Mechanisms Of Long Non-coding RNA SLNCR1 Targeting SOX5 To Promote EMT In Malignant Melanoma

Malignant melanoma(MM) is a type of highly malignant tumors of melanocytes,which are initially migrated from the neural crest to the skin,uvea,meninges,and mucous membranes during embryo development.Most melanoma cells are located at the epidermis-dermis interface of the skin.The incidence of malignant melanoma accounts for 4%of all skin cancers,but its mortality rate after metastasis accounts for 75%of skin tumors.The global incidence of melanoma has been increasing at an alarming rate in recent decades.Therefore,the control of melanoma will face significant challenges in the future.Thus,identifying critical specific biomarkers is crucial for the identification and precise clinical treatment of MM.Long non-coding RNA(LncRNA) is a new type of non-coding RNA,usually referring to RNA transcripts longer than 200 nucleotides.In addition,more and more studies have shown that lnc RNAs may participate in the development of tumors and act as oncogenes or tumor suppressors in the process of tumor evolution.SLNCR1 is a newly discovered LncRNA which is located on human chromosome 17q24.3.Recent research results show that SLNCR1 is aberrantly expressed in various tumors and promotes tumor cell migration,proliferation,and invasion.However,the specific mechanism as to how SLNCR1 involves in the development of MM remains unclear.Object:To explore the differentially expressed molecules in the progression of MM,clarify the mechanism of promoting the progression of MM,and lay the foundation for the diagnosis and treatment of MM.Methods and Results:The role of SLNCR1 in the development of MM was explored through two mechanisms in my study.Firstly,the localization of SLNCR1 in cells was determined by FISH.The results show that SLNCR1 is mainly located in the nucleus and exists in a small amount in the cytoplasm,so we hypothesized that SLNCR1 might involve in the progression of MM through two mechanisms:transcriptional regulatory and post-transcriptional regulatory(ce RNA).In my study,the following experiments were carried out:1)Transcriptional regulation.Firstly,validating differentially expressed LncRNAs in human MM tissues with RNA-seq sequencing,then verified the candidate LncRNAs expression level by q PCR in human MM tissues and cell lines.SLNCR1 was selected for the following experiments for its significant difference and higher expression level.Further statistics of the basic clinical information and pathological information of the patients confirmed that the high expression of SLNCR1 was closely related to lymph node metastasis and P⁵³.To explore the function of SLNCR1 in the development of MM cells,we knockdown the expression of SLNCR1,it was found that silencing of SLNCR1 inhibited cell proliferation,migration,and invasion,through CCK8,crystal violet staining,wound-healing assay,Transwell assay,and clone formation assay,and induction of EMT phenomenon.Then,it was discovered that SLNCR1 might interact with transcription factor SOX5 using RNA-seq sequencing,website prediction,and bioinformatics analysis.And I verified that SLNCR1 bound to the promoter region of transcription factor SOX5 through dual-luciferase experiments.Next,in order to verify the role of SOX5 in the progression of MM,I found that the silencing of SOX5inhibited cell proliferation and migration and EMT phenomenon through CCK8,crystal violet staining,scratching assay,Transwell and cloning formation assay.And the expression level of SOX5 decreased after knockdown of SLNCR1.To verify that SLNCR1 interacts with SOX5 to regulate the development of MM,I found that SOX5overexpression effectively reversed SLNCR1-induced proliferation,migration,and EMT after knockdown SLNCR1 and overexpression of SOX5.Finally,to test and verify that SLNCR1 targets and binds to SOX5 in vivo to regulate the development of MM,we established a Balb/c nude mouse subcutaneous tumor model,and found that the volume of subcutaneous tumor was smaller compared with the NC group,immunohistochemistry staining showed that silencing of SLNCR1 in vivo inhibited the expression of SOX5,Ki67 and Vimentin,which was consistent with the results of in vitro experiments.2)Post-transcriptional regulatory(ce RNA).I firstly download melanoma-related data package(GSE20994,GSE25653,GSE34460,GSE61741)from the GEO database,and performed differential analysis after ID conversion and data grouping using the limma package of R language,then obtain differentially expressed mi RNAs in MM tissues with pheatmap package.We selected 10 mi RNAs for the following experiments,including mi R-214,mi R-183,mi R-455-5p,mi R-146b-3p,mi R-429,mi R-10b,mi R-200a,mi R-211,mi R-379,mi R-132,and detected the expression levels of these mi RNAs by q PCR in the MM tissue,and found that only mi R-146b-3p was low expressed in the MM tissue at low level.After knockdown of SLNCR1,the expression level of mi R-146b-3p was increased,thus I infer that SLNCR1 might have a certain regulatory effect on mi R-146b-3p.Conclusions:LncRNA SLNCR1 promotes the EMT of MM by targeting the transcription factor SOX5.Mi R-146b-3p was expressed in MM tissue at low level,and silencing of SLNCR1 reversed the expression level of mi R-146b-3p,SLNCR1may involve in the occurrence of MM by competitively binding to mi R-146b-3p developing.