Study On The Expression Profile Of Long Non-coding RNA In Acral Lentiginous Melanoma And The Function Of Linc37877 On Melanoma Cells

Cutaneous malignant melanoma(cutaneous malignant melanoma,CMM)is described as malignancy of the melanocyte.The incidence is increasing continuously worldwide.ALM is rare in western populations but is more prevalent in China.Despite the rapid development of diagnosis and treatment of melanoma in recent years,the 5-year survival rate has not been significantly improved.Therefore,a better understanding of the molecular mechanism of tumorigenesis is still of great significance.Long non-coding RNA(lncRNA)is a kind of long transcripts longer than 200 nucleotides which can't be translated into protein.Many lncRNAs are indicated to associate with the development of melanoma.In order to find the differentially expressed lncRNAs,microarray analysis was used to analyze the expression profiles of IncRNA and mRNA.We found that linc37877 was highly expressed in both tumor tissues and melanoma cell lines.We speculate that linc37877 may play an important role in the development of melanoma as an oncogene.Part 1:Analysis of lncRNA expression profile in Acral Lentiginous MelanomaObjective:To establish Acral Lentiginous Melanoma lncRNA and mRNA expression profiles and to obtain molecular function,biological process and related disease pathway information of differentially expressed mRNA.Methods:First,six pairs of Acral Lentiginous Melanoma and adjacent non-tumor tissues were used for microarray assay to determine differentially expressed IncRNA and mRNAs.Then we conducted Gene Ontology(GO)analysis and KEGG analysis to explore the function of biological processes,cellular components,molecular functions and disease pathways involved of differentially expressed mRNA.Finally,five differentially expressed lncRNAs were selected and verified by RT-qPCR experiment.IncRNA and mRNA co-expression network as well as lncRNA-miRNA-mRNA interaction.Results:We constructed lncRNA and mRNA expression profile of Acral Lentiginous Melanoma and found 4490 differentially expressed lncRNAs and 3919 differentially expressed mRNAs by microarray assay.The differentially expressed mRNAs wereanalyzed by GO analysis and KEGG analysis.The results showed that differentiallyexpressed mRNAs were involved in events of pigmentation and melanocytes.melanosome membrane,pigment granule and melanosome,channel activity,cation channel activity and calcium channel activity,cell junction and tissue developments.KEGG pathway showed that differentially expressed mRNAs were involved in glycosphingolipid biosynthesis-ganglio series,transcriptional misregulation in cancer,toll-like receptor signaling pathway and melanogenesis,oxytocin signaling pathway,regulation of actin cytoskeleton and hippo signaling pathway.Conclusion:Construction of Acral Lentiginous Melanoma IncRNA and mRNA expression profiles which provides a data source for the subsequent research.Part II:The primary study of influence and mechanism of linc37877 expression on the biological behaviors of melanoma cells.Objective:To investigate tile influence of linc37877 expression on proliferation,apoptosis,migration and invasion of melanoma cells.Methods:First,the expression level of linc37877 in melanocytes and four melanoma cell lines(A375,A875,MV3,SK-MEL-28)was verified by RT-qPCR experiment.RNA fluorescence in situ hybridization and nuclear-cytoplasmic separation assay were used to identify the cellular localization of linc37877.In combination with ASO and siRNA techniques,the expression of linc37877 in melanoma cells was downregulated,and the interference efficiency was evaluated.The effect of linc37877 on proliferation of melanoma cells was detected by CCK8 and EDU assay,and the effect of apoptosis was detected by flow cytometry.Wound-healing assay and transwell assay were used to detect the invasion and migration ability.Western-blot was used to explore the mechanism of linc37877 on invasion and migration of melanoma cells.Results:The expression level of linc37877 in melanoma cell lines was significantly higher than that in melanocytes,among them the highest expression was found in melanoma cell line A875.Most of linc37877 was located in nucleus.The level of linc37877 was significantly decreased after downregulating its expression.Knocking down linc37877 could inhibit proliferation,migration and invasion and promote apoptosis of A875 cells.The expression level of MMP2 and MMP9 was decreased.There were no significant changes in N-catenin,E-catenin and Vimentin.Conclusion:linc37877 is mainly located in the nucleus,which can contribute to proliferation,invasion and migration of melanoma cells.The increased ability of invasion may be related to MMP2 and MMP9,but not epithelial-mesenchymal transition.