplatin alone.2.Compared with the non-target si RNA combined with cisplatin,the volume,weight and growth rate of cervical cancer subcutaneous tumors in the si IFI16 1/2combined with cisplatin group were further decreased.3.Knockdown of IFI16 followed by cisplatin stimulation for 12 h significantly reduced p65 protein expression in the nucleus of human cervical cancer cells He La compared to the group with cisplatin alone;the expression level of IL-6 and cyclin D1 m RNA,a target gene of NF-κB pathway,was reduced.4.Knockdown of IFI16 followed by cisplatin stimulation for 12 h resulted in increased expression of p53 protein in the nucleus of human cervical cancer cells He La and increased expression levels of p21 m RNA,a downstream target gene of p53,compared with the group with cisplatin alone.5.The expression levels of P-STING/STING protein expression ratio and IFN-βm RNA expression of STING signaling pathway target genes were increased after cisplatin stimulation for 12h;no significant changes in P-STING/STING protein expression ratio and IFN-β m RNA expression of STING signaling pathway target genes were observed after knockdown of IFI16 plus cisplatin stimulation for 12 h.6.After STING inhibitor H-151 pretreatment for 2h followed by cisplatin stimulation for 12 h,there was no significant change in the expression of p65 protein in the nucleus of human cervical cancer cells He La cells and no significant change in cell activity compared with the group with cisplatin alone.Conclusion:1.In cervical cancer cells He La,induction of p53 or overexpression of p53 by cisplatin treatment induced increased protein expression of IFI16 and NF-κB(p65)in the nucleus and elevated transcript levels of the NF-κB target gene IL-6 and cyclin D1.suggesting that cisplatin induction of p53 into the nucleus may also induce increased expression of IFI16 and NF-κB(p65)in the nucleus.2.Knockdown of IFI16 inhibited cisplatin-induced NF-κB(p65)entry into the nucleus and transcription of its target gene IL-6 and cyclin D1,which exacerbated cisplatin-induced mitochondrial dysfunction and increased the sensitivity of cervical cancer cells He La to cisplatin.suggesting that targeted inhibition of IFI16 may be a novel way to increase cisplatin sensitivity in cervical cancer cells.3.Knockdown of IFI16 did not affect cisplatin-induced activation of STING pathway,and inhibition of STING signaling pathway using STING inhibitor H-151 did not affect cisplatin-induced NF-κB(p65)entry into the nucleus and sensitivity of He La cells to cisplatin.suggesting that STING signaling may not be involved in the IFI16-regulated NF-κB pathway antagonizing cisplatin-induced apoptosis in cervical cancer cells.Therefore,we suggest that targeted inhibition of the IFI16 pathway can increase cisplatin sensitivity in cervical cancer cells.