Screening Of Vemurafenib-resistant Melanoma Cell Line Using Genomewide Short-chain Nonconding RNA Library And Preliminary Study On The Mechanism

Objectives:The BRAF V600E mutation is the most common genetic change in malignant melanoma,Vemurafenib,a BRAF inhibitor developed for this mutation,is the first-line drug for the treatment of malignant melanoma.However,the drug resistance is prevalent in the clinical treatment,the drug resistance mechanism has not been fully elucidated.To explore the mechanism of drug resistance,we constructed a genomewide short-chain non-coding RNA(50 bases)library,which can be used for rapid screening of the vemurafenib-resistant cells,By using this drug-resistant cell model and high-throughput sequencing,we try to explore the new drug resistance mechanism of malignant melanoma,and provide theoretical basis for seeking new therapeutic targets.Methods:Using retroviral plasmid backbone pSEB-61d as vector,The primers containing 50-base random order were synthesized by chemical method.Double-stranded DNA fragment containing 50-base random order was amplified by PCR,which used as the insertion fragment and placed under the U6 promoter by Gibson Assembly method.The library we constructed was named pSEB-U50,which is short-chain non-coding RNA library.The cloning positive rate and coverage of the RNA library were identified.The pSEB-U50 library plasmid was packaged into retrovirus and infected with melanoma cells A375 and Mel-888,which have BRAF V600E mutation.After infection with retrovirus with the RNA library,The drug-resistant melanoma cell model was established by screening with lethal dose of vemurafenib.The genomic DNA of drug-resistant cells was extracted.A 50-base fragment was specifically amplified for sequencing on Illumina HiSeq platform,try to find out the 50-base fragment enriched in the drug-resistant cells derived from RNA library screening.The expression of drug-resistant related genes in drug-resistant cells was detected by qPCR.To investigate the changes of autophagy in drug-resistant cells screened from pSEB-U50 RNA library,an autophagy flow monitoring adenovirus Ad-mRFP-EGFP-LC3 was designed and constructed,which was successfully applied to the monitoring of autophagy flow in drug-resistant cells.Other cell models and animal experiments were used to evaluate the monitoring of autophagy flow in vitro and in vivo.Results:(1)The method of Gibson Assembly to construct pSEB-U50RNA library has high efficiency.The number of library plasmids in a single assembly was about 2.5x10~6.Moreover,the introduction of bacterial suicide gene ccdB into the vector plasmid successfully increased the cloning positive rate of the library,reaching more than95%.The 40 positive clones selected randomly were sequenced after plasmid extraction.It was found that the 50 base fragments inserted in the library plasmid did not duplicate,indicating that the library had diversity and high coverage.(2)Under the cell culture condition with 2%FBS DMEM,The lethal dose of vemurafenib on A375 and Mel-888 was 10 uM and 15uM respectively.(3)A375 and Mel-888 cells infected with retroviral pSEB-U50short-chain non-coding RNA library were screened for drug resistance with lethal dose of vemurafenib.Crystal violet staining showed that A375 and Mel-888 cells infected with pSEB-U50 short-chain non-coding RNA library could survive at a drug concentration of up to30uM.It was confirmed that drug-resistant melanoma cells were successfully screened by pSEB-U50 RNA library.(4)Flow cytometry was used to detect the cell cycle of A375parental cells and vemurafenib-resistant A375-U50V cells.It was found that the cell cycle of parent cells stagnated in G1 phase after adding 5 uM vemurafenib at low concentration,while most of A375-U50V resistant cells could enter S phase and G2 phase.(5)q-PCR was used to detect the expression of resistance-related genes in A375 parental cells and A375-U50V drug-resistant cells screened from the library.The expression of multidrug resistance genes,EMT-relatedgenes,tumorstem-relatedgenesand autophagy-related genes in A375-U50V drug-resistant cells were significantly increased after 24 hours of low-concentration vemurafenib treatment.(6)AdenovirusAd-mRFP-EGFP-LC3wassuccessfully constructed and applied to the detection of autophagy in A375 parental cells and A375-U50V drug resistant cells.It was found that the autophagy activity in drug-resistant cells increased,which was consistent with the expression of autophagy genes.The application of the autophagy monitoring adenovirus in other cells and animal experiments confirmed that the adenovirus reporter could successfully monitor the whole process of autophagy flux.Using the adenovirus reporter,it can be observed that the green particles disappear or weaken,while the red particles remain unchanged.Conclusions:In this study,A375 and Mel-888 cells with BRAF V600E mutatoin were used as cell models.Through infection with retroviral pSEB-U50short-chain non-coding RNA library,rapid screening of lethal dose vemurafenib,crystal violet staining,flow cytometry and q-PCR,we confirmed that the drug-resistant melanoma cells were successfully screened.It was found that the mechanism of drug resistance might be related to the increase of cell autophagy activity,EMT and so on.In addition,autophagy monitoring adenovirus was successfully constructed and successfully applied in vitro and in vivo experiments.