Mechanism Exploration And Screening For Drug Resistance Induced By SAM-RNA In Melanoma

Objectives Although small-molecule targeted therapy for BRAF-mutated melanoma has long been approved for clinical application,more than half of the patients who are initially sensitive to such regimen quickly develop acquired drug resistance.Besides,mechanisms underlying drug resistance have not been fully understood yet.Herein,we aim to explore the molecular changes and potential regulatory mechanisms during the acquirement of small-molecule targeted therapy resistance in melanoma,or further to find molecular indicators that can be used to early predict the occurrence of drug resistance or efficacy evaluation.Methods 1.Molecular cloning was used to construct a retroviral expression library containing 19 random bases(N19),namely short artificial modulatory RNA(SAM-RNA)synthesized by chemical approach and amplified by PCR.The library diversity and properties were evaluated by PCR,whole genome sequencing.2.Based on N19 library expressing melanoma cell lines Mel-888 and Mel-624 with BRAF mutation,vemurafenib acquired drug-resistance was further detected using histological staining in vitro and in vivo.Regulatory linkages between mi RNA and single nucleotide variation(SNV),gene expression,functional enrichment were tested and analyzed by quantitative real-time PCR,whole-exon sequencing,and bioinformatics.3.Through expressing single,representative vemurafenib resistance associated transcript(VRT),morphological and histological staining was employed to monitor drug resistant phenotype.Results1.The N19 library created by the Gibson cloning approach contains as large amount of highly random transcripts as 10~6,enough to screen the human genomic function.2.The effective doses of vemurafenib within two melanoma cell lines closed to each other,range from 5 to 10μM.3.MAPK signal pathway,EMT,drug resistance-related gene levels varied in Mel-888 N19 and Mel-624 N19 library derived drug-resistant cells.The molecules NRAS,BRAF,and RAF1 related to the MAPK signaling pathway showed an up-regulation trend,while MEK1 and 2decreased slightly.The transcriptional levels of EMT markers VIM,SNAIL1,SNAIL2,TWIST1,MMP14 increased,whereas the expression such as OCLN,FOXC2,CDH2 decreased.4.Sequences BLAST showed N19 library displayed rarely identity with the human genomic sequences,more than 60%N19 sequences exhibited at least 4 base mismatch.5.Exon sequencing results showed that during the process of acquired resistance,the Mel-888 N19 and Mel-624 N19 groups carried 49 newly discovered SNV-specific genes,accompanying with 96 SNV accumulation.The distribution of the differential gene KEGG pathway(P<0.05)was significantly diversed from those of the parents,and more than half of the pathways altered.6.Bioinformatics analysis found that the binding capacity of mi RNA-SNV in N19 library cells changed during the drug resistance screening process.Single base changes within the exon promoted forming more than1000 pairs of new mi RNA-SNV regulatory coupling.The Ontology enrichment of differential mi RNA expression profile shifted from the representative Hippo signaling pathway and Oocyte meiosis to those including protein dephosphorylation and postsynaptic density.7.In vivo experiments displayed that Mel-888 N19 proliferation rate and tumorigenesis ability of Mel-888 N19 group were stronger than the parental while there is no obvious difference between Mel-624 N19 and the parental.8.Melanoma cell lines stably expressing single VRT1-12 or TOP1000fragment can obtain drug-resistant phenotypes through pressure screening,accompanying with relatively reduced cytoplasm and neurofibroblast-like shape.9.Crystal violet and Hochest 33258 staining showed that the melanoma cell lines stably expressing VRT1-12 or TOP1000 motif fragment still exhibited short-and long-term viability after obtaining the drug-resistant phenotype.Conclusion We successfully constructed a short-chain non-coding library N19 without genomic bias,and completed the prospective exploration of acquired resistance to the vemurafenib-targeted therapy within BRAF mutant melanoma.Successfully simulating the occurrence of clinical drug resistance in vitro helps clarify the potential mechanism between acquired drug resistance and the genomic SNV and mi RNA regulatory network.N19 structure or base featured non-coding sequences might be an early indicator for acquired resistance screening in melanoma.