Establishment Of A New OSCC Cell Line Derived From OLK And Identification Of Malignant Transformation-related Genome

Oral squamous cell carcinoma(OSCC)is one of the most common cancer in the world and accounts for more than 90% of oral malignancies.The prognosis is still poor with a 5-year survival rate of approximately 50%.The incidence rate is high,and in recent years there is a trend of rising and younger.OSCC is usually preceded by the oral premalignant lesions,mainly oral leukoplakia(OLK)after repeated insults of carcinogens,tobacco.Tobacco smoking is the most important etiological factor in the development of OLK and OSCC.While the main key carcinogens in tobacco smoke are B(a)p and DMBA.In the present study,for the first time we established the cancerous cell line OSCC-BD induced by B(a)P/DMBA mixture and transformed from dysplastic oral leukoplakia cell line DOK.Cell morphology,growth ability,cell cycle and invasion ability were studied and confirmed the malignant characteristics of OSCC-BD cells.Although early diagnosis and treatment are very important for the prognosis of oral squamous cell carcinoma,the pathogenic mechanism and related effective molecular markers are still rare.Through the study of gene chip technique for genome differences between the DOK cells and OSCC cells,the search of effective molecular markers has important significance for the early found,diagnosis and treatment of oral leukoplakia malignant transformation,then improve the prognosis of patients,prevent the occurrence of oral squamous cell carcinoma.Objective: In this study,we attempt to establish the malignant cell line OSCC-BD induced by B(a)P/DMBA mixture and transformed from dysplastic oral leukoplakia cell line DOK.Analysis malignant transformation-related genes in two different cell lines by gene chip technology to find effective molecular markers for predicting malignant transformation of OLK,and provide clues to find early treatment targets of malignant transformation of OLK.Methods: DOK cells were treated intermittently and gradually with 70umol/L B(a)P/DMBA mixture for four months.The transformed cells were named as OSCC-BD cells.Using the inverted contrasting microscope to observe the cell morphology.The characteristics of malignant cell was assessed by HE staining.The invasion ability of cells was assessed using transwell assays.The cell cycle change was stained with propidium iodide and measured using a flow cytometer.All these tests confirmed the malignant characteristics of OSCC-BD cells.The malignant transformation-related genes was detected by m RNA Human expression microarray chip Gene Expression Affymetrix and the results were confirmed with RT-PCR.Result: In the present study,under the induction with B(a)P/DMBA mixture,DOK cells are transformed to cancer cells OSCC-BD.OSCC-BD cells showed the uncontrolled cell division with overlapping.By HE staining OSCC-BD cells showed the malignant character,the increased ratio of nucleus and plasma,more cell division,and sporadic tumor giant cells.The flow cytometric assay confirmed the increased proliferation ability in OSCC-BD cells.OSCC-BD cells showed a significant accumulation in S phase(p < 0.05),which showed that the DNA synthesis of the OSCC-BD cells was active,and its proliferation and growth ability was significantly increased than that of DOK.Transwell assays suggested a strong invasion ability in OSCC-BD cells.The gene chip results display screen detected 5383 up-regulated genes and 5764 down regulated genes in OSCC-BD compared with DOK.Pick out ten significant fold difference genes from the genomic differences.In the cell model for preliminary validation,results suggest that,seven genes were confirmed with RT-PCR.Conclusion:In our study,we establish the OSCC-BD cell lines derived from DOK under the induction with B(a)P/DMBA mixture and confirmed the malignant characteristics.Based on the above new cell model,the malignant transformation-related genes were analyzed using the Gene Chip technology and found some new genes,the results were confirmed with RT-PCR.The study will provide a new perspective andscientific clue to the prediction of the malignant transformation of oral leukoplakia and the exploration of the key therapeutic targets.