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. 2017 Aug 3;8(49):85401-85414.
doi: 10.18632/oncotarget.19910. eCollection 2017 Oct 17.

V体育官网 - Long non-coding RNA HOTAIR acts as a competing endogenous RNA to promote malignant melanoma progression by sponging miR-152-3p

Wenkang Luan  1 , Rubo Li  2 , Liang Liu  3 , Xin Ni  4 , Yan Shi  3 , Yun Xia  1 , Jinlong Wang  1 , Feng Lu  1 , Bin Xu  1
Affiliations

Long non-coding RNA HOTAIR acts as a competing endogenous RNA to promote malignant melanoma progression by sponging miR-152-3p

Wenkang Luan (V体育官网) et al. Oncotarget. .

"V体育官网入口" Abstract

HOX transcript antisense RNA (HOTAIR) is associated with the growth and metastasis of many human tumors, but its biological roles in malignant melanoma remain unclear. In this study, we show that HOTAIR is overexpressed in melanoma tissues and cells, especially in metastatic melanoma. High HOTAIR levels correlate with poor prognosis in melanoma patients. We also determined that HOTAIR functions as a competing endogenous RNA (ceRNA) for miR-152-3p. miR-152-3p was decreased and acted as a tumor suppressor in melanoma, and c-MET was the functional target of miR-152-3p. Furthermore, HOTAIR promotes the growth and metastasis of melanoma cells by competitively binding miR-152-3p, which functionally liberates c-MET mRNA and results in the activation of the downstream PI3k/Akt/mTOR signaling pathway. We determined that HOTAIR acts as a ceRNA to promote malignant melanoma progression by sponging miR-152-3p VSports手机版. This finding elucidates a new mechanism for HOTAIR in melanoma development and provides a potential therapeutic target for melanoma patients. .

Keywords: HOTAIR; c-MET; ceRNA; malignant melanoma; miR-152-3p V体育安卓版. .

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Conflict of interest statement

