| Oral squamous cell carcinoma(OSCC),accounting for more than 90%of oral malignancies,is the sixth most common malignancy in the world.OSCC is highly invasive,tending to invade adjacent bone tissue and metastasize to local lymph nodes,and prone to local recurrence.Moreover,the highly heterogeneous biological behavior of OSCC leads to different invasiveness and prognosis in different patients.Currently,the main treatment for OSCC is surgery,chemotherapy,radiotherapy,or a combination of these treatments.Despite there is significant advances in combination therapy about OSCC,overall survival in OSCC patients is low.Finding early diagnosis markers of OSCC is help for early screening and prognosis of patients with OSCC.Targeted therapy is more effective to OSCC while avoiding the harm to normal cells.However,there is still a lack of ideal and specific OSCC-related gene.The purpose of this experiment is to study the biological functions of gene X and alternative splicings of gene Z on OSCC cells,and to provid new clues for searching OSCC biomarkers.Objective:The mechanism and biological behavior of gene X and alternative splicings of gene Z were explored in the procession of occurrence and development of oral cancer,and the molecular mechanism of oral cancer was explored further,so as to provide new clues to find the methods of early diagnosing and targeted treating oral cancer.Methods:Human tongue squamous cell carcinoma cells(CAL-27 cells)were infected with lentivirus vector carrying exon 15 of gene Z and lentivirus vector excluding exon15 of gene Z,and OSCC cell lines with stable alternative splicing transcrits of gene Z overexpression were established.OSCC cell lines with stable gene X overexpression represent as CAL-27-Gene X,and blank control cells represent as CAL-27-Ctrl,CAL-27-Z(15+)and CAL-27-Z(15-)are OSCC cell lines with stable alternative splicings transcrits of gene Z overexpression.The growing rate of stable cell lines was measured by real-time unlabeled cell function analyzer.The x CELLigence RTCA MP E-plae96 plates which were added 2000 cells per well were test by real-time cell analysis system for 96-112 hours,then we got and analysised the growth rate of each cell;The cell proliferation was detected by cloning formation assay,The 6-well plates which were added 1000 cells per well were incubated for 3 weeks,the cells were fixed,dyed,photographed,and counted;Transwell assay was used to study the migration and invasion of cells.The 2%Matrigel I glue was laid in the transwell chambers in advance when we detected the invasion of cells,the transwell chambers added 2×10~5ce Ils per well were placed in incubator.It took 16 hours to detect the invasion of cells and 12hours to detect the migration.We randomly selected 9 fields in each well for counting and statistics;Cell cycle distribution was detected by flow cytometry.The collected cells in good condition were fixed with ethanol,stained at 4?in dark for 30min,then detected by flow cytometry,and finally analyzed by Modfit software;The overexpressed cells were subcutaneously injected into nude mice to detected the ability of cells to form subcutaneous tumors.We took 4-week-old SPF BALB/C nude mice weighing about 18-20g,and injected subcutaneously 4×10~6cells per mouse at the right upper middle groin of the mice.We observed and took photos of the mice every day,and measured the volume of the tumor body with a vernier caliper.After one month,the tumor body was removed,weighed,measured and photographed,one part of them was soaked in formalin and immobilized for pathological examination,and the other part was cryopreserved for storage,then we analysised the results.Results:1.The results of real-time analysis of unlabeled cell function showed that cells with overexpression of gene X grew about twice as fast as cells of control group after 96hours of culture.Cells with overexpression of exon 15 of gene Z grew about twice as fast as cells without exon 15 of gene Z after 112 hours of culture.2.Cloning formation experiments showed that the number of clones formed by the cells with overexpression of gene X was about twice that of cells of control group at the21st day,and the difference was significant(p=0.0037).The number of clones formed by the cells with overexpression of exon 15 of gene Z was about twice that of cells without exon 15 of gene Z at the 21st day,the difference was significant(p=0.0058).3.The results of transwell assay showed that the number of migration of cells with overexpression of gene X was about 25 times that of cells of control group after 12hours of culture,and the difference was significant(p<0.0001),the number of invasion of cells with overexpression of gene X was about 21 times that of cells of control group after 16 hours of culture,and the difference was significant(p<0.0001).The number of migration of cells with overexpression of exon 15 of gene Z was about 21 times that of cells without exon 15 of gene Z after 12 hours of culture,and the difference was significant(p<0.0001),the number of invasion of cells with overexpression of exon 15of gene Z was about 26 times that of cells without exon 15 of gene Z after 16 hours of culture,and the difference was significant(p<0.0001).4.Flow cytometry assay showed that the proportion of cells with overexpression of gene X in S phase and G2/M phase of cell cycle was significantly higher than that of cells of control group,the difference was statistically significant(p=0.0074;p=0.0154).The proportion of cells with overexpression of exon 15 of gene Z in S phase was significantly higher than that of cells without exon 15 of gene Z,the difference was significant(p=0.0289).5.The results of formation of subcutaneous tumor in nude mice showed the volume of tumor formed by the cells with overexpression of gene X was about 3.2 times that of cells of control group,and the weight of tumor formed was about 2.8 times that of cells of control group after one month,all the differences were significant(p=0.038;p=0.0001).The volume of tumor formed by the cells with overexpression of exon 15 of gene Z was about 3.3 times that of cells without exon 15 of gene Z,and the weight of tumor formed was about 2.7 times that of cells without exon 15 of gene Z after one month,the differences were significant(p<0.0001).The HE staining revealed the tumor was squamous cell carcinoma.Conclusion:In this study,we successfully constructed cell lines with alternative splicings of gene Z overexpression,CAL-27-Z(15+),with exon 15 contained in gene Z,and CAL-27-Z(15-),with exon 15 excluded by gene Z.we illustrated important biology functions of gene X and alternative splicing of gene Z in the occurrence and development progression of OSCC by a series of experiments in vitro and in vivo.1.Overexpression of gene X significantly accelerated growth rate,enhanced proliferation,migration,invasion,subcutaneous tumorigenesis in nude mice of OSCC cells,the proportion of cell cycle S and G2/M increased,indicating active cell cycle of OSCC cells.2.Exon 15 of gene Z made OSCC cells grow faster,enhanced proliferation,migration,invasion,subcutaneous tumorigenesis of nude mice of OSCC cells,the proportion of cell cycle G2/M increased,indicating active cell cycle of OSCC cells. |
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