| Abstract:Objective:Using siRNA to interferent the NF-κBp65gene within oral squamous cell carcinoma cell line Tca8113, and combining with5-Fu,then observing the effects of NF-KBp65siRNA on Tca8113cells proliferation, apoptosis and the impact of drug resistance, to provide new ideas for the treatment of oral squamous cell carcinoma. Methods:The Tca8113cells were cultured in vitro and divided into three groups when they were in exponential growth phase:①blank control group;②egative control group;③experimental group.1. NF-κBp65siRNA was transiently transfected into Tca8113cells, observing the cells in each group with fluorescent microscope, and canculating the rate of transfection;2. The expression level of NF-κBp65mRNA was detected by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR);3. The expression level of NF-κBp65protein was determined by enzyme-linked immuno sorbent assay (ELISA) after transfection;4. The proliferation of Tca8113cells was detected by methyl thiazolyl tetrazolium (MTT) after transfection;5. The apoptosis of the Tca8113cells was detected by Annexin V-FTIC/PI;6. Tca8113cells were divided into transfection group and non-transfection group after transfection, then combined with different concentration of5-Fu (Omg/L,16.35mg/L,32.7mg/L,327mg/L,3270mg/L,6540mg/L), the sensitivity of Tca8113cells after cultured72hours to5-Fu was detected by MTT. Results:1. A fluorescein-labeled NF-κBp65 siRNA was used tomonitor the efficiency in Tca8113cells, demonstrating nearly61.1%transfection efficiency24hours post transfection.2. The test results of semi-quantitative RT-PCR showed that,48hours post transfection, the relative quantitative expression of NF-KBp65mRNA was0.539±0.036in cells of blank control group,0.514±0.031in cells of negative control group,0.313±0.031in cells of experiment group, the relative quantitative expression of NF-κBp65mRNA in cells of experiment group decreased significantly compared with the other tow groups, a statistically significant difference (P<0.05).3. The test results of ELISA showed that,48hours post transfection, the relative quantitative expression of NF-κBp65protein was1.165±0.074in cells of blank control group,1.182±0.049in cells of negative control group,1.002±0.055in cells of experiment group, the relative quantitative expression of NF-KBp65protein in cells of experiment group decreased significantly compared with the other tow groups, the difference was statistically significant (P<0.05).4. The MTT results displayed that, the absorbance values of cells in experiment group were lower during each timing period compared with the other tow groups (P<0.05), the proliferation of Tac8113cells was inhibited obviously, and the inhibitory effect is highest at72hours post transfection, the rate of cell inhibition was28.1%.5. The test results of Annexin V-FITC/PI showed that,72hours post transfection, the apoptosis rata was (8.50±0.92)%in blank control group,(8.39±1.03)%in negative control group,(24.58±2.31)%in experiment group, the apoptosis rata of experiment group was higher obviously than other tow groups, a statistically significant difference (P<0.05), and the early apoptosis rate was greater than late apoptosis rate.6. Tac8113cells were combined with different concentration of5-Fu after transfection, the results of MTT showed that, the proliferative activity of cells in transfection group and non-transfection group had a downward trend along with increasing concentration of5-Fu72hours post transfection, and the proliferative activity was lower in transfection group than in non-transfection group at the same concentration, a statistically significant difference (P<0.05),it meaned that the sensitivity of cells to5-Fu in ransfection group increased significantly compared with non-transfection group. Conclusion:1. NF-κBp65siRNA could effectively transfect the oral squamous cell carcinoma Tca8113cells.2. The expression level of NF-KBp65mRNA and protein within the oral squamous cell carcinoma Tca8113cells decreased obviously after transfection, this displayed that the expression of NF-KBp65gene in oral squamous cell carcinoma Tca8113cells was silenced by NF-KBp65siRNA.3. Silencing the NF-κBp65gene could inhabit the proliferation of oral squamous cell carcinoma Tca8113cells, and induce oral squamous cell carcinoma Tca8113cells apoptosis.4. After the NF-KBp65gene was silenced, the sensitivity of oral squamous cell carcinoma Tca8113cells to5-Fu was significantly improved.6. NF-κB may be emphasized as a therapeutic target for gene theary of oral squamous cell carcinomas. |
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