The Function And Mechanism Of CDKL1 Inoral Squamous Cell Carcinoma Resistarice To HCPT

ObjectivesOral squamous cell carcinoma(OSCC)is one of the most common malignant tumors in the head and neck.It is highly malignant and prone to metastasis.Hydroxycamptothecine(HCPT)has been used in the clinical treatment of OSCC and has achieved good clinical results,but there still are patients who were resistant to HCPT.This study aims to investigate the mechanism of OSCC resistance to HCPT,and to explore biomarkers that can individuallypredict the sensitivity of OSCC to HCPT,as well as to explore potential target for OSCC treatment and HCPT chemotherapy sensitization improvement.Methods1.Investigate the role of cyclin-dependent kinase-like 1(CDKL1)in OSCC cells resistance to HCPTHCPT-resistant OSCC cell lines were screened by treating with dose-increasing HCPT.Total protein in HCPT-resistant OSCC cells was extracted by RIPA lysate,and CDKL1 protein was detected by Western Blot.CDKL1 gene in OSCC cells was stably knocked-out by lentivirus infection.MTT cell proliferation assay was used to evaluate cell proliferation,and flow cytometry was applied to evaluate cell cycle progression and apoptosis.2.Investigate whether CDKL1 mediates OSCC resistance to HCPT by regulating endoplasmic reticulum stress(ER stress)The total protein was extracted by RIPA lysate,and the expression of ER stress-related protein inositol-requiring enzyme-1(IRE1)and immunoglobulin heavy chain binding protein(BIP),were detected by Western Blot.The interaction between IRE1 and BIP was evaluated by Immunoprecipitation assay(IP)using the IRE1 antibody.HCPT-resistant cells in OSCC were treated by BIP inhibitor,HA15,to reduce ER stress threshold;cell proliferation after HCPT treatment was evaluated to investigate whether reducing the ER stress threshold can increase HCPT sensitivity.CDKL1 was overexpressed in OSCC cells,and knocked down in HCPT-resistant OSCC cells;the expression of IRE1 and BIP was detected by Western Blot,and the interaction between IRE1 and BIP was detected by IP,to evaluate the regulation of CDKL1 on ER stress threshold.3.Investigate the regulation mechanism of CDKL1 expression in HCPT-resistant OSCC cel sCDKL1 protein level was detected by Western Blot,and CDKL1 mRNA expression level was measured by qRT-PCR.CDKL1 promoter activity reporter was constructed,and CDKL1 promoter activity was detected by firefly luciferase activity assay.Putativespecificity protein 1(Sp1)binding sites on the CDKL1 promoter were induced by site-directed mutagenesis.Chromatin immunoprecipitation assay was conducted to verify the binding of Spl to the CDKL1 promoter.Results1.CDKL1 overexpression mediated HCPT resistance in OSCCWe used HCPT to treat OSCC CAL-27 cells in the logarithmic growth phase,screened for drug-resistant cells,and selected two HCPT-resistant cell colonies and obtained two cell lines,CAL-27H1 and CAL-27H2,by monoclonal culture.It was confirmed by MTT cell proliferation assay that the inhibition of proliferation of CAL-27H1 and CAL-27H2 by HCPT was significantly suppressed than that of CAL-27.CAL-27H1 was named CAL-27H and used for subsequent experiments.CAL-27H and CAL-27 cells were inoculated subcutaneously into BALB/c nude mice,and intraperitoneal injection of HCPT was performed after tumor formation.The tumor volume and quality of the CAL-27H group were lower than those of the CAL-27 group.The RNA in CAL-27 and CAL-27H cells was extracted by Trizol.After reverse transcription,the expression of CDKL1 mRNA in both cells was evaluated by qRT-PCR.The results showed that the CDKL1 mRNA level in CAL-27H cells was higher than CAL-27 cells.Western Blot showed that the expression of CDKL1 protein in CAL-27H cells was higher than that in CAL-27 cells.CAL-27 and CAL-27H cells were subjected to tumor-bearing experiments in nude mice.After tumor formation,tissue sections were taken for tumor tissue,and immunohistochemical staining was performed to label CDKL1 expression in tumor tissues.The results showed that the CAL-27H group showed more positive staining than the CAL-27 group,indicating higher expression of CDKL1 in CAL-27H cells than that in CAL-27 cells.CAL-27 cells were infected with Lv-shCDKL1 to knockdown CDKL1,and the effects of CDKL1 knockdown on proliferation,apoptosis and cell cycle weretested.MTT assay showed that the