Effect Of Silencing USP9X Expression On Reversal Of Cisplatin Resistance In Nasopharyngeal Carcinoma Cells

Background:Chemotherapy combined with radiotherapy is the main treatment for locally advanced nasopharyngeal carcinoma,and cisplatin resistance and advanced metastasis are often the key reasons for the failure of nasopharyngeal carcinoma treatment.In various cancers,abnormal USP9X(ubiquitin-specific peptidase 9X-linked)expression is involved in tumor genesis,progression,apoptosis,and metastasis.However,the relationship between USP9X abnormal expression and cisplatin resistance in nasopharyngeal carcinoma(NPC)cells remains unclear.β-catenin is an important regulatory factor in the Wnt signaling pathway,which regulates various important processes in cancer progression.It is important to find biomarkers that can improve NPC metastasis and chemotherapeutic resistance.Objective:This project aims to investigate whether silencing USP9X can reverse cisplatin resistance in NPC cells.Moreover,the effects on proliferation,apoptosis,invasion and metastasis of NPC cells were also discussed.Methods:Using qRT-PCR and western blot,we detected the expressions of USP9X and β-catenin in NPC cells.The USP9X and β-catenin silenced nasopharyngeal carcinoma cell lines were constructed by siRNA transfection technique.Nasopharyngeal carcinoma parent HNE1 and cisplatin-resistant HNE1/DDP cell lines were divided into four groups(blank Control group,negative Control si NC transfection group,si USP9X silencing group,and si β-catenin silencing group)for subsequent experiments.The effects of USP9X and β-catenin on cell cisplatin resistance,proliferation,apoptosis,and metastasis were examined by CCK-8 assay,flow cytometry,wound-healing assay,and Transwell chamber assay.Co-IP assay,qRT-PCR,and western blot were performed to explore the detailed molecular mechanism of USP9X-β-catenin and its effect on the mRNA and protein levels of MDR1,MRP2,Bcl-2,Bax,MMP2,and MMP9.Results:We found that USP9X and β-catenin expressions in cisplatin-resistance cell lines(HNE1/DDP)were much higher than cisplatin-sensitive cell lines(HNE1)at both mRNA and protein levels.Compared with the parent HNE1 cell line,the mRNA expression of USP9X in HNE1/DDP cells was up-regulated by about 4.3 times(P<0.01),and β-catenin in HNE1/DDP cells was up-regulated by about 3.4 times(P<0.01).Co-IP assay demonstrated that USP9X was immunoprecipitated with β-catenin in NPC cells.Knockdown of USP9X was able to partially reverse cisplatin-resistance,increased cisplatin-induced apoptosis,and decreased the capacities of proliferation,migration and invasion.Moreover,knockdown of USP9X expression can significantly reduce the expressions of MDR1,MRP2,Bcl-2,MMP2,and MMP9,but significantly increased the expression of Bax.However,mRNA levels had no significant effect on the above genes.The silencing of β-catenin expression can act on downstream genes at both protein and mRNA levels.Conclusions:These findings indicate that USP9X high expression plays a significant part in cisplatin-resistance of NPC.Silencing of USP9X expression can reverse cisplatin resistance,increase apoptosis,and decrease metastasis of NPC cells by decreasing the expression of β-catenin and MDR1,MRP2,Bcl-2,Bax,MMP2,and MMP9.This study elucidated the possible mechanism of cisplatin resistance in NPC cells and may have implications for therapeutic reversal of cisplatin resistance.