Study On Influence And Mechanism Of Abnormal Expression And Mutation Of NOTCH On Oral Squamous Cell Carcinoma

Purpose Oral squamous cell carcinoma(OSCC)is one of the most common oral and maxillofacial malignancies.The speech,eating and facial beauty of patients with OSCC will be seriously affected.In recent years,the morbidity and mortality of OSCC have not been significantly improved,and the cause of OSCC is still unclear.Since the discovery of NOTCH signaling pathway,there have been many reports that its abnormal expression and mutation may affect the occurrence and development of tumors.In this study,NOTCH1 mutation was found to be as high as 18% in head and neck squamous cell carcinoma based on TCGA data.How does the abnormal expression of NOTCH signaling pathway affect the progression of OSCC? Is abnormal expression of NOTCH signaling pathway related to mutation of NOTCH signaling pathway? Can mutations in the NOTCH signaling pathway affect the progression of OSCC? None of these issues is clear.Therefore,the aim of this study is to investigate the effect of down-regulation of NOTCH signal pathway on the progression of OSCC,the frequency and distribution of mutations in OSCC,and the effect of NOTCH signal pathway mutants on OSCC cell function.Methods1.Effect of abnormal expression of NOTCH signaling pathway on the development of OSCCOn the one hand,the expression of NOTCH1 and NOTCH2 in OSCC cell lines were inhibited by si RNA interference method,and then the expression of NOTCH1 and NOTCH2 in OSCC cells was detected by Real-time PCR and Western blot technique respectively;the proliferation of OSCC cells was detected by CCK-8 test and colony formation test respectively;cell apoptosis was detected by Annexin V-PI double staining method and cell cycle was detected by PI single staining method;cell migration and invasive ability were observed by scratch healing test and Transwell cell experiment;and finally,the growth ability of tumor stem cells was detected by stem cell pelleting culture.On the other hand,293 T cells were treated with FLI-06 and RO-4929097 respectively,and the effect of two inhibitors on the activation of NOTCH signaling pathway was detected.Then,four kinds of OSCC cell lines CAL-27,TCA-8113,SCC-9 and WSUHN30 were treated with semi-logarithmic concentration dilution method.After 72 hours,the absorbance value at 450 nm was measured by CCK-8 method,and IC50 was calculated.Finally,two NOTCH signal pathway inhibitors were applied to tumor stem cells of OSCC,respectively to observe the effect of NOTCH signal pathway inhibitors on the growth of tumor stem cells and the osmotic damage of tumor stem cell spheres.2.Mutations in NOTCH signaling pathways associated with OSCCFirstly,15 pairs of OSCC tissues and adjacent tissues were collected for DNA extraction and whole exon sequencing,and bioinformatics analysis was carried out.Secondly,the NOTCH1 high frequency mutation sites in head and neck squamous cell carcinoma or all tumors were screened by COSMIC database.Finally,Sanger sequencing was used to detect the distribution of NOTCH mutations in a larger number of DNA samples from OSCC tissue,para-cancerous tissue,patient blood,and healthy human blood.Clinical data including sex,age,preoperative radiotherapy and chemotherapy,lymph node metastasis and clinical staging were collected to analyze the association with NOTCH mutation.Real-time PCR was used to detect the expression of NOTCH1 in mutant and wild type OSCC tissues.3.Effect of NOTCH1 site-specific mutation on cell function of OSCC cell The NOTCH1 mutant plasmid was constructed by using gene site-directed mutagenesis kit.The constructed plasmid was sequenced by Sanger.The expression of NOTCH1 in293 T cells,KYSE-150 cells of human esophageal squamous cell carcinoma and SCC-9cells of OSCC was detected by Western Blot,the expression of NOTCH1 was detected by CCK-8 assay and colony formation assay,the migration ability was detected by scratch healing assay,the activation of NOTCH1 signaling pathway was detected by double-luciferase report assay,and the expression of downstream genes in NOTCH1signaling pathway was detected by Real-time PCR.Results1.Effect of abnormal expression of NOTCH signaling pathway on the development