Regulation Mechanism Of MiR-675-3p In Cutaneous Melanoma And Its Clinical Significance

Objective: Cutaneous melanoma is one of the most malignant skin tumors,ranking first in the mortality rate of cutaneous malignant tumors,and its incidence rate is increasing faster than any other preventable skin tumors.After early diagnosis,the prognosis of surgical resection is generally good.Once metastasis occurs,there is still a lack of effective treatment,and the survival rate of patients drops sharply.Although the current application of targeted therapy and the improvement of immunotherapy strategies have improved patient survival to a certain extent,there are still tumor resistance and serious side effects of therapeutic drugs.Therefore,early diagnosis and treatment is extremely important.Recent studies have shown that many miRNAs are involved in the occurrence and development of melanoma,regulate the mechanism of drug resistance of melanoma,and can even be used as biomarkers for the diagnosis and prognosis of melanoma.This study aims to investigate the upstream and downstream regulatory mechanism of miR-675-3p in cutaneous melanoma and its potential clinical value.Methods: In this study,miRNAs sequencing(miRNA-seq)data and clinical phenotypes of cutaneous melanoma patients from the TCGA database were downloaded from the UCSC XENA website.Then,the "limma" software package in the R software was used to identify the miRNAs(DEMs)that were differentially expressed in the tissues of metastatic and primary melanoma patients in the miRNA-seq data.A total of 3significantly upregulated and 23 downregulated DEMs were identified in metastatic melanoma samples.Among the selected DEMs,the expression of miR-675-3p was the most significant in the metastatic melanoma tissue compared with the primary melanoma tissue.Furthermore,the expression of miR-675-3p in the peripheral blood of cutaneous melanoma patients was confirmed by GEO database GSE20994 dataset,and its expression in melanoma cell lines was detected by q RT-RCR.Kaplan-Meier survival analysis and Log-rank test were used to evaluate the relationship between miR-675-3p expression and survival in patients with cutaneous melanoma.The chi-square test was used to evaluate the relationship between miR-675-3p expression and clinical parameters and histological characteristics of patients with cutaneous melanoma.MEXPRESS database and Pearson correlation analysis were used to evaluate whether DNA methylation regulates miR-675-3p expression.Target genes and transcription factors of miR-675-3p were obtained by a variety of bioinformatics tools.Dual luciferase assay and Western Blot were used to verify the target genes.Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)analyses were performed to assess the biological role of miR-675-3p regulatory networks and tumor-related pathways.The overexpression of miR-675-3p in melanoma cell lines was measured by flow cytometry.The proliferation activity of melanoma cells was detected by CCK8 cell proliferation assay.Cell migration and invasion ability were observed by Transwell assay,and the expression of related signaling pathway proteins was detected by Western Blot.Results: 1)From miRNA-seq data of 353 metastatic melanoma and 97 primary melanoma tissue samples in the TCGA database,3 significantly upregulated and 23 downregulated DEMs in metastatic melanoma samples were screened.Among the selected DEMs,the expression of miR-675-3p was the most significant in the metastatic melanoma tissue compared with the primary melanoma tissue.GEO database GSE20994 data set contains microarray data of peripheral blood samples from 35 cases of melanoma and peripheral blood samples from 22 normal subjects.The results of microarray data analysis of these two groups showed that the expression of miR-675-3p in the peripheral blood of cutaneous melanoma patients was significantly up-regulated compared with the normal peripheral blood samples.In addition,q RT-PCR results showed that miR-675-3p was up-regulated in both metastatic melanoma cell lines(A2058,451LU)and primary melanoma cell lines(A375),and the expression was higher in metastatic melanoma cell lines.2)According to the median expression level of miR-675-3p,patients with available overall survival(OS)data were divided into the high expression group of miR-675-3p and the low expression group.Kaplan-Meier survival analysis was performed on the expression of miR-675-3p and the survival results of melanoma patients,and the results showed that patients with increased expression level of miR-675-3p had poor OS.X-tile software was further used to calculate the optimal truncation 8.5 of miR-675-3p expression,and the patients were again divided into two groups: the high expression and the low expression of miR-675-3p.Survival analysis results again confirmed that high expression of this miRNA was significantly associated with decreased OS in patients with melanoma.Then,the chi-square test was used to analyze the relationship between miR-675-3p expression and clinical parameters and histological characteristics.The results