Experimental Study Of Oct4 And Sox2 On The Occurrence And Proliferation Of Oral Squamous Cell Carcinoma By Affecting The Expression Of RHEBL1

Experimental study of effect of Oct4 and Sox2 on the occurrence and proliferation of oral squamous cell carcinoma by affecting the expression of RHEBL1Oral squamous cell carcinoma is a malignant tumor derived from oral mucosal epithelium.It generally grows rapidly,has strong local invasion,is prone to recurrence and regional lymph node metastasis,and has a poor prognosis.The research on the molecular mechanism related to the occurrence and development of oral squamous cell carcinoma,and the further screening and development of diagnostic and therapeutic markers and drug targets are the forefront and focus of research at home and abroad.In the previous study,the research group found that Oct4 and Sox2 double positive cells existed in precancerous lesions of oral mucosa such as leukoplakia and lichen planus.Human telomerase reverse transcriptase(hTERT)gene was also transfected into primary cultured oral mucosal epithelial cells to establish an immortalized cell line(hTERT+-OME),which is an ideal model for studying reprogramming and tumorigenesis;Further study found that hTERT+-OME cells induced by 3D suspension culture α-Tubulin expression decreased and the expression of NF-κB(p65)increased and EMT appeared.Using this cell model,the research group also confirmed that hTERT+-OME cells overexpressing Oct4 and Sox2 can be reprogrammed and induce tumorigenesis.In the previous research,the research group applied the second-generation highthroughput sequencing technology to detect the following 8 groups of cells:hTERT+OME cells,hTERT+-OME cells overexpressing Oct4,hTERT+-OME cells overexpressing Sox2,hTERT+-OME cells overexpressing Oct4 and Sox2 at the same time,Cal27 cells,Cal27 cells silencing Oct4,Cal27 cells silencing Sox2 and Cal27 cells silencing Oct4 and Sox2 at the same time,The differentially expressed mRNA after overexpression/silencing of Oct4 and Sox2 genes was screened and identified.The analysis results showed that the expression level of RHEBL1 mRNA changed significantly after overexpression or silencing of Oct4 and Sox2 genes,suggesting that rhebll may be the target gene regulated by Oct4 and Sox2 in the occurrence and development of oral squamous cell carcinoma.The purpose of this study is to clarify the role and possible mechanism of RHEBL1 in the occurrence and development of oral squamous cell carcinoma,and further study and analyze the possible regulatory effects of Oct4 and Sox2 on RHEBL1 gene.Part Ⅰ:expression changes of RHEBL1,Oct4 and Sox2 in oral squamous cell carcinomaObjective:To investigate the expression of RHEBL1 in oral precancerous lesions and oral squamous cell carcinoma,and to observe its relationship with related stem cell factors Oct4 and Sox2.Methods:40 cases of normal oral mucosa,31 cases of oral lichen planus and 46 cases of oral leukoplakia were collected.The expression of rhebll in normal oral mucosa and precancerous lesions was detected by immunohistochemical staining.The expression of rhebll,Oct4 and Sox2 in oral cancer and adjacent tissue chip(horac080pg01)was detected by multi label immunofluorescence staining to understand the relationship between rhebll and stem cell factors Oct4 and Sox2.Results:The results showed that rhebl1 was not expressed in normal oral mucosa,and the positive rates in oral lichen planus and oral leukoplakia were 45.2%and 60.9%,respectively;RHEBL1 is widely expressed in oral squamous cell carcinoma and adjacent tissues.Most of the positive cells are located in the basal layer and arranged in a linear order.There are many RHEBL1,Oct4 and Sox2 positive cells in the front of tumor invasion and the basal layer of adjacent tissues.Conclusion:The expression of Oct4 and Sox2 in oral squamous cell carcinoma and precancerous lesions is closely related to the expression of RHEBL1.Part Ⅱ:the role of RHEBL1 in the occurrence and development of oral squamous cell carcinomaObjective:To determine the effect of RHEBL1 on the occurrence and development of oral squamous cell carcinoma by regulating the expression of RHEBL1.Method:The immortalized oral epithelial cells hTERT+-OME overexpressed RHEBL1 by cas9 gene editing technology,and the hTERT+-RHEBL1+-OME cell line was obtained.The 3D suspension culture was induced by spheroidizing medium in vitro.The expression changes of RHEBL1 in immortalized oral epithelial cells under different culture conditions,the spheroidizing ability in vitro and the morphological changes of cells in each group were observed;Combined with the subcutaneous tumorigenesis experiment of NOD-SCID mice,the tumorigenicity of the two cells was observed.Human tongue squamous cell carcinoma cell line Cal27 was knocked out of RHEBL1 to obtain Cal27-RHEBL1K.O cell line.The expression of RHEBL1 in Cal27 cells before and after knockout was detected,and 3D suspension culture was induced.The subcutaneous tumorigenesis