1. IMP-3 Promotes Migration and Invasion of Melanoma Cells by Modulating the Expression of HMGA2 and Predicts Poor Prognosis in Melanoma 
Yi-Shuan Sheen, Yi-Hua Liao, Ming-Hsien Lin, Chia-Ying Chu, Bing-Ying Ho, Meng-Chen Hsieh, Pin-Chun Chen, Shih-Ting Cha, Yung- Ming Jeng, Cheng-Chi Chang, Hsien-Ching Chiu, Shiou-Hwa Jee, Min-Liang Kuo, Chia-Yu Chu 
Journal of Investigative Dermatology 
Volume 135, Issue 4, Pages (April 2015)
DOI: /jid
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2. Figure 1
IGF II mRNA-binding protein 3 (IMP-3) is expressed in malignant melanoma and is correlated with its progression and prognosis. Benign melanocytic nevi such as intradermal nevus (a) or dysplastic nevus (b) are negative for IMP-3. (c) Melanoma in situ has only weak cytoplasmic expression of IMP-3. (d) Focal IMP-3 expression found in superficial malignant melanoma (Breslow depth ≦1 mm). (e) Strong and diffuse cytoplasmic expression noted in deep malignant melanoma (Breslow depth>1 mm). (f) Metastatic melanoma expresses IMP-3 in >90% of tumor cells (bar=10 μm). (g) Poor prognosis in cutaneous melanoma is predicted by IMP-3 expression. Kaplan–Meier survival analysis of 97 primary cutaneous melanomas revealed that patients with positive IMP-3 expression have significantly worse overall survival compared with those negative for IMP-3 (P=0.001, log-rank test).
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

3. Figure 2
IGF II mRNA-binding protein 3 (IMP-3) enhanced aggressiveness of melanoma cells. (a) Melanoma cells with higher migratory/invasive abilities demonstrated higher expression of IMP-3. (b) Transient transfection of small interfering RNA (siRNA) against IMP-3 inhibited the migratory/invasive abilities of A2058 cells. (c) MeWo/IMP-3 cells showed enhanced migratory abilities. (d) Overexpression of IMP-3 in MeWo cells had significant change on cell proliferation. *P<0.05, n=3. (e) MeWo/IMP-3 xenografts were larger compared with MeWo/vec at weeks 2–6. (f) MeWo/IMP-3 cells resulted in larger popliteal lymph node (LN) metastasis compared with the MeWo/vec cells (n=10). (g) Upper panel shows representative HMB-45 immunostaining for lymph nodes taken from the two groups. The MeWo/IMP-3 group had positive HMB-45 staining in all collected 10 lymph nodes, whereas only 2 of the 10 mice in MeWo/vec group had positive staining. T, tumor; Vec, vector.
Journal of Investigative Dermatology  , DOI: ( /jid )
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4. Figure 3
HMGA2, c-myc, and TGF-β2 mRNAs are potential RNA targets for IGF II mRNA-binding protein 3 (IMP-3). (a) Six upregulated genes in MeWo/IMP-3 cells are known to be involved in migration/invasion/metastasis. (b) Quantitative real-time reverse-transcriptase–PCR analysis found that IMP-3 overexpression in MeWo cells was associated with elevated HMGA2, c-myc, and TGF-β2 mRNA levels relative to control cells. (c) Western blot analysis showed elevated HMGA2, c-myc, and TGF-β2 protein levels in MeWo/IMP-3 cells compared with MeWo/vec cells. CCL20, chemokine (C–C motif) ligand 20; HMGA2, high mobility group AT-hook 2; TES-85, leucine zipper protein 4/CT-8/HOM-TES-85; TGF-β2, transforming growth factor-β2; TGM2, transglutaminase 2; WISP2, Wnt-1-inducible signaling pathway protein-2. Data are presented as relative changes to the control and are shown as mean±SD of three independent experiments. *Student’s t-test, P<0.05.
Journal of Investigative Dermatology  , DOI: ( /jid )
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5. Figure 4
HMGA2 is the key mRNA that regulated by IGF II mRNA-binding protein 3 (IMP-3) and correlated with migratory/invasive abilities of melanoma cells. (a) Anti-human IMP-3 antibody precipitated HMGA2, c-myc, and TGF-β2 mRNAs, whereas the control IgG did not precipitate these RNAs. Real-time quantitative real-time reverse-transcriptase–PCR-based quantitation found a 32.4-fold enrichment of HMGA2 mRNA in A2058 cells upon immunoprecipitation with anti-IMP-3. (b) A2058 cells with high IMP-3 expression demonstrated increased high mobility group AT-hook 2 (HMGA2) expression compared with MeWo cells. Compared with MeWo cells, A2058 cells did not express higher c-myc, TGF-β2, CCL20, and WISP2 mRNA levels. (c) Overexpression of IMP-3 in MeWo cells did not change intracellular or secreted IGF-2 levels. (d) IMP-3 overexpression had no significant effect on IGF-2 leader-3 or leader-4 mRNA levels compared with the mock treatment. *P<0.05, n=3.
Journal of Investigative Dermatology  , DOI: ( /jid )
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6. Figure 5
HMGA2 is involved in IGF II mRNA-binding protein 3 (IMP-3)-mediated migration/invasion of melanoma cells. (a) Co-transfection of shRNA for HMGA2 (shHMGA2) with IMP-3-expressing plasmid knocked down the high mobility group AT-hook 2 (HMGA2) expression and also (b) attenuated the IMP-3-mediated melanoma migration. (c) Co-transfection of HMGA2-expressing plasmid with IMP-3 siRNA (siIMP-3) in A2058 cells enhanced HMGA2 expression, which also (d) restored the inhibitory effects of siIMP-3 on A2058 migration/invasion. Immunohistochemical examination of human melanoma tissues demonstrated that tumor cells with high IMP-3 levels (e) also had high HMGA2 expression (f), whereas those with low IMP-3 levels (g) had weak or negative HMGA2 expression (h). Bar=5 μm. Data are presented as relative changes to the control and are shown as mean±SD of three independent experiments. *Student’s t-test, P<0.05. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Scr, scramble; vec, vector.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions