The Effect And Mechanism Of Slug In Promoting The Invasion And Metastasis Through Epigenetic Regulation Of IGFBP3 In Malignant Melanoma

Research backgroundMalignant melanoma,referred to as MM,originates from neuroectodermal melanocytes and can migrate widely throughout the body,including skin,mucosa,uvea,inner ear and rectum,etc.It is one of the most aggressive and deadliest malignant tumors,and is the main cause of mortality associated with skin cancer.Early diagnosis of MM can achieve better therapeutic effect and prognosis through surgical resection,but the prognosis of patients with advanced metastasis is extremely poor.In recent years,the emergence of immunotherapy and molecular targeted therapies has significantly improved the prognosis,but their application in patients with MM is widely deficient in drug resistance and severe immune-related adverse effects,so further clarifying the molecular mechanism of MM is important to find new therapeutic targets to improve the clinical prognosis of patients.Epithelial mesenchymal transition(EMT)is an important cellular process in which cells lose their characteristic epithelial polarity,tight junctions and associated adhesive capacity and undergo a complex series of biological processes to transform into cells with a mesenchymal phenotype,eventually acquiring the ability to invade and metastasize.Three major transcription factors,the SNAIL family,the ZEB family and the TWIST family,are activated early in the EMT process and are closely associated with EMT.Among them,the SNAIL family including Snail1 and Slug,which are zinc-finger transcriptional repressor that regulate the EMT process during embryogenesis and tumor progression,can inhibit the expression of target genes through epigenetic methods,and closely related to tumor recurrence,metastasis and drug resistance mechanisms.However,the role and specific mechanism of Slug in MM have not been clarified.PurposeTo investigate the role and molecular mechanism of Slug in the invasion and metastasis of MM,to further elucidating the specific mechanism of MM development,and to provide a theoretical and experimental basis for finding more effective therapeutic targets for MM.Methods and ResultsFirst,it was found that the high expression of Slug in EMT transcription factors is significantly positively correlated with poor prognosis in patients with MM by TCGA database analysis,and is significantly correlated with clinical staging.Next,the expression of Slug at m RNA level was detected in MM tissues by q PCR method,and it was found that Slug was significantly overexpressed in MM tissues.Meanwhile,the relationship between the expression of Slug and patient prognosis was analyzed using the MM data in TCGA,confirming a significant correlation between high expression of Slug and poor prognosis in MM patients.Next,the expression level of Slug were detected in various MM cell lines,and stable overexpression of Slug was constructed in A375 cell lines with lower Slug expression,and stable knockdown Slug cell line in Mewo cell line with higher Slug expression.The effects of Slug on proliferation,migration and invasion ability of MM cells were detected through CCK8 experiments,scratch experiments,Transwell migration,and invasion experiments.It was found that Slug can promote the occurrence of EMT phenomenon in MM cells and enhance their migration and invasion ability in vitro and in vivo.In order to investigate the molecular mechanism of Slug promoting the migration and invasion of MM,transcriptome sequencing was performed on the A375 cell line overexpressed by Slug,and the sequencing results were analyzed by bioinformatics.Through screening and q PCR verification,it was found that IGFBP3 expression was reduced in the cells overexpressed by Slug,and significantly increased in the cells knocked down by Slug.It was speculated that IGFBP3 might be the key gene that Slug promotes the development of MM.To verify our hypothesis,through a response experiment,the plasmid transfected with IGFBP3 was overexpressed in Slug overexpressed cell lines,and si RNA of IGFBP3 was transfected into Slug knockdown cell lines for interference.The results showed that overexpression of IGFBP3 significantly inhibited Slug’s promotion of black migration and invasion,and IGFBP3 interference weakened the inhibitory effect of Slug knockdown on black migration and invasion.Next,we examined the expression of key proteins in the PI3K/Akt/GSK3β pathway and found that Slug-IGFBP3 promoted MM invasion and metastasis through the PI3K/Akt/GSK3β pathway.In order to further explore the molecular mechanism of Slug inhibiting the expression of IGFBP3,we conducted dual-Luciferase reporter gene analysis,immune coprecipitation assay and chromatin immunoprecipitation assay.The results showed that Slug interacted with HDAC3 in MM,inhibited the activity of IGFBP3 promoter through deacetylation and delactylation,and significantly promoted the migration and invasion of Slug on MM in vitro after overexpression of HDAC3,the addition of HDAC3 inhibitors significantly inhibited the migration and invasion of Slug on MM cells in vitro.Indicating that Slug recruited HDAC3 and co recruited it in the IGFBP3 promoter region,by deacetylation and delactylation modification of its histones,it suppressed the transcription and expression of IGFBP3,ultimately promoting the migration and invasion of MM.ConclusionSlug is highly expressed in MM and is significantly associated with poor prognosis in MM patients.Slug can promote the migration and invasion of MM both in vitro and in vivo,while Slug promotes the migration and invasion of MM by inhibiting IGFBP3.Slug inhibits IGFBP3 by promoting PI3K/Akt/GSK3 βThe signaling pathway promotes the migration and invasion of MM.Slug collaborates with HDAC3 to reduce H3K4 acetylation and H4K5 lactylation modifications in the promoter region of IGFBP3,inhibiting IGFBP3 transcription and promoting the invasion and metastasis of MM.