Study On The Mechanism Of Cisplatin Changes Oral Squamous Cell Carcinoma Stem Cell Self-renewal Ability Through Notch4 Signaling Pathway

Objective:As one of the head and neck tumors,oral squamous cell carcinoma is the sixth most common type of cancer in the world.At present,one of the first-line treatment methods for oral squamous cell carcinoma is cisplatin chemotherapy,but clinical recurrence is easy.One of the important reasons is the presence of tumor stem cells,which have strong selfrenewal ability and can increase the probability of tumor recurrence and drug resistance.The abnormally highly expressed Notch signaling pathway can promote tissue growth and self-renewal as well as maintain the function of undifferentiated stem cells,and is also closely related to the self-renewal of tumor stem cells in vivo.Most previous studies have focused on the effect of Notch4 signaling pathway and epithelial mesenchymal transformation,and few studies have been conducted on the self-renewal ability of tumor stem cells.Therefore,the scientific question to be answered in this study is the relationship between Notch4 signaling pathway and the self-renewal ability of head and neck tumor stem cells.Methods:1.CCK-8 experiment was used to determine the optimal concentration of cisplatin by adding gradient concentration cisplatin to treat oral squamous cell lines PJ15 and PJ41;2.In order to verify whether cisplatin could affect the expression level of CSCs.AnnexinV-FITC staining was used to detect the proportion of CSCs by flow cytometry,and the changes in protein expression levels of CSCs specific genes(Nanog,Bmi-1,Oct4)and gene transcription levels before and after the addition of cisplatin were detected by Western Blot and q PCR;3.TCGA Head and Neck Cancer(HNSC)was selected from UCSC Xena database and four receptors of Notch pathway were input to perform gene table difference analysis.Then the expression levels of Notch pathway related genes(Notch1-4,HES1,HEY2,etc.)were detected from molecular and gene transcription by Western Blot and q PCR;4.At the same time,in order to prove the connection between Notch pathway and oral squamous cell carcinoma stem cells,Notch pathway inhibitor DAPT was added to both cell lines,and protein expression levels of CSCs specific genes,Notch pathway related genes and gene transcription levels were detected after Notch pathway was blocked by Western Blot and q PCR;5.In order to further detect the role of Notch4 signaling pathway,the self-renewal ability was detected by tumor stem cell sphere-forming experiment after the Notch4 gene was knocked down by si-RNA transfection technology;6.After the Notch4 gene was knocked down,added cisplatin,the proportion of ALDH1-positive cells was detected by flow cytometry,and the expression of CSCs specific markers Nanog,Bmi-1 and other genes was detected by Western Blot to verify whether the knock down of Notch4 gene affects the expression of CSCs increased by cisplatin.Results:1.According to the CCK-8 experiment,the optimal concentration of cisplatin was determined to be 2μmol/L;2.After cisplatin was added,the proportion of CSCs were detected by flow cytometry and PJ15s’ increased from 4.94% to 11.8%,PJ41 s’ increased from 4.08% to 11.3%.Western Blot and q PCR results showed that the expressions of Nanog,Bmi-1 and Oct4 in the two cell lines were on the rise under the action of cisplatin at gradient concentration,which was statistically significant(P < 0.05);3.From the TCGA head and neck cancer database of normal tissue and tumor samples are compared,and Notch pathway related gene expression quantity found Notch4 compared with other receptors,the amount of gene expression in tumor group was obviously higher than that of normal group(P = 0.046),Western Blot and q PCR results showed that compared with other receptors,Notch4 has a higher level of protein expression and Notch4 ’s m RNA expression quantity rising trend is more obvious(P < 0.05);4.After the added Notch pathway inhibitor DAPT,the expression levels of Nanog,Bmi-1 and downstream genes HES1 and HEY2 of Notch pathway were decreased(P < 0.05);5.The tumor stem cell sphere-forming experiment also showed that after knocked down Notch4,compared with the control group,the tumor stem cell sphere-forming ability of CSCs in both cell lines was reduced;6.After Notch4 gene was knocked down,added cisplatin,compared with the group without knocked down Notch4,Western Blot results showed that the protein expression levels of Nanog,Bmi-1 and other genes were reduced.Conclusion:In oral squamous cell carcinoma,Cisplatin-induced enhancement of the self-renewal ability of CSCs is mediated through the Notch4 signaling pathway,Notch4 signaling can regulate the self-renewal of CSCs.