Effect Of Hyperthermia On Id-1 Expression Of Cal27 Cells In Oral Squamous Cell Carcinoma

Objective:To investigate the influence of hyperthermia on the expression level of Id-1 in oral squamous cell carcinoma,by observing the change of the proliferation activity,migration ability and related cytokines in Cal27 cells.Methods:1.The Cal27 cells were cultured in 10% fetal bovine serum and 1640 medium at37℃ in a 5% CO2 incubator.The Cal27 cells were digested with 0.25% trypsin(containing EDTA).The cells were stored with 1ml of cryopreserved solution.The morphology of the cells was observed by inverted microscope.2.The optical density(OD)of the experimental group and the control group were measured at the time of 0h,2h,4h,8h and 12 h after the hyperthermia at 43℃ for 1 hour by CCK-8 experimental methods with 450 nm,and marking the cell growth curve.The t test was used to calculate whether the experimental group and the control group were statistically significant at each time point.3.The migration of the experimental group and the control group was observed at 0h,6h,12 h and 24 h after the hyperthermia at 43℃ for 1 hour.The area of migration was calculated by Image J software and the line chart was drawn.The t test was used to calculate whether the experimental group and the control group were statistically significant at each time point.4.The logarithmic growth cells of the experimental group were incubated in a 43℃incubator for 1 hour and then incubated for 4 hours under standard conditions for PCR experiments.The control group did not accept any treatment.Total RNA was extracted from Trizol lysate.The total RNA was reverse transcribed into c DNA,and then amplified with Id-1 and HSF1 primers,and β-actin was used as internal reference.Real-time fluorescent dyes were added to the amplified body.The expression of Id-1 and HSF1 m RNA in the cells before and after hyperthermia was examined by PCR.Results:1.Cal27 cells morphology: All cells were adherent growth,polygonal,nuclear large,round,nucleolus clearly visible,the cytoplasm whose particles depth ranging from dark tolight,cell division phase being more common,and multi-level nuclear division.When arranged closely like paving stone,the cells were too high density that they could be overlapping growth.2.Result of CCK-8 experiment: Hyperthermia can reduce Cal27 cells activity and inhibit cell proliferation.The P value of the experimental group and the control group were P2=0.036,P4=0.007,P8=0.002,P12=0.001 after the hyperthermia at 2h,4h,8h,12 h.The cell proliferation activity of the hyperthermia group and the experimental group was statistically significant.3.Result of scratch test: Hyperthermia can inhibit the migration ability of Cal27 cells.The use of t test to observe the experimental group and the control group after the hyperthermia at 6h,12 h,24h,48 h,and the P value were P6=0.028,P12=0.038,P24=0.012,P48=0.001,all of which were statistically significant.4.Result of q RT-PCR: The m RNA expression level of Id-1 was decreased after hyperthermia treatment(P<0.05).Hyperthermia can activate HSF1 m RNA expression(P<0.05).Conclusion:Through CCK-8 experiment and scratch test,hyperthermia were found that it could inhibit cell proliferation activity and migration ability.And through the experimental method of q RT-PCR,hyperthermia were found that it could decrease Id-1 m RNA expression.At the same time,the HSF1 m RNA expression was activated.This results suggested that hyperthermia may affect cell proliferation and migration by reducing the expression level of Id-1.