USP10 Targeting POLR2A Deubiquitination Promotes SLC7A11 Inhibition Of Ferroptosis In Head And Neck Squamous Cell Carcinoma

Chapter 1 Systematic analysis of prognosis-related ferroptosis-regulated genes in head and neck squamous cell carcinomaObjective:The continuous improvement of the treatment model of head and neck squamous cell carcinoma has not significantly improved the prognosis of patients.It is still very important to discover new markers for head and neck squamous cell carcinoma and develop effective prognostic assessment models.Ferroptosis is a new way of programmed cell death.The targeted induction of ferroptosis in cancer cells is a new cancer treatment strategy.Key genes in the regulation of ferroptosis in head and neck squamous cell carcinoma remain unclear.We aim to systematically analyze the prognostic relevance of ferroptosis-regulated genes based on CRISPR-Cas9 genome-wide screening in head and neck squamous cell carcinoma.We aim to construct and validate the prognostic evaluation model of ferroptosis-regulated genes,and reveal their potential biological roles and tumor immunity.Methods:1.The ferroptosis-regulated genes screened by CRISPR-Cas9 in GEO were used to analyze the prognosis of head and neck squamous cell carcinoma,and multiple databases were used to construct and validate the ferroptosis-regulated gene prognosis evaluation model of head and neck squamous cell carcinoma.2.Gene enrichment was used to analyze potential biological function differences between high-risk and low-risk groups of head and neck squamous cell carcinoma.3.We evaluated the immune score and tumor immune cell infiltration in head and neck squamous cell carcinoma samples by ESTIMATE and CIBERSORT algorithms to explore the relationship between risk score and tumor immune score and immune cell infiltration.4.The expression levels of risk model constitutive genes in clinical tissues were detected by RT-q PCR to verify their expression differences.Results:1.Seven ferroptosis-regulated genes related to the prognosis of head and neck squamous cell carcinoma were obtained,namely ACAT1,HSP90B1,MRPL10,PGK1,PPFIBP2,USP10 and WNT7A.Based on these seven genes,a risk model of head and neck squamous cell carcinoma was constructed,and the reliability of the model was verified with multiple databases.2.Multiple metabolic pathways were significantly activated in the high-risk group of head and neck squamous cell carcinoma,including the glutathione metabolic pathway.3.The high-risk group of head and neck squamous cell carcinoma and the low-risk group showed a low immune score.The proportion of CD8~+T cell infiltration were decreased.4.ACAT1,HSP90B1,MRPL10,PGK1,USP10 and WNT7A were highly expressed in head and neck squamous cell carcinoma,while PPFIBP2 was lowly expressed.Conclusion:The prognosis of patients can be well evaluated by the risk model constructed from the seven prognostic-related ferroptosis-regulated genes screened in head and neck squamous cell carcinoma.High risk scores suggest activation of multiple metabolic pathways and poor tumor immunosuppression and CD8~+T cell infiltration.Chapter 2 The role of USP10 in ferroptosis in head and neck squamous cell carcinomaObjective:In the previous chapter,we screened out the ferroptosis regulators associated with the prognosis of HNSCC.Ubiquitination is one of the most common post-translational modifications,and the ubiquitin-proteasome system regulates a wide range of biological processes.Currently,the role of the ubiquitin-proteasome system in HNSCC ferroptosis remains unclear.We mainly observed the effect of USP10 on ferroptosis in head and neck squamous cell carcinoma through in vitro and in vivo experiments in this chapter.Methods:1.The protein expression levels of USP10 in immortalized epithelial cells DOK and 7 head and neck squamous cell carcinoma cells were detected by Western Blot.2.Fadu and SAS cells were infected with lentiviral sg RNA vector to construct stable USP10 knockout cell lines.The stable overexpression cell line of USP10 was constructed by infecting Cal27 cells with lentiviral overexpression vector.Knockout or overexpression efficiency of USP10 was detected by RT-q PCR and Western Blot.3.CCK8,BODIPY-C11 probe staining and flow cytometry,GSH and GSSG detection kit were used to analyze the effect of USP10knockout and overexpression on ferroptosis induced by ferroptosis inducers Erastin and RSL3.The cells in each group were observed by clone formation.4.We used the Fadu cell lentivirus USP10 stable knockout strain and the control group for subcutaneous tumor formation and intraperitoneal injection of ferroptosis inducers in nude mice.Results:1.In head and neck squamous cell carcinoma cells,the expression of USP10 was higher in Fadu and SAS cells,but lower in Cal27cells.2.The USP10 knockout efficiency was high in Fadu and SAS USP10stable knockout cell lines.The USP10 was upregulated by about 30 times in m RNA and nearly 4 times in the protein in Cal27 cells USP10 stable overexpression cell lines.3.Cell viability and GSH were decreased compared with the control group after knockdown of USP10 in head and neck squamous cell carcinoma cells.ROS level and GSSG were increased compared with the control group after knockdown of USP10 in head and neck squamous cell carcinoma cells.Conversely,cell viability and GSH were increased compared with the control group after overexpression of USP10 in head and neck squamous cell carcinoma cells.ROS level and GSSG were decreased compared with the control group after overexpression of USP10 in head and neck squamous cell carcinoma cells.4.Oncogenic inhibition effect of ferroptosis inducers can be enhanced or weakened by knockdown or overexpression of USP10 in head and neck squamous cell carcinoma,respectively.In the nude mouse subcutaneous