Study on the mechanism of demethylase JMJD6 affecting the formation of drug resistance in BRAF mutant melanoma
Objective To explore the possible mechanism of demethylase JMJD6 affecting the formation of drug resistance in BRAF mutant melanoma.Methods In vivo model of BRAF mutant melanoma with stable drug-resist-ant phenotype against BRAF inhibitor(Dabrafenib)and MEK inhibitor(Trametinib)was established by intrap-eritoneal administration in tumor-bearing mice.TCGA,Oncomine and OSskcm database analysis,Western blot and immunohistochemistry were used to study the expression and prognostic value of JMJD6 in melanoma.At the same time,after down-regulating its expression level,TUNEL apoptosis kit and in vivo tumorigenesis experiment were used to detect the reversal of drug-resistant phenotype in vitro and in vivo.Finally,bioinformatics analysis,shRNA interference,Western blot and immunofluorescence experiment were used to verify whether transcription factor E2F2 was a potential target of JMJD6.Results After establishing three generations of drug-resistant models in vivo,the models A375-R and SK-Mel-28-R of BRAF mutant melanoma with stable drug-resistant phenotypes were successfully obtained.The databases such as TCGA,Oncomine and OSskcm all showed that the expression of JMJD6 in melanoma was significantly higher than that in normal healthy people,with a worse prognosis(P＜0.05).At the same time,Western blot and immunohistochemical results showed that JMJD6 was expressed higher in drug-resistant BRAF mutant melanoma.After the expression level of JMJD6 was down-regulated by shRNA,the apoptosis level and tumorigenicity of A375-R and SK-Mel-28-R were significantly decreased under drug pressure(P＜0.05),showing the phenomenon of drug resistance phenotype reversal in vitro and in vivo.Through the analysis of Genemania and GEPIA databases,it was found that there was a correlation between the expression of JMJD6 and E2F2(P＜0.05).At the same time,Western blot and immunofluorescence proved that down-regulation of JMJD6 could inhibit E2F2 expression and RNA polymerase Ⅱ binding,and down-regula-tion of E2F2 expression by shRNA verified that it was located at the downstream of the molecular mechanism of JMJD6.Conclusion JMJD6 affects the resistance of BRAF mutant melanoma to BRAF and MEK inhibitors by regulating the expression of E2F2.
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