BMI1 Induces Cisplatin Resistance Of Oral Squamous Cell Carcinoma Cells Via NOTCH Signaling Pathway

Objective:The oral squamous cell carcinoma(OSCC)cell line CAL27 with knockdown and overexpression of the BMI1(B cell-specific moloney murine leukemia virus insertion site 1)gene was constructed by lentiviral transfection.And the expression levels of NOTCH pathway genes were examined to analyze the activation of the pathway induced by BMI1.Different concentrations of cisplatin(CDDP),the NOTCH pathway inhibitor DAPT and CDDP+DAPT were then used to investigate the effect of BMI1 on cisplatin resistance in CAL27 oral squamous cell carcinoma cells via the NOTCH pathway.The preliminary investigation of the mechanism of action of CDDP resistance will provide a basis for the further development of drug resistance reversal agents.Methods:(1)The OSCC cell line CAL27 with puromycin-resistant knockdown and overexpression of the BMI1 gene was constructed by lentivirus.The CAL27 knockdown group shBMI1 and overexpression group BMI1 stable cell lines and their corresponding negative control cell lines were constructed.The infection efficiency of shBMl1-1,shBMI1-2,shBMI1-3,shNC,BMI1 and NC was observed by inverted fluorescence microscope.(2)RT-qPCR and Western blot were used to detect the changes of BMI1 mRNA and protein expression levels in CAL27 cells after infection,and the BMI1 stable cell line with the best interference effect was selected for the next experiment.(3)Western blot and immunofluorescence staining were used to detect the expression of NOTCH1,DDL1,JAG1 and HES1 genes in the NOTCH pathway in CAL27 cells after infection.(4)The cells were cultured with DMSO,CDDP,DAPT and CDDP+DAPT,respectively.CCK-8 assay,Wound healing assay,Transwell invasion assay,colony formation assay and flow cytometry were used to detect proliferation,migration,invasion,clone and apoptosis of each group.(5)Western blot was used to detect the expression of BMI1 and NOTCH signaling pathway genes in Blank(CAL27),CAL27-CDDP and CAL27+DAPT cells.CAL27 and CAL27-CDDP cell lines were treated with DMSO,CDDP,DAPT and CDDP+DAPT to observe differences in cell proliferation,migration,invasion,cloning and apoptosis.Results:(1)Construction of CAL27 stable cell lines with BMI1 gene knockdown and overexpression;the efficiency of cell infection was 90%under inverted fluorescence microscope.Then,according to the results of RT-qPCR and Western blot,it was confirmed that BMI1 overexpression and knockdown were significant,and the expression of shBMI1-2 cell line in the knockdown group was the lowest(P<0.05)for subsequent experiments.(2)Western blot was used to detect the expression of NOTCH signaling pathway genes NOTCH1,DLL1,JAG1 and HES1 protein in each cell line.The results showed that BMI1 could induce the activation of NOTCH signaling pathway and increase the expression level of NOTCH signaling pathway gene protein(P<0.05).In immunofluorescence staining,the expression of NOTCH1 showed the same trend as in Western blot.(3)DMSO,CDDP,DAPT and CDDP+DAPT were added to intervene in each group of cells.The experimental results showed that DAPT inhibited cell activity and promoted apoptosis after inhibiting the NOTCH pathway(P<0.05).The sensitivity to cisplatin of the BMI1 group was lower than that of the shBMI1 group,the cell viability,invasion,migration and colony formation ability of the cells were increased,and apoptosis was decreased(P<0.05).In addition,compared with the CDDP group and the CDDP+DAPT group,the apoptosis rate increased significantly after drug combination,and the sensitivity of cancer cells to cisplatin increased(P<0.05).(4)Western blot showed that the expression levels of BMI1 and NOTCH signaling pathway genes were increased in the CAL27-CDDP group compared with the Blank(CAL27)group.The immunofluorescence staining trend of NOTCH1 was the same as that of Western blot(P<0.05).The growth,invasion,migration and plate cloning ability of CAL27-CDDP cells were stronger than those of Blank.The drug sensitivity of drug-resistant strains to CDDP and DAPT was reduced.When CDDP+DAPT was used in combination,the level of apoptosis was still significantly increased(P<0.05).Conclusions:(1)BMI1 enhances the resistance of oral squamous cell carcinoma CAL27 to CDDP by activating the NOTCH signaling pathway and promotes the proliferation,migration and invasion of OSCC cells.(2)NOTCH pathway inhibitor DAPT can reduce the cisplatin resistance of oral squamous cell carcinoma cell line CAL27,and CDDP+DAPT has synergistic cytotoxicity and promotes apoptosis of OSCC cells.