| BackgroundMelanoma is a highly malignant tumor derived from the basal layer of melanocytes in the epidermis.The tumor is predominant in the skin or in the skin adjacent to the skin and is the most aggressive and lethal type of skin cancer,with a dramatically increased global incidence over the past few decades.The main cause of melanoma-related deaths is the metastatic phenotype.It is highly malignant,progresses rapidly and metastasizes early.Moreover,there are still no satisfactory treatments for patients with advanced melanomas.Accumulating evidence has demonstrated that the development of melanoma involves complex changes in multiple genes,multiple signaling pathways and gene regulation.Therefore,identifying some potential target molecules in melanoma,including oncogenes and tumor suppressors,might show promise to serve as therapeutic targets for the treatment of it.microRNAs(miRNAs),a class of small(19-24 nucleotides in length),non-coding RNA molecules,regulate gene expression at the post-transcriptional level by directly binding to seed sequences in the 3'-untranslated regions(3'-UTRs)of their targets which ultimately induce the targets degradation or translational suppression.A single miRNA can regulate the expression of more than 100 genes;meanwhile,a gene may be targeted by several miRNAs.Currently,given the large number of miRNA annotated in the human genome,30%?80%of human genes are predicted to be regulated by miRNAs.Specifically,deregulation of miRNAs expression profiling between tumor cells and cells derived from normal tissue have been observed in various cancer types,which may be a result of epigenetic alterations of miRNA promoters,the altered activity of transcriptional factors or dysregulated enzymes involved in miRNA biogenesis.Furthermore,evidence is emerging that altered miRNA expression contributes to the occurrence,development and progression of many cancers by down-regulating tumor suppressor genes or up-regulating tumor-promoting oncogenes.Thus,miRNAs are considered as key regulator during the development of melanoma.Collagen triple helix repeat containing 1(CTHRC1),initially found in a screen for differentially expressed genes in balloon-injured versus normal rat arteries,is widely detected in human solid cancers,especially cancers of the gastrointestinal tract,lung,breast,thyroid,ovarian,cervix,liver,and pancreas.Nevertheless,the detailed function of CTHRC1 in melanoma remained to be not elucidated.Additionally,we also predicted that miR-155 might be an up-stream regulator of CTHRC1 by bioinformatics analysis.miR-155,produced from the processing of the B-cell integration cluster(BIC),exerts an important role in various physiological and pathological processes,especially in tumorigenesis.Hence,in the current study,we aimed to further investigate the interaction between miR-155 and CTHCR1 and to clarify the roles of miR-155 and CTHCR1 in the pathogenesis and progression of melanoma which might provide new biomarkers for the aggressive melanoma,or novel therapeutic targets in treating melanoma.Part One:Expressions of miR-155 and CTHRC1 in melanoma tissuesObjective To identify whether miR-155 is an up-stream miRNA of CTHR1 gene,and to explore the expressions of miR-155 and CTHRC1 in melanoma patients'tissues.Methods Bioinformatics methods were used to predict the up-stream miRNAs of CTHRC1 gene and identify the key miRNAs which might regulate CTHRC1.qRT-PCR was utilized to detect the expressions of miR-134,miR-155,miR-30c and miR-630 in tumorous tissues and matched adjacent non-tumorous normal tissues of melanoma patients.Moreover,the expression of miR-155 and CTHR1 was further examined by qRT-PCR in melanoma patients with their tumorous tissues and matched adjacent non-tumorous normal tissues.Results Bioinformatics analysis showed that seven miRNAs databases co-predicted that only four miRNAs could target CTHRC1,namely miR-134,miR-155,miR-30c and miR-630.Subsequently,two patients with melanoma were selected for the four miRNAs examinations by qRT-PCR.The relative expression of miR-134,miR-30c and miR-630 was up-regulated in one patient's cancer tissues and down-regulated in the corresponding adjacent normal tissues.But the three miRNAs were down-regulated in the other 1 patient's cancer tissues and up-regulated in the corresponding adjacent normal tissues,thereby these results indicated that the three miRNAs presented different expressed trend in different patients.However,the relative expression level of miR-155 was decreased and increased in both 2 patients'cancer