| Background:Palytoxin（PLTX）belongs to the polyether toxin of marine biotoxins.It is widely distributed in nature and can accumulate high concentration in fish,crabs,mussels and other aquatic products through bioenrichment effect,and even spread to the air to form aerosol pollution.The main manifestations of poisoning are dyspnea,convulsion,movement disorder,lethargy,collapse,vomiting,extensive gastrointestinal bleeding,shock,and even death.These facts make PLTX a serious threat to human health and aquaculture.The clear mechanisms of action of PLTX are to bind and block Na+-K+-ATP pump and open Na+channels,leading to intracellular changes of H+and Ca2+with interacting with Ca2+channels,resulting in cell depolarization.Therefore,PLTX has strong neurological and cardiovascular activities,and is the strongest known vasoconstrictor.However,the known mechanisms of action do not fully explain the complex and multiple acute and chronic toxic effects of PLTX.Aptamers are a class of single-stranded nucleotide sequences with a length of about 10-100 nt,obtained by systematic evolution of ligands by exponential enrichment（SELEX）.Because of its high affinity and specificity with the target,as well as convenient screening,synthesis,and other advantages,it is referred to as a chemical antibody,which plays an important role in environmental monitoring,clinical testing,and drug research and development.At present,aptamers have extensive research basis in the field of marine biological toxin monitoring.Coincidentally,these synthetic aptamers have similar or even the same sequences in the genomes of various organisms.Therefore,we speculate that there are aptamer sequence regions that bind toxins in the genomes,which are named the natural aptamers of toxins by us.In this study,a natural aptamer of PLTX modified by methylation was obtained by One-round Genomic SELEX（OG-SELEX）method,and the interactional mechanism between the aptamer and PLTX was analyzed by molecular docking and molecular dynamics simulation.Finally,from the perspective of the interaction between PLTX and the aptamer sequence in the genome,a new mode of action of PLTX was identified by a variety of cell and molecular experiments.Objectives:1.To obtain the natural aptamers that are methylated and bind to PLTX with high affinities.2.To analyze the interactional mechanisms of the aptamers and PLTX and the role of methylated modification.3.To identify a new mode of action for the toxic effects of PTLX.Methods:1.After PLTX intervention,CCK8 assay was used to detect the survival rate of different cells.Then we selected HaCaT and CHO-K1 cell lines which had appropriate sensitivity to PLTX,and extracted the genomes,obtaining nucleic acid libraries with a sequence length of about 200-300 nt after ultrasonic breakage,thermal denaturation and quenching treatment.They were set into the blank group,the unmodified group and the methylated group,with 3 repeat libraries in each group.The libraries of blank group were incubated with empty magnetic beads,and the libraries of unmodified and methylated groups were incubated with PLTX-coupled magnetic beads.The bound ss DNA was recovered and high-throughput and methylation sequencing were performed.The candidate aptamer sequences were obtained by bioinformatics analysis and their affinities were determined by BLI technology.2.BLI and MST multi-concentration fitting methods were used to accurately determine the KD values of the aptamers,then RNAfold was used to predict the secondary structures,which were verified by CD experiments.GROMACS and Rosetta were used to construct the three-dimensional structures of PLTX and the aptamers,and molecular docking and molecular dynamics simulation were carried out to analyze the interactional mechanisms between the aptamers and the toxin and to determine the target of PLTX.3.We used databases such as miRanda and miRDB to predict ce RNA networks.The bioinformatics analysis determined that the downstream of the target of PLTX was the LINC01250/miR-3646/GSK3B signaling pathway.The interactions of LINC01250 with miR-3646 and miR-3646 with GSK3B 3’UTR were verified by dual luciferase reporter gene assay system.Subsequently,After LINC01250,or LINC01250 and miR-3646 were overexpressed or down-regulated in HaCaT cells,the phenotypic changes of the cells were analyzed by CCK8,cell scratch,and cell migration and invasion experiments,and the expression changes of relevant RNA and proteins in the cells of each group were analyzed and compared by RT-q PCR,WB,and other experiments,verifying the effects of PLTX binding its target and the biological functions of the downstream signaling pathway.Finally,PLTX intervention was performed on each group of cells,and the changes of the above indexes were analyzed and compared,and a new mode of action of PLTX was identified.Results:1.IC50 values of HaCaT,Caco-2,CHO-K1 and 3T3-Swiss cells treated with PLTX for 24 h were 8.8×10-4,3.2×10-3,7.8×10-5 and 3.2×10-10μg/m L,respectively.The efficiency of coupling PLTX by magnetic beads was 90.38%,the nucleic acid recovery rates of all parallel screening libraries were between 1.0‰and 2.0‰,and the genome comparison rates were all above 80%.2.A batch of natural aptamers with high affinities to PLTX were obtained by OG-SELEX screening method.The KD values of the unmodified natural aptamers H127,H128,H12,C1293 and C1732 were 834,119,40.9 and 103 n M,respectively.The methylated natural aptamers m C525 and mC1512 had KD values of 199 and 12.0 n M,respectively,and their corresponding unmethylated aptamers C525 and C1512 could also bind PLTX,but the affinities were somewhat reduced,and the KD values were 838 and 139n M,respectively.Among them,mC1512,C1293 and C1512 had the strongest binding