experiment of NOD-SCID mice was carried out to observe the tumorigenesis of the two cells.Result:After hTERT+-OME overexpressed RHEBL1,hTERT+-RHEBL1+-OME cells increased significantly in vitro compared with other groups(p<0.05);The expression of RHEBL1 increased spontaneously in hTERT+-NC-OME cells in 3D suspension culture induced by spheroidizing medium;By observing the overexpression group cells and control group cells under different culture conditions,it was found that the morphology of hTERT+-RHEBL1+-OME cells in 3D suspension culture was changed,mostly in long spindle type;Both groups of cells could not form tumors under the skin of mice,but hTERT+-RHEBLl+-OME could form tumor like lesions under the skin of mice.After rhebll knockout,the in vitro balling ability of Cal27 cells decreased significantly with RHEBL1 knockout(p<0.01);The average tumor volume(168mm3)and average tumor mass(0.146g)of RHEBL1 knockout group were significantly lower than those of non knockout group(390.1mm3 and 0.300g)(p<0.01).Conclusion:RHEBL1 can promote the occurrence and proliferation of oral squamous cell carcinoma.Part Ⅲ:Study on the regulatory mechanism of Oct4 and Sox2 on RHEBL1Objective:To investigate the regulatory mechanism of Oct4 and Sox2 on RHEBL1 expression.Methods:NCBI database was used(https://www.ncbi.nlm.nih.gov/)RHEBL1 promoter sequence was queried and UCSC database was used(http://www.genome.ucsc.edu/)Compare and verify.Using Jaspar database(http://jaspar.genereg.net/)The binding sites of Oct4\Sox2 and RHEBL1 promoter region were predicted.The regulatory mechanism of Oct4\Sox2 on RHEBL1 was verified by double luciferase reporter gene experiment and ChIP-PCR experiment.Results:Both Oct4 and Sox2 could activate the transcription of RHEBL1,p<0.05.The simultaneous action of Oct4 and Sox2 had the strongest transcriptional activation of RHEBL1,p<0.01.Oct4 can combine with Sox2 to form a complex.There are two potential binding sites between Oct4 Sox2 complex and RHEBL1 promoter region(Location:-1716～-1702,sequence:tattctgaagctaag;Location:-1372～-1358,sequence:cgttaagatgaaat).The analysis of these two potential binding sites showed that the transcriptional activation of RHEBL1 gene by Oct4 and Sox2 decreased significantly(p<0.05).Oral squamous cell carcinoma cells(Cal27,HN4)were subjected to chromatin immunocoprecipitation with Oct4 and Sox2 antibodies,respectively.According to the information of the two potential binding sites,primers were designed and the chromatin coprecipitate was amplified.The analysis showed that the two potential binding site fragments could be amplified in ChIP products(p<0.005).Conclusion:Oct4 and Sox2 can jointly promote the transcription and expression of rhebll gene.Part Ⅳ:Study on the mechanism of RHEBL1 promoting the occurrence and development of oral squamous cell carcinomaObjective:To explore the mechanism of RHEBL1 promoting the occurrence and development of oral squamous cell carcinoma.Methods:The expression of pathway proteins in cells of each group after regulation of RHEBL1 was detected by pathway protein chip,the differentially expressed proteins were screened,the function was enriched by KEGG database,the differential signal pathways were screened,and the function was verified by Western blot and annexin V-FITC/PI double staining.Results:Compared with the control group,the immortalized oral epithelial cells overexpressing RHEBL1 screened two signal pathways with the most obvious differential expression and strong functional enrichment NF-κB and p53 signaling pathways.In NF-κB signal path,high NEMO(IKKγ),increased expression,The increase of phosphorylation level of IκBα and p65 expression can promote the transcription of Bcl-XL,increase its expression and inhibit cell apoptosis;the decrease of p53 expression and Bax transcription level in p53 signal pathway and the weakening of its inhibition of Bcl-XL can increase the expression of Bcl-XL and inhibit cell apoptosis.Compared with the control group,the oral squamous cell carcinoma cells knockout rhebll were also screened to obtain NF-κB and p53 signaling pathways are differentially expressed in NF-κB signal path The expression of NEMO(IKKγ)decreased,IκBα The level of phosphorylation decreased,the expression of Bcl-XL decreased,which promoted cell apoptosis;in the p53 signal pathway,the expression of p53 increased,promoted the expression of Bax transcription,inhibited the expression of Bcl-XL,and promoted cell apoptosis.Western blot in vitro verified that the expression trend of each protein was the same as that of chip detection.Annexin V-FITC/PI double staining showed that the immortalized oral epithelial cells overexpressing rhebll The apoptosis rate decreased significantly(p<0.01),and the apoptosis rate of oral squamous cell carcinoma cells knockout of rhebl1 increased significantly(p<0.01).Conclusion:RHEBL1 may affect NF-κB and p53 signaling pathway,and then affect the proliferation and apoptosis of oral squamous cell carcinoma cells.