Mouse Melanoma(B16) Transcriptional?lncRNA Expression Analysis And Construction Of B16 Nanolibrary

Melanoma is one of the most aggressive malignant skin cancer,which deeply harms people's health.Scientific research shows that the incidence of melanoma in the last 20 years is increasing year by year.It may be related to global climate change and environmental pollution.There have been many studies on malignant melanoma,and remarkable results have been achieved.But so far the etiology is not clear,the incidence of melanoma maybe be related to race,ultraviolet radiation,endocrine,stimulation,trauma,Virus,heredity,immune deficiency,chemical carcinogen and other factors related.The human genome project found that protein encoding genes only accounted for about 2%of the overall human genes,most of the genes encoding proteins have no ability,but to the transcription of genes has reached a staggering 75%,which found in the human gene in most of the non encoding gene,and these two types of miRNA and lncRNA gene its unique function has attracted extensive attention of researchers.Heavy chain antibody,a special structure of small antibodies in the sera of alpaca,camel,and cartilaginous fish.Compared with the traditional antibody,the heavy chain antibody only contains two heavy chains.The structure of the heavy chain antibody is very simple,but it still has the same activity as the traditional antibody.Based on the heavy chain antibody,researchers have developed a simpler,more stable and smaller molecular weight VHH antibody(nano antibody)compared to traditional antibodies.Because of its excellent characteristics,nano antibody has broad application prospects in the diagnosis and treatment of diseases.This experiment adopts the second generation sequencing technology on isolated from C57BL-6J mice normal melanocytes and melanoma cells(B16),respectively to detect the transcriptomic differences of two cell gene,encoding non differential expression of RNA(miRNA and lncRNA),the selection of specific expression of gene function research,occurrence to explore the mechanism of melanoma disease.At the same time,the B16 cell full protein nanoscale antibody library is established,which provides a basis for the follow-up research and development of B16 diagnosis and treatment of antibodies.1.Normal melanocytes and B16 cell transcriptome sequencing analysis:mRNA sequencing analysis using differential multiple(Fold Change)is greater than or equal to 2,and false detection rate(FDR<0.05)as a screening standard.The sequencing data were selected and the error rate of screening using the EBSeq software,there are 4086 genes,compared with normal melanocytes,differences in melanoma cell;KEGG database for annotation of 4086 genes,4086 genes found the difference belong to the 146 pathways;according to the annotation results of randomly selected 9 differentially with cancer related genes were verified by quantitative PCR(qRT-PCR)assay,and the results were consistent with the sequencing results;selected the relative genes of melanoma and melanoma cell migration and proliferation to performed q-RT-PCR and Western blotting assay to verify the expression.2.LncRNA sequencing analysis of normal melanocytes and B16 cells:the data after sequencing were assembled by StringTie.After data integration,gffcompare method was used to annotate and select possible IncRNAs.The lncRNA sequencing analysis used the difference multiplier(Fold Change)greater than 2 and the error detection rate(FDR<0.05)as the screening standard.The difference ratio and error rate were obtained 373 differentially expressed IncRNAs sequencing data from two samples of the Communist Party of China,one of the 136 IncRNAs expression in B16 cells was up-regulated,the expression of 237 IncRNAs in B16 cells was significantly reduced;quantitative PCR to detect the expression of 6 IncRNAs the most significant difference in the amount of test results,with the sequencing results of the same trend;two kinds of prediction methods(1ncRNA-cis-target and IncRNA-lucTar-target)of target gene prediction of 373 differentially expressed IncRNAs,were screened 467 potential target gene.The KEGG database of 467 notes to the target gene,we found that 43 IncRNAs and the target gene and the formation of melanin and melanoma pathway;screening theory can be found in the IncRNA-13317.1 targeting BRCA1,by double fluorescent reporter vector experiment confirmed this result,and the BRCAI gene in the repair process of cell injury after plays an important role in the Inc-13317.1 damage repair process in cells;cell proliferation experiments showed that overexpression of Inc-MSTRG.13317.1 can inhibit the proliferation of melanoma cells3.According to the sequencing data analysis found that the lnc-MSTRG.54238.1 abnormal expressed in B16 cells may be the precursor of miR-143-5p.In previous research,miRNA sequencing of normal mice and B16 cells shows that miR-143-5p plays a very important role in the proliferation of melanoma cells.MiRBase data was used to predict the target gene of miR-143-5p,the result showed TAK1 have a site which could can be combined with miRNA targets.According to the sequencing data anlysis found that TAK1 also was abnormal expressed in B16 cell.However,the mechanism of miR-143-5p in normal melanoma cells has not yet been studied.In this experiment,the luciferase assay was used to determine the targeted regulatory relationship between miR-143-5p and TAK1 Overexpressing miR-143-5p in alpaca normal melanocyte found that the melanin production of melanocytes in alpaca was significantly down-regulated.Cell proliferation assay showed that miR-143-5p could up-regulate the proliferation of melanocytes and promote the migration of melanocytes in alpaca.4.Repeated freeze-thaw and sonication of B16 cells total protein of B16 cells,immune adult alpaca,is used to construct B16 protein nano antibody cDNA phage display pCANTAB5e library technology,gradient dilution detection capacity of 5.76 x 1010,gradient dilution detection of the library abundance reached 3.65x1010/ml,randomly selected 50 monoclonal of cDNA insert the rate of detection results in 50 samples of only two samples not inserted VHH fragments of cDNA insert rate reached 96%,the above results showed that the constructed B16 protein nano antibody library of good quality,can be used in the subsequent development of B16 nano antibody cellsConclusion:In this study,the difference expression of mRNA and IncRNA between normal melanocytes and B16 cells were analyzed by deep sequencing and the biological functions of miR-143-5p and Inc-MSTRG.54238.1 were verified by molecular biology experiments;The whole protein nano-antibody library of B16 cells was constructed by phage display technology,which laid a foundation for the subsequent development of B16 nano-antibody.