Hyperthermia Combined With Silencing Id-1 Gene Reverse Epithelial-mesenchymal Transition In Tongue Squamous Cell Carcinoma

Background:Hyperthermia not only has the advantage of protecting normal tissue structure and improving patient quality of life compared with conventional surgery,radiotherapy and chemotherapy,but also can reduce the postoperative recurrence rate and distant metastasis rate of malignant tumor by reversing the epithelial-mesenchymal transformation process of tumor cells.However,hyperthermia alone is not effective in treating tumors,in clinic,it is usually combined with surgery,radiotherapy,chemotherapy and molecular targeted therapy to enhance the efficacy,among which molecular targeted therapy has become a research hotspot in recent years.Purpose:This topic try to silencing Inhibitor of differentiation/DNA binding-1（Id-1）in the form of molecular targeted therapy,and joint compatibility with hyperthermia,to explore the possibility of treatment for tongue squamous cell carcinoma,especially in observational studies whether hyperthermia and silencing Id-1 gene can reverse the EMT process of the tongue squamous cell carcinoma（TSCC）,and the molecular mechanism of their combined action on the TSCC EMT is preliminarily discussed.Methods:1.Select the appropriate TSCC lines.RT-PCR assay was performed to detect the expression level of Id-1 mRNA in SCC4,SCC9,SCC25 and CAL-27,and the cell lines with the highest expression of Id-1 mRNA were selected as the follow-up study cells of this subject.2.Verify the ability of siRNA to silence the expression of Id-1 gene.The cells were randomly divided into 3 groups,respectively the blank control group（normal）,the negative control group（NC）and the small interfering RNA group（Id-1-siRNA）.RT-PCR assay was performed to detect the mRNA expression level of Id-1 after siRNA transfection of TSCC for 36 hours.3.Observe the morphological changes of TSCC.SiRNA transfection TSCC after36h,cells at 42.5℃5%CO₂ incubator 40min,back in 37℃5%CO₂ incubator 12 h,under inverted microscope to observe cell morphological changes before and after the experiment.4.CAL-27 cells were divided into 4 groups.Normally cultured CAL-27 cells were treated as control group.In silencing Id-1 group（Id-1-siRNA）,siRNA was used to silence the expression of Id-1 in the CAL-27 cells,and the mRNA expression of Id-1 was detected by RT-PCR.In hyperthermia group（HT）,CAL-27 cells were placed into constant temperature incubator at 42.5℃for 40 minutes.In combined group（HT+Id-1-siRNA）,hyperthermia combined with silencing Id-1 gene were on CAL-27cells.（1）After the cells of the HT+Id-1-siRNA group were treated for 24h by hyperthermia,the mRNA expression levels of Id-1,TGF-β1,E-cadherin and Vimentin of the four groups were detected by RT-PCR.（2）After the cells of the HT+Id-1-siRNA group were treated for 48h by hyperthermia,the protein expressions of Id-1,TGF-β1,E-cadherin and Vimentin in four groups of TSCC were detected by western blotting.（3）When the HT+Id-1-siRNA group were treated with hyperthermia for 12h,24h and 36h,the migration ability of TSCC in the four groups were detected by scratch repair experiment.（4）When the HT+Id-1-siRNA group were treated with hyperthermia for 6h,12h and 24h,the invasion ability of TSCC in the four groups were detected by Transwell chamber test.Results:1.RT-PCR results showed that the expression level of Id-1 mRNA in CAL-27 cells was significantly higher than that of SCC4 cells,SCC9 cells and SCC25 cells.Therefore,the CAL-27 cell line of TSCC was selected for this study.2.Compared with the normal group,the mRNA expression level of Id-1 in the silencing group decreased by 83.29%.On the other hand,compared with the NC group,the mRNA expression level of Id-1 in silencing group was decreased by 70.39%.3.After the combined effect of siRNA-Id-1 and hyperthermia on CAL-27 cells,the cell morphology changed from spindle-like to pebbled,and the cell arrangement became compact and orderly from loose and disorderly.4.TSCC showed good sensitivity to hyperthermia and silencing Id-1 gene expression.Compared with either hyperthermia or silencing Id-1 gene alone,co-administration of both treatment could make the decrease of migration and invasiveness of TSCC is more obvious.Besides,it can activate E-cadherin gene and inhibit Vimentin gene by down-regulating the expression of TGF-β1.Conclusion:This founding suggest us that concurrent treatment with silencing Id-1 gene enhances the process of hyperthermia reverses the EMT of TSCC,whither by down-regulating the expression of TGF-β1.This may be regarded as a noel strategy for treatment of TSCC.