The Mechanism And Toxicity Of Antisense Oligonucleotide Targeting PBX1 G-quadruplex In Melanoma

Melanoma is a kind of malignant skin cancer with poor prognosis.Over the past half-century,the global incidence of melanoma is growing rapidly each year and its incidence has increased faster than that of any other cancer.Meanwhile,the annual costs of treatment have also risen rapidly.Over the past years,clinical and basic research has developed various therapeutic strategies for melanoma,but the therapeutic effect is still unsatisfactory,especially for metastatic melanoma.The occurrence of melanoma is associated with the abnormal regulation of many genes and signal pathways.Thus,it is very important to explore the molecular mechanism of the malignant progression of melanoma and to find potential therapeutic targets for melanoma.Research background:Pre-B-cell leukemia homeobox transcription factor 1(PBX1)was first identified as part of a fusion protein resulting from the chromosomal translocation t(1;19)in pre-B cell acute lymphoblastic leukemias,which belongs to the PBX1-4 family and is an important transcription factor.PBX1 involves in the regulation of important developmental processes and its dysexpression is also associated with multifactorial diseases,including cancer.Increasing studies have shown that PBX1 is abnormally expressed in many cancers and its dysexpression is associated with poor prognosis in patients with cancer.Meanwhile,PBX1 is closely related with sustaining proliferative signaling,activating invasion and metastasis,angiogenesis,resisting cell death,and deregulating cellular energetics etc.cancer hallmarks.It has been reported that PBX1 expression is abnormally upregulated in melanoma cells.However,the molecular mechanism of its dysexpression in melanoma,its effects on melanoma development and whether PBX1 can be used as a novel biomarker and therapeutic target for melanoma need to be further explored.G-quadruplexes(G4s)are 4-stranded structures formed in guanine(G)-rich DNA or RNA strands.Due to its important biological function and special conformation,the structural and functional properties of G4 have become an important research field in chemistry,biology and pharmacy.It has been found that G4 s widely exist in the genomic regulatory regions and in various types of RNAs,such as oncogene promoters,splice and recombination sites,telomeric ends,micro RNA,m RNA and long-non coding RNA.Due to high representation of G4 s in human oncogene promoters and the 5’ untranslated region(5’ UTR)of m RNAs,their involvement in tumorigenesis and tumor progression is being widely investigated.Worth to note,PBX promoter and transcript are extremely rich in guanines,suggesting that G4 s may form in PBX promoter and transcripts and involve in regulating the transcription and translation processes of PBX1.Research purpose:In this study,we investigated the expression of PBX1 in human melanoma tissues.By using primary melanocytes and melanoma cells B16-F10 and A375,we investigated the roles of PBX1 in the tumorigenesis and progression of melanoma.Furthermore,we clarified the relationship between G4 structures and the dysexpression of PBX1 in melanoma,explored the possibility of G4 structure and PBX1 as biomarkers and therapeutic targets for melanoma,screened the drugs targeting PBX1 and G4 structure and performed the toxicological studies of the candidates,evaluated their value of clinical application,which would a new avenue for developing novel melanoma therapeutic strategies.Research methods:A total of 47 melanoma tissues and matched adjacent non-tumor skin tissues,48 melanoma tissues with complete follow-up information and 15 normal tissues were enrolled in this study.The 47 pairs of melanoma tissues were used for quantification of PBX1 expression levels and correlation analyses between the PBX1 expression levels and clinicopathologic features of patients with melanoma.The 48 melanoma tissues with complete follow-up information and 15 normal tissues were used for quantification of PBX1 expression levels and used for survival analyses.By performing CCK8,melanosome,migration and invasion assays,we investigated the effects of PBX1 on the primary melanocytes and mouse melanoma cells B16-F10.By performing RNA transcriptome sequencing,we examined the genes expression changes in human melanoma A375 cells and determined the downstream signaling pathway regulated by PBX1 by enrichment analysis.Bioinformatics analysis was used to predict potential G4 sequences in the PBX1 promoter region and m RNA transcripts.By performing circular dichroism(CD)measurements,thermal melting assays,fluorescence spectrum assays,gel mobility shift assays and chromatin immunoprecipitation(Ch IP),we investigated whether the potential G4 sequences can fold into stable G4 structure in vitro and in vivo.Quantitative real-time polymerase chain reaction(q RT-PCR),Western blotting(WB),dual luciferase reporter gene and EGFP reporter system were used to determine the effect of PBX1G4 s formation on PBX1 gene transcription and protein expression.The software for predicting transcription factors was used to analyze the transcription factors targeting the G4 sites of PBX1 promoter regions,and Ch IP assays were used to determine the effect of PBX1 G4 on the binding of transcription factors to the PBX1 promoter regions.By performing CCK8 assays,colony formation assays,wound scratch assays,migration and invasion assays as well as animal models of melanoma,we investigated the effects of PDS,TMPy P4 and antisense oligonucleotide(ASO)targeting PBX1 G4 on the growth and metastasis of melanoma.We performed the acute and chronic toxicity tests of TMPy P4 and ASO r G1,and the possibility of their clinical transformation value was evaluated by blood routine test,liver and kidney function,coagulation function index test and histopathology.Research result:1.The function and clinical significance of PBX1 in melanoma.By detecting the PBX1 expression in the collected melanoma tissues,we found that PBX1 is highly expressed in melanoma tumors.Kaplan-Meier analysis of Cohort 2 and TCGA revealed that high PBX1 expression in melanoma tissues correlates with reduced overall survival and metastasis-free survival,suggesting that PBX1 can be used as a potential prognostic biomarker in