Based On Bioinformatics Analysis To Verify The Role And Function Of MiR-510-5p And MiR-513c-5p In Skin Melanoma

Objective:Cutaneous melanoma(CM)is a very malignant type of tumor with strong invasive ability.Metastasis may occur in the early stage,and once it occurs,the patient's survival time is usually very short.The incidence of CM in our country is lower than that in Western countries.However,our country has the characteristics of a large population base,and the number of patients is increasing year by year,the overall number of patients is relatively high.At present,early surgical treatment is still the only way to cure skin malignant melanoma.With the development of science,the application of targeted therapies to the clinic has prolonged the survival time of patients who have undergone metastasis.However,tumor recurrence and drug resistance have become common clinical problems.In the past ten years,the rapid development of genome sequencing technology has produced a large amount of high-throughput information and biological information data.This brings up two follow-up questions: The first,the storage and output of high-throughput data;the second,the analysis and processing of these data.The establishment of a large number of biological information databases solves the first problem.Researchers around the world can download patient clinical data and organize sequencing information through public databases.For example,GEO(the Gene Expression Omnibus)database(The database contains expression profile information of different types of diseases),TCGA(the Cancer Genome Atlas)database(The database contains clinical and expression profile information from various cancers)etc.,the various tumor information stored in these two databases are available for free download.The application of a variety of big data analysis methods solves the second problem,and bioinformatics as an independent discipline has been extensively developed.The information obtained by public databases has the characteristics of large sample size and high reliability.These samples can be blood of clinical patients,paraffin-embedded diseased tissues and frozen pathological tissues,etc.,which greatly reduces research costs.The application of bioinformatics data analysis methods in the prognosis prediction of diseases(tumors,etc.),molecular screening,mutation gene detection,drug resistance gene screening,drug target determination and other fields has achieved good results.Humans have never stopped "struggling" against various tumors including melanoma of the skin,and there are still many problems that have not been resolved.The collection and analysis of medical information big data,with its own advantages,can help human achieve greater success in defeating the lethal disease of tumors in the future.Micro RNAs(miRNAs)are a type of short non-coding RNAs that are widely present in various organisms.They are widely involved in the occurrence and development of various diseases by affecting the function of m RNA.Abnormal expression of miRNAs in skin melanoma is very common,but many mechanisms are still unclear.Therefore,based on the database information,we further explore the abnormal expression of miRNAs in patients with cutaneous melanoma,in order to find unreported miRNAs molecules that can affect the occurrence or development of the disease,which can provide more effective methods for future clinical treatment.Methods: In this study,we used differential expression analysis of the transcript data of skin malignant melanoma tissue and pigmented nevus tissue,combined with prognostic analysis,obtained several miRNAs related to the prognosis of the disease,and a prognostic signature of skin melanoma based on 5 miRNAs was established.Among these 5 miRNAs,the coding sites of miR-510-5p and miR-513c-5p are located on the X chromosome,and no relevant reports on the functions and effects of these two miRNAs in cutaneous melanoma have been seen,so as the miRNAs we are interested in,we have done further research on the functions of miR-510-5p and miR-513c-5p.The detailed method is as follows: 1.According to the screening criteria(the number of patients reaches or exceeds 40 and also contains sample information of skin melanoma and pigmented nevus),search the GEO database for the expression data set of miRNAs in cutaneous melanoma and pigmented nevus tissue as a test set,and then we perform differential analysis to obtain differentially expressed miRNAs(DEMs);We downloaded the clinical information of cutaneous melanoma patients and the expression profile information of the above DEMs in the TCGA database,and performed Kaplan–Meier survival analysis to screen out DEMs with survival significance.The Kaplan-Meier survival analysis method was also used to establish a prognostic signature based on multiple miRNAs,then unilateral and multilateral regression analysis were performed to verify the reliability of the prognostic signature;We downloaded a new data set(including the expression profile data of cutaneous melanoma and skin pigmented nevus tissues)in the GEO database as a validation set,and conducted differential expression analysis to verify whether the differential expression of miRNAs in the prognostic model is consistent with the results in the test set.We verify and analyze miR-510-5p and miR-513c-5p in the 5-miRNAs signature from multiple aspects: First,we performed target gene prediction on the mi Walk website(miRWalk,http://mirwalk.umm.uni-heidelberg.de/)to obtain the target gene information of miR-510-5p and miR-513c-5p,downloaded target gene expression data from TCGA database,then applyed these two miRNAs and the expression profile of target genes for correlation analysis(the screening criteria are |R|?0.3,P <0.05).We performed further KEGG and GO