abilities,and their KD values were 25.1,551 and 112 n M detected by BLI multi-concentration fitting accurately.The KD values detected by MST multi-concentration fitting were 31.4,606 and 241 n M,respectively.3.The CD spectra of the aptamers mC1512,C1293 and C1512 all showed positive and negative absorption peaks at 280 nm and 245 nm,confirming that they contained B-type double-stranded structures.Their ss DNA properties indicated that some nucleotides had base complementary pairing within the molecule,forming a stem-ring structure.The results were consistent with the secondary structure prediction.4.Molecular docking results showed that the hydrogen bonds formed by aptamer C1512 and PLTX were mainly located in G9,C10,C12,T13,C14 and A15,while the hydrogen bond sites formed by aptamer mC1512 abandoned C12,but accommodated C8,and G9 produced more hydrogen bonds to form a more stable binding site with PLTX.Molecular dynamics simulation results showed that RMSF values of aptamer mC1512decreased by about 0.2（?）after methylated modification,which made it more stable than C1512,and more hydrogen bonds were formed with PLTX.At the same time,mC1512could form the stronger overall non-bonded interaction with PLTX,suggesting mC1512sequence as the key target of PLTX.5.After PLTX intervention,the proliferation,post-injury repair,migration and invasion abilities of HaCaT cells were inhibited,and the inhibition level increased with the elevation of intervention time and toxin concentration within 48 h.After PLTX intervention for 24 h,the expressions of LINC01250 and GSK3B in HaCaT cells were significantly increased,and the degree of improvement increased with the elevation of PLTX concentration in a certain range.6.Overexpression of miR-3646 significantly decreased the luciferase activity of reporter plasmid containing wild-type LINC01250,but did not decrease the luciferase activity of reporter plasmid containing mutant LINC01250-Mut.The results confirmed a complementary base pairing mode between LINC01250 and miR-3646,establishing their mutual targeting interaction;overexpression of miR-3646 significantly decreased the luciferase activity of the wild-type GSK3B 3’UTR reporter plasmid,but did not decrease the luciferase activity of the mutant GSK3B 3’UTR-Mut reporter plasmid.The results confirmed a complementary base pairing mode between miR-3646 and GSK3B 3’UTR,establishing their mutual targeting interaction.7.After the overexpression of LINC01250 in HaCaT cells,the phosphorylation level of GSK3B was decreased,mediating the degradation ofβ-catenin,inhibiting the downstream expressions of c-Myc,cyclin D1 and PPARδ,resulting in the inhibition of cell proliferation,migration and invasion.After the subsequent overexpression of miR-3646,the changes of protein expressions and cell phenotypes exhibited partial recovery.After the interference with LINC01250,the phosphorylation level of GSK3B increased,inhibiting the degradation ofβ-catenin,increasing the downstream expressions of c-Myc,cyclin D1and PPARδ,resulting in the promotion of cell proliferation,migration and invasion.After the subsequent interference with miR-3646,the changes of protein expressions and cell phenotypes exhibited partial recovery.8.After 24 h of PLTX intervention,the overexpression of LINC01250 could further reduce the phosphorylation level of GSK3B,reduce the expressions ofβ-catenin,c-Myc and PPARδ,and reduce the expression of cyclin D1with a decreasing tendency,which further inhibited cell proliferation,migration and invasion.After the subsequent overexpression of miR-3646,the changes of protein expressions and cell phenotypes exhibited partial recovery.The interference with LINC01250 could increase the phosphorylation level of GSK3B,increase the expressions ofβ-catenin,c-Myc,cyclin D1and PPARδ,and improve the inhibition state of cell proliferation,migration and invasion.After the subsequent interference with miR-3646,the changes of protein expressions and cell phenotypes exhibited partial recovery.Conclusions:In this study,the established OG-SELEX method was used to screen and obtain the first methylated natural aptamer of PLTX.Using the improved computer simulation method,the 3D structure of PLTX was successfully constructed.We analyzed the structural basis of improving the affinity of the aptamer by methylation,identified a new target of action of PLTX,and explained the new mode of toxic effects,providing a new method to follow for obtaining naturally modified aptamers with high affinity.At the same time,it also provides a complete new strategy for exploring the new mechanisms of marine biotoxins,and further expands the application of aptamers from environmental monitoring,disease detection and drug research and development to the field of mechanism analysis of bioactive molecules.The specific conclusions of this study are as follows:1.Natural aptamers for PLTX were screened and obtained:The OG-SELEX method can be applied to the screening of chemically modified natural aptamers for marine biotoxins.Taking PLTX as an example,the methylated aptamer mC1512 was successfully obtained.2.Methylation facilitated the binding of aptamers to PLTX:Methylated modification could improve the stability of aptamer mC1512,increase the number of hydrogen bonds formed with PLTX,increase the overall non-bonded interaction between the molecules,and improve the affinity,suggesting mC1512 sequence as the key target of PLTX.3.mC1512 was involved in mediating certain functions of PLTX:PLTX could interact with mC1512 sequence in the genome to promote the transcription of gene LINC01250,and play an important regulatory role in the proliferation,migration and invasion of HaCaT cells through the downstream LINC01250/miR-3646/GSK3B signaling pathway. |
