Genome Wide Screening Identifies Small Noncoding RNAs That Drive The Development Of Vemurafenib-resistance In Human Melanomas

Objective: It is a difficult issue for medical profession that it’s easy to develop drug resistance to targeted drugs of melanoma patients.Library has high through,save time,economy etc advantage.The genomic scale library which we generated is used to screen targeted drug(Vemurafenib)resistance of melanoma cells.Melanoma cells resistant to Vemurafenib was explored by lethal dose determined,drug resistant cells generating,high-throughput sequencing identification small non-coding RNA lead to drug resistance,biological information analysis and so on several aspects.It is hoped that mechanism of drug resistance in melanoma can be elaborated from the RNA regulation level.Methods: Lethal dose of Vemurafenib for melanoma cell was determined in cell culture.A lot of retrovirus was packaged at the same time,and a lagre number of melanoma cells were infected.Inorder toincrease the diversity of small non-coding RNAs in the virus library,and reduce the number of viruses infecting the same cell.It is easy to verify and analyze single RNA.Multi-wheel pressure screening was performed by adding Vemurafenib.Crystal violet staining,IC50 analysis,migration assays,flow cytometry,q PCR was used to confirm resistance cell lines established.The small non-coding RNA was identified by Illumina Hi Seq of the second-generation high-throughput sequencing platform,and single segment was validated by importing into cells.The mechanism of melanoma cells resistant Vemurafenib was preliminarily explored through bioinformatics analysis and q-PCR.Results:(1)The lethal dose of Vemurafenib for Mel-888,Mel-624 and A375 melanoma was 15~25 μM.In order to facilitate the follow-up experiment,we set the lethal dose to 15~25 μM.(2)After multiple rounds of pressure screening,the crystal violet staining results showed that the cells infected with the Library could survive in high concentrations of Vemurafenib.Whether it’s a short time or a long time drug added culture,the number of cell survival that Mel-888 v,Mel-624 v and A375 v was higher than its parental cells.(3)WST-1 results showed that the IC50 of the cells that infected Library was significantly higher than its parental cells.The IC50 of Mel-624 v,Mel-888 v cells more than ten thousand times its parental cells.(4)The wound healing results showed that Mel-888 v,Mel-624 v and A375 v cell’s lateral migration ability is better than that of its parental cells.(5)Transwell results showed that Mel-888 v,Mel-624 v and A375 v cell’s also been promoted in terms of longitudinal mobility.(6)Flow cell cycle showed that all the parental cells were blocked in the G1 phase after Vemurafenib adding.But there are more cells enter S phase and G2 phase.(7)q-PCR results showed that the cell contain Library became drug-resistant,the majority of gene expression in the random selection of drug-resistant genes,EMT related genes and BRAF pathway was significantly increased.