melanoma patients.Compared with the control groups,the proliferation ability and melanosome formation ability of primary melanocytes were significantly enhanced.While PBX1 knockout inhibited the proliferation,migration and invasion ability of mouse melanoma B16-F10 cells,indicating that PBX1 plays an oncogene role in melanoma.By performing RNA transcriptome sequencing,we identified 666 up-regulated genes and 305 down-regulated genes,respectively.Furthermore,we performed Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Gnomes(KEGG)on the changed genes.The analysis showed that the significantly overrepresented biological processes include NF-κB signaling pathway,cytokine and chemokine pathway,chemokine receptor pathway and TNF signaling pathway.Through subsequent experiments,we found that PBX1 activates NF-κB signaling pathway by promoting the nuclear translocation of NF-κB p65,resulting in the activation of cytokine and chemokine signaling pathways.These results indicated that PBX1 plays an oncogene role in melanoma by activating the NF-κB signaling pathway.2.The identification and function of PBX1 G4 structures.By combing 2 independent G4 prediction software,we identified 2 potential DNA G4s(named as d G1 and d G2)in PBX1 promoter region and 3 potential RNA G4s(named as r G1,r G2 and r G3)in the transcript of PBX1.Furthermore,c Gc C,G4 H and G4 NN algorithms were used to score the five candidate sequences for evaluating their G4 formation ability.We found that the candidate sequences d G1 located in the promoter region and r G1 located in the 5’ UTR region of the m RNA had the strongest ability to form stable G4 structures.Moreover,we found that these two candidate sequences are highly conserved,suggesting that they may play important regulatory roles.By performing circular dichroism(CD)measurements,thermal melting assays,fluorescence spectrum,gel mobility shift and Ch IP assays,we found that d G1 and r G1 can indeed form stable G4 structure in vitro and in live cells.As well-known G4 stable ligands,TMPy P4 and PDS could bind and stabilize the G4 structure formed by d G1 and r G1.The effects of PBX1 G4 s on the gene transcription and protein expression were detected by double luciferase reporter gene and EGFP reporter system assays.First,d G1 and r G1 sequences were constructed into the promoter of double luciferase reporter gene and the 5’ UTR of EGFP m RNA,respectively.Then,PBX1 G4 s formation was induced by treating with TMPy P4 and PDS.TMPy P4 and PDS treatments significantly inhibited the transcription of luciferase reporter gene and the expression of EGFP protein,suggesting that PBX1 G4 involved in the regulation of gene transcription and protein translation processes.Further,the effects of PBX1G4 s on PBX1 transcription and protein expression were detected by q RT-PCR and WB assays.We found that the formation of PBX1 G4 s significantly reduces the RNA and protein levels of PBX1,indicating that PBX1 G4 s can inhibit the transcription and protein expression of PBX1 gene.By using transcription factor prediction software,we predicted the potential transcriptional factors binding to the d G1 site in PBX1 promoter region.And we investigated the effects of PBX1 d G1 G4 on the binding of transcription factors to the PBX1 promoter regions by performing Ch IP assays.We found that transcription factor Zic2 can bind to the PBX1 promoter region and activate the transcription of PBX1.In addition,PDS or TMPy P4 treatment causes a significant reduction in the strength of Zic2 binding at the promoter of PBX1,resulting in the inhibition of PBX1 transcription.3.The effects of G4 specific ligands on the progression of melanoma.By performing CCK8 assays,colony formation assays,wound scratch assays,migration and invasion assays as well as animal models of melanoma,we found that PBX1 G4 s formation induced by TMPy P4 and PDS treatment can significantly inhibit the proliferation,colony formation,migration and invasion ability of melanoma cells,as well as the growth and metastasis of melanoma in animal models.However,animal studies had shown the toxicity of TMPy P4,limiting its clinical application.4.The anti-melanoma activity and toxicological study of antisense oligonucleotide(ASO)targeting PBX1 G4Further,ASO molecules that can specifically target PBX1 r G1 were designed and synthesized,and we found that ASO treatment can induce the formation of PBX1 r G1 G4.Meanwhile,ASO treatment inhibited the proliferation of melanoma cells in vitro and the growth of melanoma in patient-derived tumor xenograft(PDX)models.These results suggested that PBX1 G4 can be used as a potential therapeutic target for melanoma.By performing acute toxicity and chronic toxicity,we found that the ASO targeting PBX1 G4(ASO r G1)only showed certain toxicity in high-dose acute toxicity assays,but no toxicity in subchronic toxicity and chronic toxicity assays,suggesting that ASO r G1 is relatively safe and may be developed as a potential clinical drug for melanoma therapy in the future.Research conclusion:1.PBX1 expression is significantly up-regulated in melanoma,and its high expression predicts the poor prognosis of melanoma patients.Furthermore,PBX1 promotes the growth and metastasis of melanoma.Knockdown of PBX1 can effectively inhibit the melanoma progression.These results indicate that PBX1 is a potential biomarker and novel therapeutic target for melanoma.2.The PBX1 promoter region and the m RNA 5’ UTR region contain the potential G4 formation sequences.3.G4 ligands TMPy P4 and PDS can induce the formation of PBX1 G4 s,resulting in the inhibition of the transcription and translation of PBX1.4.Transcription factor Zic2 can bind to the promoter region of PBX1 to activate the transcription of PBX1.PBX1 d G1 G4 structure blocks Zic2 occupancy in PBX1 promoter regions,resulting in the inhibition of PBX1 transcription.5.G4 ligands TMPy P4 and PDS as well as designed ASO molecules(ASO r G1)can inhibit melanoma growth and metastasis in vitro and in vivo by inducing PBX1G4 s formation.TMPy P4 and PDS are characterized by low specificity,low drug-like properties and high cytotoxicity,which limit its clinical application.ASO molecule(ASO r G1)is characterized by high specificity and low toxicity,and can be used as a potential clinical drug for melanoma therapy in the future.