showed that the increased expression of miR-675-3p was significantly correlated with tumor histological grade and Clark level,but not with age,gender,TNM stage,family history of cancer,and pathological grade.These results suggest that miR-675-3p may serve as a potential clinical prognostic biomarker for patients with melanoma.3)The relationship between DNA methylation and miR-675-3p expression was evaluated using the MEXPRESS database,and the results showed no significant differences between miR-675-3p expression level(low/high),tumor type(primary/metastatic),tumor stage or Clark level and DNA methylation.Further Pearson correlation analysis showed that the expression level of miR-675-3p was slightly negatively correlated with nine major Cp G loci(CG03175030,CG07342901,CG14937069,CG15269875,CG15963714,CG19943238,CG21167159,CG25852472 and CG26857192)in the promoter region,but these differences were "not significant" statistically.4)47 and 79 transcription factors(TFs)that may regulate the expression of miR-675-3p were identified in the TransmiR v2.0 and h TFtarget databases,respectively,and 32 of them overlapped between the two datasets,so they were considered as possible transcriptional regulators of miR-675-3p expression.A total of 10 overlapping miR-675-3p target genes were predicted by Target Scan Human V7.1,miRDB and miRTARbase.Then,a comprehensive miR-675-3p regulatory network containing these 32 TFs and 10 target genes was constructed in this study.Notably,this network suggests that EGR1 may play a feedback role in regulating the expression of miR-675-3p,while ZBTB7 A may inhibit the expression of this miRNA.Analysis based on network topology reveals a subnetwork containing seed genes and top neighbors.Pearson correlation analysis showed that miR-675-3p was significantly positively correlated with the expression of ERG1 in TCGA database,and negatively correlated with the expression of ZBTB7 A and IGF1 R.5)miR-675-3p was overexpressed in melanoma cell lines,and the m RNA and protein levels of ERG1,IGF1 R and OPCML were detected by q RT-PCR and WB,respectively.The results showed that EGR1 was significantly up-regulated in m RNA and protein levels,while IGF1 R and OPCML were significantly down-regulated.Further dual luciferase reporting experiments confirmed that OPCML was the direct binding target of miR-675-3p.6)Go and KEGG Pathway enrichment analysis was performed using TFs and target genes to further understand the potential biological role of miR-675-3p.GO analysis showed that some genes are involved in key biological processes and molecular functions related to carcinogenesis.KEGG Pathway analysis further showed that miR-675-3p may regulate cell cycle,cancer transcriptional dysregulation,TGF-? and HIF-1 signaling pathways.Flow cytometry cell cycle analysis showed that overexpression of miR-675-3p decreased the proportion of G0/G1 cells and increased the proportion of G2/M cells.CCK8 assay showed that miR-675-3p could promote the proliferation of A375 cells.Transwell assay showed that miR-675-3p promoted the migration and invasion of melanoma cells.Western Blot results showed that TGF?2,Smad2/3,Smad4,and HIF1 A protein levels associated with the TGF-?/Smad and HIF-1 signaling pathways were significantly increased and TGF?1protein levels were significantly down-regulated compared to the negative control group.Conclusions: 1)The expression of miR-675-3p was up-regulated in tissues,peripheral blood samples and melanoma cell lines of cutaneous melanoma patients,and the expression was higher in metastatic samples.These results suggest that miR-675-3p may play an oncogenic role in cutaneous melanoma.2)The high expression of miR-675-3p was significantly correlated with decreased OS in patients with cutaneous melanoma,and miR-675-3p was significantly correlated with the histological grade and Clark level of cutaneous melanoma,but not with age,gender,TNM stage,family history of cancer,and pathological grade.These data suggest that miR-675-3p may serve as a clinically important prognostic biomarker in patients with cutaneous melanoma.3)DNA methylation was not significantly correlated with the expression and clinical characteristics of miR-675-3p,and the expression of miR-675-3p was not significantly correlated with the Cp G site of the promoter region,suggesting that the expression of miR-675-3p in cutaneous melanoma was not affected by DNA methylation.4)Bioinformatics predicted 32 upstream transcription factors and 10 downstream target genes of miR-675-3p,among which ERG1 was significantly positively correlated with miR-675-3p,while ZBTB7,IGF1 R and OPCML were negatively correlated with miR-675-3p expression.5)The OPCML gene is directly targeted by miR-675-3p.6)miR-675-3p affected the cycle distribution of melanoma cells and promoted the proliferation activity,migration and invasion ability of melanoma cells.In addition,TGF-?/Smad signaling pathway TGF?2,Smad2/3,Smad4 proteins,HIF-1 signaling pathway HIF1 A protein expression can be up-regulated,TGF-?/Smad signaling pathway TGF?1 protein expression can be down-regulated.