PHF20 Inhibition Promotes Apoptosis And Cisplatin Chemo-sensitivity Via OCT4-p-STAT3-MCL1 Signaling Pathway In Hypopharyngeal Squamous Cell Carcinoma

Part ⅠPHF20 is upregulated in cisplatin-resistant HSCC cellsObjective:Hypopharyngeal squamous cell carcinoma(HSCC)is one of the most aggressive types of head and neck cancer,cisplatin-based chemotherapy is an important option for patients with recurrent and metastatic HSCC,but nearly all patients will die within a year because of resistant to cisplatin The underlying molecular mechanism requires further investigation.PHF20,also known as glioma-expressed antigen 2(GLEA2)is a putative transcriptional factor.However,whether the expression of PHF20 may be involved in the cisplatin resistance of HSCC have not to be elucidated.Methods:FaDu cells were treated with various concentrations(0,1,2,4,8,16 and 32μM)of cisplatin for 2 days,and the half maximal inhibitory concentration(IC50)was determined.FaDu cells were treated with lower concentrations(0.25 μM).The concentrations of cisplatin were gradually increased,and cisplatin-resistant FaDu cells were generated for 6 months.Cell survival assay(CCK-8 assay)determined the cell viability.The apoptotic proteins were detected by Western blot.RNA sequencing was performed to analyze the genes with altered expression levels in FaDu cells due to cisplatin resistance.The expression levels of PHF20 were validated by RT-PCR and western blot.Results:FaDu cells were treated with cisplatin,and the IC50 was determined.The IC50 of FaDu cells was 2.370 μM with a 95%confidence interval(CI)of 2.228-2.529 μM.FaDu cells were treated with increasing doses of cisplatin for 6 months to establish a cisplatin-resistance model.The IC50 of cisplatin-resistant FaDu cells was 6.937 μM with a 95%CI of 6.379-7.543 μM with a resistance index(RI)of 2.927.The sensitivity of cells to cisplatin was detected via CCK-8 assay,and a stable cell population resistant to cisplatin(FaDuCIS-R)was obtained compared with parental FaDu cells(FaDuCIS-S).Cell viability was significantly lower in FaDuCIS-S+Cis cells compared with that in FaDuCIS-R+Cis cells.In addition,at the protein level,the expression levels of MCL1 decreased,whereas the levels of cleaved caspase-3 and PARP1 increased in FaDuCIS-s+Cis cells compared with those in FaDuCIS-s cells indicating apoptosis.The RNA sequencing data are available at the Sequence Read Archive database of NCBI(accession nos.SRR13805665,SRR13805664,SRR13805661,SRR13805657,SRR13805656 and SRR13805655).With fold-change>2 and P<0.001 used as the screening criteria in gene detection,the transcriptome sequencing analysis identified 24,361 differently expressed genes between FaDuCIS-R and FaDuCIS-S,including 14,487 upregulated and 9,874 downregulated genes.Among the differentially regulated genes between the two cell types analyzed in the present study,PHF20 was remarkably upregulated in FaDuCIS-R cells.The expression levels of PHF20 were validated by RT-PCR and western blot.FaDuCIS-R cells exhibited higher expression levels of PHF20 compared with those in FaDuCIS-s cells.Conclusions:Therefore,our findings reveal that PHF20 is upregulated in cisplatin-resistant HSCC cells and the PHF20 expression may be associated with chemoresistance in HSCC cells.Part ⅡPHF20 inhibition promotes apoptosis and cisplatin chemosensitivity via the OCT4-p-STAT3-MCL1 signaling pathway in hypopharyngeal squamous cell carcinomaObjective:PHF20 has been found to be highly expressed in several cancer types,and the function of PHF20 is not exactly the same in different tumors.PHF20 is related to cisplatin resistance of hypopharyngeal cancer cells and cisplatin resistance is closely related to apoptosis.However,the biological behavior of PHF20 involved in HSCC has not to be illustrated.The signaling pathways associated with PHF20 are not exactly the same in different tumors.However,the molecular mechanisms need to be further elucidated.Methods:The FaDu cell line was used as the HSCC model.To verify the efficiency of knockdown on PHF20 expression,we examined the relative protein and mRNA levels of PHF20 expression in LV-PHF20-RNAi(shPHF20)and scramble shRNA(Scr)FaDu cells by Western blot and RT-PCR analyses.After confirming that PHF20 expression level was down-regulated,cell survival assay(CCK-8 assay)determined the cell viability,the invasion and migration ability of FaDu cells were detected by Transwell chamber test.EDU labeling assay and