proliferation activity of CDKL1-knockdown cells was significantly weakened.Cell cycle assay showed that the proportion of cells inthe G0/G1 phase was significantly higher in CDKL1-knockdown cells,suggesting that knocking down CDKL1 can arrest thecell cycle inthe G0/G1 phase.Annexin V/PI flow cytometry was used to detect the proportion of apoptotic cells in CDKL1-knockdown and wild-type CAL-27 cells.The results showed that the apoptosis rate was significantly increased in CDKL1-knockdown cells.We overexpressed CDKL1 in CAL-27 cells by lentivirus infection,and treated CDKL1-overexpressing and wild-type cells by HCPT.MTT assay showed that HCPT inhibited both groups of cells,but inhibition on proliferation in CDKL1-overexpressing cells was significantly weaker than wild-type cells.Annexin V/PI flow cytometry showed that HCPT inducedimproved apoptosis of CAL-27 cells and CAL-27H cells.After HCPT treatment,the apoptosis rate of CAL-27H was lower than that of CAL-27 cells.It is suggested that overexpression of CDKL1 enhanced the resistance of cells to HCPT.2.CDKL1 mediated OSCC resistance to HCPT through ER stressCAL-27 cells were treated with HCPT.Totalprotein was extracted,and IRE1 was detected by Western Blot.The results showed that HCPT up-regulated the expression of IRE1 in CAL-27 cells,suggesting that HCPT may activate ER stress.The total protein of CAL-27 and CAL-27H cell wasextracted,and BIP expression was detected.The BIPprotein level in CAL-27H cells was significantly higher than that of CAL-27 cells.In addition,IP usingthe IRE1 antibody showed significantly higher BIP in CAL-27H cells than in CAL-27 cells,indicating strongerBIP-IRE1 binding in CAL-27H cells,suggesting higher threshold of ER stress activation in CAL-27H cells than that of CAL-27 cells.After treating CAL-27H cells with BIP inhibitor,HA15,HCPT was added to the cells.The results showed that the proliferation of HA15-pretreated cellswas more inhibited by HCPT than cells without HA 15,suggesting inhibition of BIP and lowering the ER stress threshold can increase the sensitivity of CAL-27 cells to HCPT.We extracted the total protein in CDKL1-overexpressing and wild-type CAL-27 cells,and detected the expression of BIP in both cells by Western Blot.It was found that overexpression of CDKL1 could up-regulate the expression of BIP in cells.The effect of CDKL1 overexpression on BIP and IRE1 interactionin cells was evaluated by IP,which showed that BIP and IRE1 interaction in CDKL1-overexpressing CAL-27 cells was stronger compared to wildtype cells.CDKL1 knockdown was associated with down-regulated BIP and weakened binding of BIP and IRE1.These findings suggested that CDKL1 improved the ER stress threshold of CAL-27 cells.3.Spl up-regulated CDKL1 expression in OSCCWe constructed the CDKL1 promoter activity reporter gene pGL3-CDKL1,which was transfected into CAL-27 cells and HCPT-resistant OSCC cells,i.e.,CAL-27H cells.The CDKL1 promoter activity in the cells was detected by firefly luciferase activity assay,which showed that the CDKL1 promoter activity in CAL-27H cells was higher than that in CAL-27 cells.The expression of transcription factor Spl in both cells was examined by qRT-PCR,which showed that the CDKL1 mRNA level in CAL-27H cells was higher than that of CAL-27 cells.Protein level detection by Western Blot showed that CDKL1 was improved in CAL-27H cells but not in CAL-27 cells.We overexpressed Spl in CAL-27 cells,and the CDKL1 promoter activity was detected.We found that overexpression of Spl up-regulated CDKL1 promoter activity,while knockdown of Spl down-regulated CDKL1 promoter activity.There are five putative Spl recognition sites on the CDKL1 promoter.We induced individual point mutations to detect the activity of each mutant reporter gene.It is found that mutation of sites A or C can down-regulate the CDKL1 promoter activity,and the mutation of site C can blocking the upregulation of SpK1 on CDKL1 promoter activity.ConclusionIn OSCC,Spl upregulates CDKL1 expression at the transcriptional level by regulating CDKL1 promoter activity.Increased expression of CDKL1 can upregulate BIP,elevatethe ER stress threshold,and mediate OSCC resistance to HCPT.Spl/CDKL1/BIP axis can be used as tumor markers for predicting the sensitivity of OSCC to HCPT and as potential targets for OSCC treatment and the development of HCPT sensitizing drugs.