analysis,protein network interaction(https://www.string-db.org/)and survival analysis on the relevant target genes obtained in the above steps to obtain more meaningful target genes.2.We used RT-PCR method to detect whether there is a difference in the expression of miR-510-5p and miR-513-5p in the melanoma A375 cell line,A2058 cell line and normal melanoma cell line.The MTS method and the Transwell chamber were used to determine the effect of overexpression of miR-510-5p and miR-513c-5p on the viability,migration and invasion of A375 cells.We used the RT-PCR method to compare whether the predicted target genes(SEMA6A,SEMA6 B,EFNB2,EPHA3,EPHB2,BDNF)are differentially expressed in A375,A2058 melanoma cell lines and normal melanoma cell lines.Using the A375 cell line,after overexpressing miR-510-5p and miR-513c-5p,we tested whether the expression levels of the predicted target genes(SEMA6A,SEMA6 B,EFNB2,EPHA3,EPHB2,BDNF)changed,and initially verified downstream target genes of miR-510-5p and miR-513c-5p.Finally,we used dual luciferase to verify whether there is a binding site between the miRNAs and the target gene.Result: 1.The data set obtained from the GEO database shows that GSE35579 meets the screening criteria.The differential expression analysis of miRNAs in skin malignant melanoma and skin pigmented nevus tissues obtained a total of 185 DEMs,of which 80 were up-regulated and 105 were down-regulated.Combining the survival information of cutaneous melanoma patients and the expression of DEMs in the TCGA database for survival analysis,a total of 8 miRNAs closely related to the survival of cutaneous melanoma patients were obtained,and then a five-miRNAs signature(miR-25,miR-204,miR-211,miR-510,miR-513c)were established.Survival analysis indicated that the survival time of skin melanoma patients was negatively correlated with the expression of the five-miRNAs signature(P = 0.001).The results of unilateral and multilateral regression analysis(HR = 0.605,P = 0.006)indicate that this signature can be used as an independent predictor of the survival of patients with cutaneous melanoma.In the validation set GSE18509,the results of differential expression analysis of miRNAs in skin melanoma and pigmented nevus tissues suggest that,except for miR-25,the remaining 4 miRNAs in the prognostic model have differential expression.We downloaded the target gene prediction results of miR-510-5p and miR-513c-5p,and analyzed the correlation between these two miRNAs and their target genes.A total of220 target genes were obtained.KEGG and GO analysis results suggest that 220 target genes are mainly concentrated in PI3K-Akt,cancer,local adhesion,axon guidance and other signaling pathways.We performed PPI network interaction on these target genes and find a gene function cluster(SEMA6A,SEMA6 B,EFNB2,EPHA3,EPHB2,BDNF),which is mainly involved in axon guidance.Prognostic analysis of the meaningful target genes in the KEGG and GO analysis,a total of 16 target genes with survival significance were obtained,of which SEMA6A(HR = 1.23,P = 9.17E-06)is most likely to be related to the survival of cutaneous melanoma patient.2.The RT-PCR results indicate that miR-510-5p and miR-513c-5p have decreased expression in melanoma cell lines(A375,A2058)compared with normal melanoma cell lines(PIG1).After transfection of miR-510-5p mimics and miR-513c-5p mimics,compared with transfected NC mimics,the proliferation,migration and invasion ability of A375 cells was weakened.RT-PCR results showed that the predicted target genes(SEMA6A,SEMA6 B,EFNB2,EPHA3,EPHB2,BDNF)all had different levels of expression differences in the A375?A2058cell lines compared with the PIG1 cell line,and the differential expression of SEMA6 A was the most obvious.The A375 cell line was used to overexpress miR-510-5p and miR-513c-5p respectively,RT-PCR results showed that the expression of SEMA6 A was down-regulated,while the expression of other target genes(SEMA6B,EFNB2,EPHA3,EPHB2,BDNF)did not change.Mi R-513c-5p(P = 0.0009)has a more significant regulatory effect than miR-510-5p.The dual luciferase experiment of miR-513c-5p and SEMA6 A found that the regulatory element miR-513c-5p has an inhibitory effect on the target SEMA6 A,which is achieved by binding to the gene coding site(CDS site).Conclusion: 1.Abnormal expression of miRNAs in skin melanoma tissue is very common.2.We have established a prognostic signature based on 5 miRNAs(miR-25,miR-204,miR-211,miR-510,miR-513c),which can predict the survival of patients with cutaneous melanoma 3.Compared with PIG1 normal melanoma cell lines,the expressions of miR-510-5p and miR-513c-5p in A375 and A2058 melanoma cell lines are all decreased,and their target genes are mainly involved in cancer-related signal pathways.4.Overexpression of miR-510-5p and miR-513c-5p can inhibit the viability,migration and invasion of A375 melanoma cells.5.Compared with the PIG1 cell line,the predicted axon guidance function cluster genes(SEMA6A,SEMA6 B,EFNB2,EPHA3,EPHB2,BDNF)have varying degrees of abnormal expression in the A375 and A2058 cell lines.Among them,the gene closely related to the survival of skin melanoma patients is SEMA6 A,and the expression of SEMA6 A in melanoma cell lines shows a downward trend.5.miR-510-5p and miR-513c-5p can inhibit the expression of SEMA6 A to varying degrees,and the inhibitory effect of miR-513c-5p is more obvious.6.The inhibitory effect of miR-513c-5p on the expression of SEMA6 A is achieved by combining with the coding site(CDS site)of the SEMA6 A gene.In summary,the prediction and the experimental verification result are consistent.It can be seen that downloading big data from the Internet and bioinformatics analysis can save experimental resources.The experimental results we obtained can provide a basis for deeper research.