of OSCCAfter the down-regulation of NOTCH1 expression by si RNA,the proliferation ability of OSCC cells were inhibited,and the cell cycle was blocked in the G2 phase.The migration and invasion ability of cells decreased with the down-regulation of NOTCH1 expression,and the proliferation ability of OSCC tumor stem cells was also inhibited.When the expression of NOTCH2 decreased,the proliferation ability of OSCC cells was also inhibited,the cell apoptosis increased,the migration and invasion ability of cells decreased,and the proliferation ability of tumor stem cells decreased.NOTCH signaling pathway was used to inhibit OSCC cell lines.The IC50 values of FLI-06against OSCC cell lines were 3.8-7.8μM for Cal-27,3.1-3.8μM for TCA-8113,5.0-7.7μM for WSU-HN30,0.6 4.3μM for SCC-9.RO-4929097 had no inhibitory effect on the proliferation of OSCC cells.However,the two inhibitors could inhibit the proliferation of OSCC stem cells at low concentrations.FLI-06 could also penetrate the tumor stem cell spheres and destroy the integrity of the tumor stem cell spheres,while RO-4929097 had a poor ability to penetrate and destroy the tumor stem cell spheres.2.Mutations in NOTCH signaling pathways associated with OSCCWhole exome sequencing results showed that one Notch1 missense mutation site Notch1-G484 D,one Notch2 missense mutation site Notch2-N2265 K,one Notch3missense mutation site Notch3-T1751 A was detected in 15 pairs of OSCC samples,respectively.Notch1-T194 P,Notch1-T311 P,Notch1-D573 A,Notch1-L1600 P and Notch1-L1678 P were selected from COSMIC tumor mutation database.Sanger sequencing analysis of the above 8 mutation sites in OSCC tissues and paracancerous tissue samples with a larger sample size showed that there were G >D,G >A,G>C,G>R and G>S at Notch1-484 sites in OSCC.The mutation rate of NOTCH1-484 was about 11.0% in OSCC,and 2.3% in paracancerous tissues.No mutation was detected in blood samples of OSCC patients or normal blood samples.Mutation rate of NOTCH1-L1678 P was17% in OSCC and1.2% in paracancerous tissues.No mutation was detected in blood samples from patients but 2.8% in normal blood samples.The other NOTCH1 sites,NOTCH2-N2265 K and NOTCH3-T1751 A sites had no more corresponding mutations were found in OSCC samples.The mutation rate of NOTCH1-484 was increased in patients who received preoperative radiotherapy.The m RNA expression level of NOTCH1 in the OSCC tissues with NOTCH1-484 and NOTCH1-L1678 P mutations was slightly higher than that in the wild-type OSCC tissues,but the difference was not statistically significant.3.Effect of NOTCH1 site-directed mutation on the function of OSCC cells NOTCH1-G484 A,G484C,G484 D,G484R,G484 S and L1678 P site mutant plasmids were successfully constructed.The expression of wild-type and mutant plasmids in293 T cells,KYSE-150 cells and SCC-9 cells was detected by Western blot.Both wildtype and mutant NOTCH1 promoted the proliferation of 293 T,KYSE-150 and SCC-9cells,but the proliferation promotion effect of NOTCH1 on 293 T,KYSE-150 and SCC-9 cells was no significantly different between mutant and wild-type.The wound and healing assay shown there was no significant difference in the migration ability of KYSE-150 cells between the wild-type and mutant types.Dual-luciferase reporting assay showed that NOTCH signaling pathway was activated after NOTCH1-L1678 P mutation,and NOTCH1-484 mutation was inhibited.Real-time PCR showed that Hes5expression of NOTCH signaling pathway was increased after NOTCH1-L1678 P mutation.ConclusionNOTCH1 and NOTCH2 act as oncogenic in OSCC.NOTCH1-G484 A NOTCH1-G484 C,NOTCH1-G484 D,NOTCH1-G484 R,NOTCH1-G484 S and NOTCH1-L1678 P were detected in OSCC tissues with high mutation rates,which may influence the occurrence and development of OSCC.NOTCH1-L1678 P is an active mutation,which can activate NOTCH signaling pathway through NOTCH-Hes5 axis.Mutations at NOTCH1-G484 A,NOTCH1-G484 C,NOTCH1-G484 D,NOTCH1-G484 R,NOTCH1-G484 S sites have no effect on NOTCH1 signaling pathway activation.Mutations in the Notch signaling pathway are closely related to the occurrence and development of oral squamous cell carcinoma.Notch mutation can affect the activation of the signaling pathway,and the mechanism needs to be further explored.