tissues and their corresponding adjacent normal tissues,respectively.Thus,miR-155 was considered to be the most likely up-stream miRNA for CTHRC1.Finally,18 cases of melanoma patients were selected for miR-155 and CTHRC1 using qRT-PCR detection.The results showed that in all 14 cases except the 4 cases,the relative expression of miR-155 was declined in cancerous tissues and elevated in the corresponding adjacent normal tissues.Finally,these 14 cases of cancer tissues and adjacent non-cancerous tissues were used to test CTHRC1 expression and the results displayed that the relative expression of CTHRC1 was ascended in the cancer tissues of patients,and descended in the adjacent non-cancerous tissues of patients.Conclusion The lower expression of miR-155 suggested that miR-155 might exert an important role in the pathogenesis of melanoma.Moreover,the expression pattern of miR-155 and CTHRC1 presented an inverse trend,indicating that there might be a target interaction between miR-155 and CTHRC1.Part Two:The target regulatory role of miR-155 on CTHRC1Objective To investigate the target regulatory interaction between miR-155 and CTHRC1.Methods psiCHECKTM-2 vector was used to construct the reporter plasmids of CTHRC1-WT and CTHRC1-Mutant.Gel electrophoresis and sequencing technology were adopted to identify the reporter plasmids of CTHRC1-WT and CTHRC1-Mutant.Dual-luciferase reporter assay was utilized to verify the target interaction between miR-155 and CTHRC1.qRT-PCR and WB were performed to further confirm the regulatory role of miR-155 on CTHRC1.Results After amplification of the fragments of CTHRC1-3'-UTR,running the gel,excising and recycling the geldouble enzyme digestion,ligation,transformation,DH5a culture,plasmid extraction,identification by restriction analysis,sequencing,site-specific mutagenesis,the psiCHECK-CTHRC 1-WT reporter plasmid and psiCHECK-CTHRC1-Mutant reporter plasmid were successfully obtained.The results of dual-luciferase reporter assay revealed that the fluorescence intensity in miR-15 5+CTHRC1-WT group was significantly lower than that in Blank+CTHRC 1-WT group,but the fluorescence intensity in miR-155 inhibitor+CTHRC 1-WT group was notably higher than that in miR-155+CTHRC1-WT group.Furthermore,there were no differences in NC+CTHRC1-WT,NC inhibitor+CTHRC 1-WT and Blank+CTHRC 1-WT groups.Eventually,qRT-PCR and WB data exhibited that miR-155 over-expression remarkably inhibited the expression of CTHRC1 in mRNA and protein levels,while miR-155 down-regulation markedly promoted the the expression of CTHRC1 in mRNA and protein levels.Conclusion miR-155 could directly target CTHRC1.Part Three:miR-155 involved in the regulation of the biological behaviors,including cell proliferation,apoptosis,cell cycle,migration and invasion,probably via targeting CTHRC1 in melanomaObjective To explore the effects of miR-155 and CTHRC1 on the cell proliferation,apoptosis,cell cycle,migration and invasion in melanoma.Methods miR-155 mimic,CTHRC1 mimic and si-CTHRC1 were transfected into M21 cells.Subsequently,CCK8 and colony-forming assays were used to examine the roles of miR-155 and CTHRC1 on cell proliferation.Flow cytometry was utilized to test the roles of miR-155 and CTHRC1 on apoptosis and cell cycle.Transwell assay was adopted to detect the roles of miR-155 and CTHRC1 on cell migration and invasion.Results CCK8 assay found that the proliferation rate of M21 cells was gradually ascended with the culture time in each group.Forced miR-155 expression and inhibited CTHRC1 expression obviously reduced M21 cells proliferation,but over-expression of CTHRC1 apparently elevated M21 cells proliferation.Colony-forming assays discovered that enhanced miR-155 expression and suppressed CTHRC1 expression dramatically declined the colony number of M21 cells,while rising CTHRC1 expression sharply augmented the colony number of M21 cells.Flow cytometry revealed that apoptosis was increased and cell cycle was retarded in miR-155 and si-CTHRC1 groups,whereas apoptosis was decreased and cell cycle was improved in CTHRC1 group.Ultimately,Transwell assay uncovered that the number of cell migration and invasion was significantly down-regulated in miR-155 and si-CTHRCl groups,and the number of cell migration and invasion was notably up-regulated in CTHRC1 group.Conclusion miR-155 could participate in the regulation of the biological behaviors,including cell proliferation,apoptosis,cell cycle,migration and invasion,probably through regulating CTHRC1 in melanoma. |
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