Establishment Of Cisplatin Resistant Cell Model Of Tongue Squamous Cell Carcinoma And Analysis Of Differentially Expressed MicroRNAs

Objective: By establishing the cisplatin resistant cell model of human tongue squamous cell carcinoma and comparing the differentially expressed miRNAs between drug-resistant cells and parental cells.This study aimed to the key miRNAs regulating drug resistance of tongue carcinoma.Methods: 1.The cisplatin resistant oral squamous cell carcinoma model OSC-19 / CDDP was established by giving gradually incremental doses of CDDP administration in cell culture medium.And the method of CCK-8 and Western blot were used to identify the cell model;2.The miRNAs expression profiles of cisplatin resistant cell OSC-19 / CDDP and its parent cell OSC-19 were measured by microarray scanning.And then the results of the test was verified by RT-qPCR;3.According to above-mentioned analysis results,up-regulate the miR-935 and miR-514a-3p in OSC-19 by chemical transfection,and the transfection effect was verified by RT qPCR.Lastly,the change of cisplatin resistance of OSC-19 cells was detected by CCK-8.Results: 1.OSC-19 cells were continuously cultured by cisplatin concentration gradient method,and finally the cell survived steady in the medium with 0.8 μg/ml CDDP.Western blot showed that MDR1 was highly expressed in OSC-19/CDDP.The IC50 of OSC-19 cells was 0.68 μg/ml calculated by CCK-8 method,while the IC50 of OSC-19/CDDP reached to 6.50μg/ml,and the RI of OSC-19/CDDP was about 9.56.2.The expression level of miRNAs between OSC-19 / CDDP and OSC-19 were measured by microarray scanning.And 264 differentially results of miRNAs were showed between OSC-19 / CDDP and OSC-19,of which 15 miRNAs were more than 20 times different,including 6 down-regulated miRNAs and 9 up-regulated miRNAs;3.The results of RT-qPCR showed that the expression of miR-486-3p、miR-514a-3p and miR-935 in OSC-19/CDDP was significantly higher than them in OSC-19 cells,while the expression of miR-146b-5p was significantly higher in OSC-19.It is consistent with the microarray scanning results;4.The expression of mir-935 and mir-514a-3p in OSC-19 was increased after transfection,and the cell’s tolerance to cisplatin was significantly improved.Conclusion: 1.The model of cisplatin resistant OSC-19/CDDP can be established by giving gradually incremental doses of CDDP administration in cell culture medium;2.There were a large number of miRNAs that expressed differently between OSC-19/CDDP and OSC-19.And miR-935 and miR-514a-3p may be the key regulating cisplatin resistance.