Identification of miR-200a-5p targeting the peptide transporter TAP1 and its association with the clinical outcome of melanoma patients

Figures & data

Figure 1. Characterization of the expression of HLA-I APM components in melanoma cell lines.

Melanoma cell lines (n = 42) have been characterized for the expression of the different HLA-I APM components TAP1, TAP2, TPN, β₂-m and HLA-ABC at the mRNA (A) and protein level (B) by RT-qPCR and Western blot analysis, respectively. An APM score was calculated by adding the expression levels of the 5 APM components upon normalization to their expression levels in melanocytes. Surface expression of HLA-ABC was determined by flow cytometry (C). Shown are the values for each individual cell line (left) as well as a pie chart providing the percentage of high, intermediate and low expressors (right).

Figure 2. Correlation of TAP1 and HLA-A expression with the survival probability in metastatic melanoma patients.

Kaplan Meier estimation curves for survival probability of individual metastatic melanoma patients based on the expression of TAP1 (A) and HLA-A (B) were generated by using the “R2: Tumor Melanoma – Joehnsson – 214 – custom – ilmnht12v4“ dataset. Shown are the curves both for disease specific (left) and distant metastasis free survival probability (right) for the two genes. The raw p-values were based on log-rank tests and calculated for every graph with the web database (http://r2.amc.nl). (C) Correlation of TAP1 and HLA-A mRNA expression in the metastatic melanoma patients using the same dataset.

Figure 3. Identification of miR-200a-5p interaction with the 3ʹ-UTR of TAP1.

(A) Graphic representation of the TAP1 3ʹ-UTR (NM_000593.5) with the in silico predicted binding site for miR-200a-5p (red box). (B) Sequence alignment, secondary structure and free energy (mfe = −26.3 kcal/mol) for predicting the interaction of TAP1 3ʹ-UTR (red) and miR-200a-5p (green) were obtained using the free online data base RNAhybrid (https://bibiserv.cebitec.uni-bielefeld.de/rnahybrid). (C) The dual luciferase reporter assay was performed with HEK293T cells as described in Materials and Methods. Briefly, HEK293T cells were transfected with the miR-200a-5p or miR mimic negative control (NC) together with the plasmid encoding for the Firefly luciferase (FFL) cloned downstream the TAP1 3ʹ-UTR in its wild type form (TAP1 3ʹ-UTR wt) or upon deletion of the predicted binding site for miR-200a-5p (TAP1 3ʹ-UTR del). FFL activities were internally normalized to Renilla luciferase activities yielding relative light units (RLU). Shown are the mean ± SE from 3 to 6 independent experiments upon normalization to the miR mimic NC. * p < .05 in un-paired t-test. (D) Alignment of the TAP1 3ʹ-UTR wt and the TAP1 3ʹ-UTR del upon plasmid sequencing. The gray box highlights the deletion of the binding site of miR-200a-5p.

Figure 4. Basal expression of miR-200a-5p and TAP1 in different melanoma cell lines.

The different melanoma cell lines were analyzed by RT-qPCR for the expression of miR-200a-5p and TAP1. Shown are the expression levels upon normalization to melanocytes as well as the linear regression equation and its R².

Figure 5. Effect of miR-200a-5p overexpression on TAP1 expression in HEK293T cells.

HEK293T cells were either left untreated (parental) or transiently transfected with 2 μg/mL empty pmRm-cherry vector (mock) or with miR-200a-5p plasmid (miR-200a-5p). After 48 h miR-200a-5p overexpression in absolute copy number (A) as well as the mRNA expression levels of TAP1 were determined by RT-qPCR (B). Protein expression was evaluated by Western blot (C) and for quantification of the results, the relative band density (A.U., arbitrary units) of transfectants was calculated to the respective parental cells and normalized to GAPDH expression (D). Shown are the normalized mean ± SE from a minimum of three different biological replicates, *p < .05 in un-paired t-test.

Figure 6. Effect of miR-200a-5p overexpression on the expression of HLA-I APM components in melanoma cell lines.

BUF1379, FM3 and FM81 melanoma cells were left untreated (parental) or transiently transfected with RNAimax alone (control) or together with 30 nM miR mimic negative control (NC) or miR-200a-5p mimic (miR-200a-5p). After 48 h miR-200a-5p overexpression (A) as well as the mRNA expression levels of the indicated APM components were determined by RT-qPCR (B – F). Protein expression was evaluated by Western blot (G – H) and flow cytometry (I – K). For quantification of Western blot results, the relative band density (A.U., arbitrary units) of transfectants was calculated to the respective parental melanoma cells and normalized to GAPDH expression. Shown are the normalized mean ± SE from a minimum of 3 different biological replicates and one representative Western blot, *p < .05, **p < .01, ***p < .001 in un-paired t-test.

Figure 7. Increased recognition of miR-200a-5p overexpressing FM3 melanoma cells by NK cells.

NK cells from healthy donors were co-incubated with FM3 cells, either left untreated (parental), transfected with the negative control (NC) or with miR-200a-5p mimics. After 4 h the percentage of CD107a expressing NK cells was determined by flow cytometry. Shown are the normalized mean ± SE from 3 independent experiments. *p < .05 in paired t-test.

Figure 8. Inverse correlation of miR-200a-5p expression with TAP1 levels and survival probability in melanoma patients.

(A-D) Paraffin-embedded tissue sections from 26 primary melanoma patients were analyzed for miR-200a-5p expression levels by RT-qPCR and scored for TAP1, CD8 and CD163 expression as high or low by immunohistochemical staining. Comparison between TAP1 high (n = 16) and TAP1 low (n = 10) melanoma lesions is shown for miR-200a expression (A) and CD8⁺ (B) and CD163⁺ cell infiltration (C). An overview of TAP1, CD8 (left y-axis) and miR-200a-5p expression (right y-axis) for each individual patient among the TAP1 high (left) and TAP1 low (right) group is provided (D). (E-G) The “R2: Tumor Melanoma – Jöhnsson – 214 – custom – ilmnht12v4“ dataset was employed to evaluate the correlation between miR-200a expression and patients’ disease specific survival probability by Kaplan Meier estimation curve (E) as well as with TAP1 (F) and HLA-A expression (G) The raw p-values were based on log-rank tests and calculated for every graph with the web database. (A: *p < .05 in paired t-test, B: *p < .05 in Fisher’s exact test)