Ki67 assay assessed cell proliferation.Flow cytometry detected cell cycle phases and apoptotic cells.The changes of PARP1 and MCL1 expression at the level of protein were detected by Western blot.Silencing PHF20 combination with cisplatin in FaDu cells.apoptotic-associated proteins were detected by Western blot,at the same time apoptotic cells were assessed by flow cytometry assay.The mRNA expression levels of PHF20 and OCT4 were detected by RT-PCR when PHF20 was inhibited.The protein expression levels of PHF20.OCT4,MCL1,STAT3 and p-STAT3 were determined by western blot assay in shPHF-and Scr-transfected cells.Immunofluorescence assay was used to detect the expression and localization of PHF20,p-STAT3 and STAT3 in shPHF20-and Scr-transfected cells.To further examine whether the OCT4 was involved in PHF20 associated signaling pathways,FaDu cells were co-transfected with lentivirus-mediated PHF20 shRNA and OCT4 overexpression plasmid.Western blot analysis was used to detect the changes of p-STAT3,PARP1 and MCL1 proteins expression levels.RT-PCR and western blot assays detected OCT4 three main isoforms,namely OCT4A,OCT4B and OCT4B1.To investigate the role of PHF20 in vivo,shPHF and control cells were subcutaneously injected into nude mice in order to establish an animal xenograft model.Histological analysis and TUNEL assays were performed on the tumor tissue sections from the mice in the two groups.Results:Western blot and RT-PCR analyses showed that the expression of PHF20 in shPHF20 group FaDu cells was decreased compared with Scr group,suggesting effectively suppression of PHF20 expression by LV-PHF20-RNAi(P<0.001).After silencing PHF20 expression,the cell viability was decreased(P<0.001),however the migration and invasion ability of FaDu cells in shPHF20 group was no changed compared with the control.EDU labeling assay and Ki67 assay indicated that there was no difference when PHF20 was inhibited.Flow cytometry documented that silencing PHF20 did not result in cell cycle phases change but apoptotic cells increased(P<0.001)compared with the control.Western blot results showed that the expression of cleaved PARP1 was increased and MCL1 was decreased at the protein.Moreover,we further found that PHF20 knockdown had a synergistic effect with Cisplatin in treating HSCC.Counting Kit-8 assay revealed that the cell viability in the shPHF+Cis group significantly decreased compared with those in the shPHF and Scr+Cis groups.Western blot assay results demonstrated that the protein expression levels of MCL1 decreased in shPHF cells treated with cisplatin compared with those in the shPHF and Scr+Cis groups,whereas the levels of cleaved PARP1 and caspase-3 increased in the same conditions,Flow cytometry showed that the proportion of apoptotic cells was significantly higher in the shPHF+Cis group compared with that in the Scr+Cis and shPHF groups.RT-qPCR assay revealed that the mRNA expression levels of OCT4 were markedly decreased following PHF20 knockdown compared with those in the Scr-transfected cells.Western blot assay demonstrated that the protein levels of OCT4,MCL1 and p-STAT3 were downregulated in the shPHF group compared with those in the Scr group.Immunofluorescence assay revealed that p-STAT3 expression was lower in FaDu cells following PHF20 knockdown comparison with that in the shPHF group.Western blot assay showed that OCT4 overexpression restored the PHF20 knockdown-mediated reduction in the levels of p-STAT3,MCL1 and cleaved PARP1.Reverse transcription-quantitative PCR assay demonstrated that the mRNA expression levels of OCT4A and OCT4B1 were decreased,whereas the expression levels of OCT4B were not affected by PHF20 knockdown compared with those in the Scr group.Western blot analysis revealed that the relative protein expression levels of OCT4A and OCT4B1 were decreased in the shPHF group compared with those in the control cells.Conclusions:Our study demonstrated that knockdown of PHF20 could promote apoptosis in FaDu cells.Furthermore,silencing PHF20 could effectively sensitize cisplatin-induced apoptosis in combination with cisplatin in FaDu cells.PHF20 inhibition may promote apoptosis in FaDu cells via the OCT4-p-STAT3-MCL1 signaling pathway and sensitize FaDu cells to cisplatin treatment.OCT4A and OCT4B1 may mediate PHF20 knockdown-induced apoptosis by regulating the levels of p-STAT3 and MCL1.therefore,PHF20 may provide a potential target for the treatment of HSCC patients.