CONFLICTS OF INTEREST The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1. HOTAIR levels are elevated in melanoma and association with poor prognosis of melanoma patients
(A) HOTAIR levels were analyzed in sixty malignant melanoma tissues and adjacent normal tissues. (B) The level of HOTAIR was analyzed in primary melanoma tissues and metastatic melanoma tissues. (C) The overall survival curves of melanoma patients with high HOTAIR expression and low HOTAIR expression. (D) The HOTAIR expression profile in human melanoma cell lines (A375, A875, SK-MEL-1, SK-MEL-5 and SK-MEL-28) and human epidermal melanocytes (HEMa-LP and HEMn-LP). *P < 0.05, **P < 0.01, ***P<0.001.
Figure 2
Figure 2. The biological functions of HOTAIR in melanoma
(A) Transfection efficiency of si-HOTAIR was determined by PCR. (B and C) The proliferative ability of A375 and A875 cells was measured by CCK8 assay after the cells were transfected with si-HOTAIR or NC. (D and E) The role of si-HOTAIR in inhibiting proliferation of melanoma cells was confirmed by colony formation assay. (F and G) The effect of si-HOTAIR on the migratory ability of melanoma cells was assessed by scratch wound assay. (H and I) The effect of si-HOTAIR on the invasive capacity of melanoma cells was assessed by transwell assay. (J) Western blot assays showed the levels of epithelial cell marker (E-cadherin) and the mesenchymal marker (N-cadherin) following transfection with si-HOTAIR or NC; GAPDH was used as a control. *P < 0.05, **P < 0.01, ***P<0.001.
Figure 3
Figure 3. Identification of miRNAs that bind to HOTAIR
(A) The expression levels of miR-152-3p in A375 and A875 cells following transfection with si-HOTAIR or NC. (B) miR-152-3p levels were analyzed in sixty malignant melanoma tissues and adjacent normal tissues. (C) The heat map shows that miR-152-3p is negatively correlated with HOTAIR in sixty malignant melanoma tissues. (D) The Pearson correlation of miR-152-3p and HOTAIR expression in sixty malignant melanoma tissues was negative. (E) The expression levels of HOTAIR in A375 and A875 cells following transfection with miR-152-3p mimic or NC. *P < 0.05, **P < 0.01, ***P<0.001.
Figure 4
Figure 4. HOTAIR directly binds to miR-152-3p
(A) The putative binding sites of miR-152-3p on the HOTAIR transcript, as predicted by starbase 2.0. (B) Over-expression of miR-152-3p led to a marked decrease in luciferase activity of pMIR-HOTAIR-WT, without any change in luciferase activity of pMIR-HOTAIR-MUT in melanoma cells. (C and D) Amount of HOTAIR bound to Ago2 or IgG measured by RT–qPCR after RIP. (E and F) Melanoma cells transfected with biotin-labeled miR-152-3p oligos (miR-152-3p-Bio) or mutated oligos (miR-152-3p-Bio-mut) or biotinylated NC (NC-Bio), assayed by biotin-based pull down after transfection. HOTAIR levels were analyzed by RT–qPCR. *P < 0.05, **P < 0.01, ***P<0.001.
Figure 5
Figure 5. c-MET is the functional target of miR-152-3p that affect proliferation, invasion and migration abilities of melanoma cells
(A) The miR-152-3p expression profile in human melanoma cell lines (A375, A875, SK-MEL-1, SK-MEL-5 and SK-MEL-28) and human epidermal melanocytes (HEMa-LP and HEMn-LP). (B) Transfection efficiency of miR-152-3p mimic was determined by PCR. (C and D) Effect of miR-152-3p on the proliferative ability of melanoma cells was assessed by CCK-8 assay, c-MET plasmid reversed the effect of miR-152-3p. (E and F) The effect of miR-152-3p on the migratory ability of melanoma cells was assessed by the scratch wound assay, the effect of miR-152-3p was largely abrogated by the c-MET plasmid. (G and H) The effect of miR-152-3p on the invasive capacity of melanoma cells was assessed by transwell assay, and c-MET plasmid reversed this effect. (I) The binding sites of miR-152-3p within the 3′-UTR of c-MET were predicted by miRanda. (J) Overexpression of miR-152-3p suppressed luciferase activity in melanoma cells with the pMIR-c-MET-WT, but did not cause a significant change in melanoma cells with the pMIR-c-MET-MUT. (K) The expression of c-MET mRNA in melanoma cells transfected with miR-152-3p or miR-152-3p combination with the c-MET plasmid. (L) Western blots identifed c-MET protein expression and its downstream PI3k/Akt/mTOR signaling pathway changes following transfection with miR-152-3p alone or in combination with c-MET. *P < 0.05, **P < 0.01, ***P<0.001.
Figure 6
Figure 6. HOTAIR promotes melanoma cell growth and metastasis by acting as a ceRNA
(A) Luciferase activity of indicated groups in melanoma cells. (B) The expression of miR-152-3p and c-MET mRNA in melanoma cells transfected with si-HOTAIR or si-HOTAIR combination with miR-152-3p inhibitor. (C) Western blots identifed c-MET protein expression and its downstream PI3k/Akt/mTOR signaling pathway changes following transfection with si-HOTAIR or si-HOTAIR combination with miR-152-3p inhibitor. (D and E) miR-152-3p inhibitor reversed the effect of si-HOTAIR on the proliferative ability of melanoma cells. (F and G) miR-152-3p inhibitor reversed the effect of si-HOTAIR on the migratory ability of melanoma cells. (H and I) The effect of si-HOTAIR on the invasive ability of melanoma cells was largely abrogated by miR-152-3p inhibitor. *P < 0.05, **P < 0.01, ***P<0.001.
Figure 7
Figure 7. Inhibition of endogenous HOTAIR expression by shRNA inhibits melanoma growth in vivo
(A) Tumor formation in nude mice and the excision tumor of A375 xenografts. (B) Difference in tumor volume between the NC group and the shRNA-HOTAIR group. (C) The tumor weight of excision tumor. (D) RT–qPCR identified miR-152-3p and HOTAIR expression changes. (E) The expression of c-MET, Ki-67, E-cadherin and N-cadherin was examined by Immunohistochemical staining of sections from melanoma xenograft model in nude mice. *P < 0.05, **P < 0.01, ***P<0.001.
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