Dataset Information

Identification of microRNAs Targeting the Transporter Associated with Antigen Processing TAP1 in Melanoma.

ABSTRACT: The underlying molecular mechanisms of the aberrant expression of components of the HLA class I antigen processing and presentation machinery (APM) in tumors leading to evasion from T cell-mediated immune surveillance could be due to posttranscriptional regulation mediated by microRNAs (miRs). So far, some miRs controlling the expression of different APM components have been identified. Using in silico analysis and an miR enrichment protocol in combination with small RNA sequencing, miR-26b-5p and miR-21-3p were postulated to target the 3' untranslated region (UTR) of the peptide transporter TAP1, which was confirmed by high free binding energy and dual luciferase reporter assays. Overexpression of miR-26b-5p and miR-21-3p in melanoma cells downregulated the TAP1 protein and reduced expression of HLA class I cell surface antigens, which could be reverted by miR inhibitors. Moreover, miR-26b-5p overexpression induced a decreased T cell recognition. Furthermore, an inverse expression of miR-26b-5p and miR-21-3p with TAP1 was found in primary melanoma lesions, which was linked with the frequency of CD8+ T cell infiltration. Thus, miR-26-5p and miR-21-3p are involved in the HLA class I-mediated immune escape and might be used as biomarkers or therapeutic targets for HLA class Ilow melanoma cells.

SUBMITTER: Lazaridou MF

PROVIDER: S-EPMC7563967 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

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Identification of microRNAs Targeting the Transporter Associated with Antigen Processing TAP1 in Melanoma.

Lazaridou Maria-Filothei MF Massa Chiara C Handke Diana D Mueller Anja A Friedrich Michael M Subbarayan Karthikeyan K Tretbar Sandy S Dummer Reinhard R Koelblinger Peter P Seliger Barbara B

Journal of clinical medicine 20200820 9

The underlying molecular mechanisms of the aberrant expression of components of the HLA class I antigen processing and presentation machinery (APM) in tumors leading to evasion from T cell-mediated immune surveillance could be due to posttranscriptional regulation mediated by microRNAs (miRs). So far, some miRs controlling the expression of different APM components have been identified. Using in silico analysis and an miR enrichment protocol in combination with small RNA sequencing, miR-26b-5p a ...[more]

PMID: 32825219

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Project description:Transporter associated with antigen processing 1 (TAP1) is a transporter protein that represent tumor antigen in the MHC I or HLA complex. Any defect in the TAP1 gene resulting in inadequate tumor tracking. TAP1 influences multidrug resistance (MDR) in human cancer cell lines and hinders the treatment during chemotherapeutic. The association of TAP1 in cancer progression remains mostly unknown and further study of the gene in relation with cancer need to conduct. Thus, the study has designed to analyze the association between the TAP1 with cancer by computationally. The expression pattern of the gene has determined by using ONCOMINE, GENT2, and GEPIA2 online platforms. The protein level of TAP1 was examined by the help of Human Protein Atlas. Samples with different clinical outcomes were investigated to evaluate the expression and promoter methylation in cancer vs. normal tissues by using UALCAN server. The copy number alteration, mutation frequency, and expression level of the gene in different cancer were analyzed by using cBioPortal server. The PrognoScan and KM plotter platforms were used to perform the survival analysis and represented graphically. Additionally, pathway and gene ontology (GO) features correlated to the TAP1 gene were analyzed and presented by bar charts. After arranging the data in a single panel like correlating expression to prognosis, mutational and alterations characteristic, and pathways analysis, we observed some interesting insights that emphasized the importance of the gene in cancer progression. The study found the relationship between the TAP1 expression pattern and prognosis in different cancer tissues and shows how TAP1 affects the clinical characteristics. The analytical data presented in the study is vital to learn about the effect of TAP1 in tumor tissue, where previously studies showing contradicting expression of TAP1 in cancer tissue. The analyzed data can also be utilized further to evade the threats against chemotherapy. Overall, the study provided a new aspect to consider the role of TAP1 gene in cancer progression and survival status. KEY MESSAGES: • This study demonstrated, for the first time, a correlation between the TAP1 gene and tumor progression. • An upregulation of TAP1 mRNA was demonstrated in various cancer types. • This study reported a significant negative correlation for TAP1 gene expression and the survival rate in different cancer types.

Project description:Tumor escape is often associated with abnormalities in the surface expression of the human leukocyte antigen class I (HLA-I) antigens thereby limiting CD8+ cytotoxic T cell responses. This impaired HLA-I surface expression can be mediated by deficient expression of components of the antigen processing and presentation machinery (APM) due to epigenetic, transcriptional and/or post-transcriptional processes. Since a discordant mRNA and protein expression pattern of APM components including the peptide transporter associated with antigen processing 1 (TAP1) has been frequently described in tumors of distinct origin, a post-transcriptional control of APM components caused by microRNAs (miR) was suggested. Using an in silico approach, miR-200a-5p has been identified as a candidate miR binding to the 3' untranslated region (UTR) of TAP1. Luciferase reporter assays demonstrated a specific binding of miR-200a-5p to the TAP1 3'-UTR. Furthermore, the miR-200a-5p expression is inversely correlated with the TAP1 protein expression in HEK293T cells and in a panel of melanoma cell lines as well as in primary melanoma lesions. High levels of miR-200a-5p expression were associated with a shorter overall survival of melanoma patients. Overexpression of miR-200a-5p reduced TAP1 levels, which was accompanied by a decreased HLA-I surface expression and an enhanced NK cell sensitivity of melanoma cells. These data show for the first time a miR-mediated control of the peptide transporter subunit TAP1 in melanoma thereby leading to a reduced HLA-I surface expression accompanied by an altered immune recognition and reduced patients' survival.AbbreviationsAb: antibody; ACTB: β-actin; APM: antigen processing and presentation machinery; ATCC: American tissue culture collection; β2-m: β2-microglobulin; BSA: bovine serum albumin; CTL: cytotoxic T lymphocyte; FCS: fetal calf serum; FFL: firefly luciferase; FFPE: formalin-fixed paraffin-embedded; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; HC: heavy chain; HLA: human leukocyte antigen; HLA-I: HLA class I; HRP: horseradish peroxidase; IFN: interferon; im-miR: immune modulatory miRNA; LMP: low molecular weight protein; luc: luciferase; MFI: mean fluorescence intensity; MHC: major histocompatibility complex; miR: microRNA; NC: negative control; NK: natural killer; NSCLC: non-small cell lung carcinoma; OS: overall survival; PBMC: peripheral blood mononuclear cells; RBP: RNA-binding proteins; RL: Renilla; RLU: relative light units; TAP: transporter associated with antigen processing; tpn: tapasin; UTR: untranslated region.

Project description:BackgroundAutoimmunity has been implicated in the destruction of melanocytes from vitiligo skin. Major histocompatibility complex (MHC) class-II linked genes proteasome subunit beta 8 (PSMB8) and transporter associated with antigen processing 1 (TAP1), involved in antigen processing and presentation have been reported to be associated with several autoimmune diseases including vitiligo.ObjectivesTo explore PSMB8 rs2071464 and TAP1 rs1135216 single nucleotide polymorphisms and to estimate the expression of PSMB8 and TAP1 in patients with vitiligo and unaffected controls from Gujarat.MethodsPSMB8 rs2071464 polymorphism was genotyped using polymerase chain reaction- restriction fragment length polymorphism (PCR-RFLP) and TAP1 rs1135216 polymorphism was genotyped by amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) in 378 patients with vitiligo and 509 controls. Transcript levels of PSMB8 and TAP1 were measured in the PBMCs of 91 patients and 96 controls by using qPCR. Protein levels of PSMB8 were also determined by Western blot analysis.ResultsThe frequency of 'TT' genotype of PSMB8 polymorphism was significantly lowered in patients with generalized and active vitiligo (p = 0.019 and p = 0.005) as compared to controls suggesting its association with the activity of the disease. However, TAP1 polymorphism was not associated with vitiligo susceptibility. A significant decrease in expression of PSMB8 at both transcript level (p = 0.002) as well as protein level (p = 0.0460) was observed in vitiligo patients as compared to controls. No significant difference was observed between patients and controls for TAP1 transcripts (p = 0.553). Interestingly, individuals with the susceptible CC genotype of PSMB8 polymorphism showed significantly reduced PSMB8 transcript level as compared to that of CT and TT genotypes (p = 0.009 and p = 0.003 respectively).ConclusionsPSMB8 rs2071464 was associated with generalized and active vitiligo from Gujarat whereas TAP1 rs1135216 showed no association. The down-regulation of PSMB8 in patients with risk genotype 'CC' advocates the vital role of PSMB8 in the autoimmune basis of vitiligo.

Project description:IntroductionInterferon alpha (IFN?) is routinely used in the clinical practice for adjuvant systemic melanoma therapy. Understanding the molecular mechanism of IFN? effects and prediction of response in the IFN? therapy regime allows initiation and continuation of IFN? treatment for responder and exclusion of non-responder to avoid therapy inefficacy and side-effects. The transporter protein associated with antigen processing-1 (TAP1) is part of the MHC class I peptide-loading complex, and important for antigen presentation in tumor and antigen presenting cells. In the context of personalized medicine, we address this potential biomarker TAP1 as a target of IFN? signalling.ResultsWe could show that IFN? upregulates TAP1 expression in peripheral blood mononuclear cells (PBMCs) of patients with malignant melanoma receiving adjuvant high-dose immunotherapy. IFN? also induced expression of TAP1 in mouse blood and tumor tissue and suppressed the formation of melanoma metastasis in an in vivo B16 tumor model. Besides its expression, TAP binding affinity and transport activity is induced by IFN? in human monocytic THP1 cells. Furthermore, our data revealed that IFN? clearly activates phosphorylation of STAT1 and STAT3 in THP1 and A375 melanoma cells. Inhibition of Janus kinases abrogates the IFN?-induced TAP1 expression. These results suggest that the JAK/STAT pathway is a crucial mediator for TAP1 expression elicited by IFN? treatment.ConclusionWe suppose that silencing of TAP1 expression provides tumor cells with a mechanism to escape cytotoxic T-lymphocyte recognition. The observed benefit of IFN? treatment could be mediated by the shown dual effect of TAP1 upregulation in antigen presenting cells on the one hand, and of TAP1 upregulation in 'silent' metastatic melanoma cells on the other hand. In conclusion, this work contributes to a better understanding of the mode of action of IFN? which is essential to identify markers to predict, assess and monitor therapeutic response of IFN? treatment in the future.

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Identification of microRNAs Targeting the Transporter Associated with Antigen Processing TAP1 in Melanoma.

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Identification of microRNAs Targeting the Transporter Associated with Antigen Processing TAP1 in Melanoma.

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