tumorigenesis model,the tumor growth in the USP10 knockout group was significantly lower than that in the control group after the premise of using ferroptosis inducers.Conclusion:The susceptibility of head and neck squamous cell carcinoma cells to ferroptosis can be inhibited by USP10.USP10 knockout enhances the growth inhibition of head and neck squamous cell carcinoma cells by ferroptosis inducers.Chapter 3 The molecular mechanism of USP10 regulating ferroptosis in head and neck squamous cell carcinomaObjective:Our studies have verified that USP10 can inhibit the susceptibility of head and neck squamous cell carcinoma cells to ferroptosis through in vitro and in vivo experiments.We aim to clarify the specific molecular mechanism of USP10 regulating ferroptosis in head and neck squamous cell carcinoma in this chapter.Methods:1.We used whole-genome transcriptome high-throughput sequencing to analyze the differential changes in transcriptional profiles of head and neck squamous cell carcinoma cells after USP10 knockout.2.The potential biological function of USP10 in head and neck squamous cell carcinoma cells was revealed by KEGG pathway and GO analysis.3.We used the String database to predict the interacting proteins of USP10.4.The UCSC(University of California Santa Cruz)database was used to query the transcription factors and promoter-binding proteins found by Ch IP-seq.JASPAR software were used to predict transcription factors.5.The m RNA and protein expression levels of target molecules were detected by RT-q PCR and Western Blot.The regulatory relationship between molecules was analyzed by rescue experiments.6.The direct interaction between transcription factors or promoter-binding proteins and promoters was verified by CHIP-PCR,RT-q PCR and luciferase reporter gene experiments.7.CO-IP and Western Blot were used to confirm the interaction of proteins.Results:1.Whole transcriptome sequencing showed that there were458 differentially expressed genes,of which 196 were down-regulated and262 were up-regulated.2.KEGG pathway analysis showed that ubiquitin-proteasome system-related pathways were enriched,and GO analysis also found that ubiquitination-related pathways were enriched.3.Taking the intersection of 196 differential genes down-regulated after USP10knockout and known ferroptosis endogenous core antagonist genes,the results showed that knocking out USP10 can reduce the transcription level of ferroptosis core antagonist SLC7A11.USP10 only regulated the m RNA expression of SLC7A11,but could reduce the protein levels of SLC7A11and GPX4 by RT-q PCR and Western Blot.Rescue experiments suggested that USP10 can inhibit ferroptosis sensitivity by regulating SLC7A11.4.POLR2A are intersected by the transcription factor and its promoter-binding protein of SLC7A11 and the interacting proteins of USP10,which interacts with USP10 and directly regulates the m RNA transcription of SLC7A11.USP10 could not change the m RNA level of POLR2A,but could regulate its protein level by RT-q PCR and WB.Rescue experiments suggested that USP10 can inhibit ferroptosis sensitivity by regulating POLR2A.5.POLR2A could bind to SLC7A11 promoter by CHIP-PCR.The result of luciferase reporter gene assay showed that POLR2A could enhance the SLC7A11 promoter activity.6.The result of Co-IP assay confirmed that USP10 can bind POLR2A,and overexpression of USP10can reduce the ubiquitination level of POLR2A.Conclusion:USP10 can target the deubiquitination of POLR2A to increase the level of POLR2A protein,which promote the transcription of SLC7A11.Then,SLC7A11 finally inhibit the cell sensitivity of ferroptosis in head and neck squamous cell carcinoma.Chapter 4 Expression of USP10 in head and neck squamous cell carcinoma and its clinical significanceObjective:We aim to clarify the expression level of USP10 in head and neck squamous cell carcinoma and its correlation with the prognosis and tumor immunity of head and neck squamous cell carcinoma in this chapter.Methods:1.Unpaired and paired differential analysis was performed using the m RNA expression levels of head and neck squamous cell carcinoma tissues and adjacent control tissues in the TCGA database.2.RT-q PCR and immunohistochemical techniques were used to detect the expression of USP10 in head and neck squamous cell carcinoma samples and adjacent tissues.3.The relationship between USP10 expression level and patient prognosis was analyzed by Kaplan-Meier survival curve.4.The immune score and tumor immune cell infiltration in head and neck squamous cell carcinoma samples were evaluated by ESTIMATE and CIBERSORT algorithms to explore the relationship between USP10expression and tumor immunity.Results:1.The m RNA expression level of USP10 was higher than that in adjacent tissues in head and neck squamous cell carcinoma tumor tissues in TCGA database.2.The m RNA and protein expression levels of USP10 in cancer tissues were significantly increased by RT-q PCR and immunohistochemical.3.Patients with high USP10 expression in cancer tissue had poor overall survival rate(P=6.258e-5)and poor disease-free survival rate(P=1.401e-2)by Kaplan-Meier survival analysis.4.The immune score of the high USP10 expression group in head and neck squamous cell carcinoma was increased,and the USP10 expression level was significantly negatively correlated with the immune score(R=-0.19,P<0.001)in the TCGA dataset.The proportion of CD8~+T cells infiltrating HNSCC samples from the high-risk group was reduced compared to the low-risk group by CIBERSORT analysis of the TCGA dataset.Conclusion:USP10 is highly expressed in head and neck squamous cell carcinoma tissues.High expression of USP10 in HNSCC cancer tissues predicts poor prognosis and may be related to tumor immunosuppression.Figures:18Forms:10Text references:138Review references:196...