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. 2013 Dec;7(6):1043-55.
doi: 10.1016/j.molonc.2013.08.003. Epub 2013 Aug 16.

Tumor-targeting TRAIL expression mediated by miRNA response elements suppressed growth of uveal melanoma cells

Jia Liu  1 , Leina Ma, Caixin Li, Ziyu Zhang, Guanghua Yang, Wenwei Zhang
Affiliations

Tumor-targeting TRAIL expression mediated by miRNA response elements suppressed growth of uveal melanoma cells

Jia Liu et al. Mol Oncol. 2013 Dec.

Abstract

Malignant uveal melanoma severely damages eye function and is prone to metastasize to other organs. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising agent to treat uveal melanoma because of its induction of apoptosis in cancer cells both at primary and metastatic sites. However, TRAIL therapy lacks tumor specificity in the current delivery systems for uveal melanoma treatment, thereby causing cytotoxiciy to normal tissues. To improve uveal melanoma specificity of adenovirus-based TRAIL introduction, we used miRNA response elements (MREs) of miR-34a, miR-137 and miR-182, which have been shown to have reduced expression in uveal melanoma cells, to regulate its expression. miR-34a, miR-137 and miR-182 all had lower expression levels in uveal melanoma cell lines, compared with normal cells. MREs-regulated luciferase activity was reduced in normal cell lines, but not significantly attenuated in uveal melanoma cells. The infection of MRE-regulated TRAIL-expressing adenoviral vector (Ad-TRAIL-3MREs) led to high level of TRAIL expression in uveal melanoma cell lines, but not in normal cells. Strong expression of TRAIL had a high anti-tumor capacity by inducing apoptosis in uveal melanoma cells. In contrast, Ad-TRAIL-3MREs had no cytotoxicity to normal cell lines. Animal experiments further confirmed tumor-suppressing effect of Ad-TRAIL-3MREs on uveal melanoma xenografts and its biosafety to hepatic tissues. Collectively, we constructed an MRE-directed TRAIL-expressing adenoviral vector and provided evidence that this vector possessed high anti-tumor activity and uveal melanoma specificity.

Keywords: Adenovirus; TRAIL; Uveal melanoma; miRNA.

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Figures

Figure 1
Figure 1
Uveal melanoma‐specific gene expression due to MREs of miR‐34a, miR‐137 and miR‐182. (A) Northern blotting was performed to detect the abundance of miR‐34a, miR‐137 and miR‐182 in ARPE‐19, L‐02, SP6.5 and OM431 cell lines. U6 was used as a loading control. (B) The levels of miR‐34a, miR‐137 and miR‐182 were also detected by qPCR in uveal melanoma and normal cell lines. Means ± SD of three independent experiments were shown. (C) Luciferase activity was detected 48 h after psiCheck2, psiCheck2‐miR‐34a, psiCheck2‐miR‐137 and psiCheck2‐miR‐182 transfection of normal and uveal melanoma cell lines. These experiments were performed in triplicate and means ± SD was presented. (D) Cells were also transfected with psiCheck2‐3MREs, followed by quantification of luciferase activity.
Figure 2
Figure 2
MREs‐regulated and adenovirus‐mediated TRAIL expression exhibited uveal melanoma specificity. (A) 2 copies of miR‐34a, miR‐137 and miR‐182 MREs were inserted immediately after the stop codon of a TRAIL‐coding open reading frame on a replication‐defect adenoviral vector Ad‐TRAIL to generate Ad‐TRAIL‐3MREs. Ad‐EGFP was used as a control. (B) Normal cells (ARPE‐19 and L‐02) and uveal melanoma cells (SP6.5 and OM431) were treated with Ad‐EGFP, Ad‐TRAIL and Ad‐TRAIL‐3MREs of 10 MOI. Proteins were extracted 48 h after adenovirus infection and detected for TRAIL expression with GAPDH as loading control. (C) Total RNA was also extracted from the same cells. TRAIL mRNA was subsequently quantified by qPCR with GAPDH as endogenous reference. (D) The production of excreted TRAIL protein was also evaluated in the same cells by ELISA assay. qPCR and ELISA assays were both repeated in triplicate and means ± SD were shown. (E) L‐02 and SP6.5 cells were transfected with mixed miRNA mimics or control (30 nM), followed by indicated adenovirus infection (10 MOI). 48 h later, TRAIL expression was determined with GAPDH as endogenous reference by immunoblotting.
Figure 3
Figure 3
Ad‐TRAIL‐3MREs reduced the survival of uveal melanoma cells without significant cytotoxicity to normal cells. ARPE‐19, L‐02, SP6.5 and OM431 cells were transduced with Ad‐TRAIL, Ad‐TRAIL‐3MREs or Ad‐EGFP at indicated MOIs. The viability was measured by MTT assay 7 days after adenovirus infection. Means ± SD of three independent experiments were shown. ** P < 0.01.
Figure 4
Figure 4
Ad‐TRAIL‐3MREs infection induced apoptosis in uveal melanoma cells. (A) Cleaved fragments of caspase 3, 8 and 9, and PARP, as well as truncated Bid, was detected by immunoblotting assays, in order to confirm the activation of apoptotic pathway. GAPDH was selected as endogenous controls. (B) Apoptotic rates were determined in ARPE‐19, L‐02, SP6.5 and OM431 cells 48 h after infection with Ad‐TRAIL, Ad‐TRAIL‐3MREs or Ad‐EGFP of 10 MOI by FACS‐based detection of Annexin V positivity. Means ± SD of three separate experiments were represented. (C) Apoptosis was detected in L‐02 and SP6.5 cells with Hoechst 33342 staining (×200). Arrows indicated the apoptotic cells with nuclear condensation.
Figure 5
Figure 5
Ad‐TRAIL‐3MREs suppressed the growth of uveal melanoma xenotransplants by inducing apoptosis. (A) 5 × 106 SP6.5 uveal melanoma cells was inoculated into the flanks of male BALB/c nude mice (4 group; n = 6). 3 × 109 pfu of Ad‐TRAIL, Ad‐TRAIL‐3MREs, Ad‐EGFP or PBS were administrated via tail vein injection. The diameters of tumors were periodically measured until Day 35. Means ± SD of tumor sizes were shown. (B) The representative photographs of the SP6.5 tumor‐bearing mice treated with Ad‐TRAIL, Ad‐TRAIL‐3MREs, Ad‐EGFP or PBS were shown. The arrows indicated the tumors. (C) TRAIL expression was investigated in the tumor samples from the mice treated with indicated adenoviruses by immunohistological staining. TUNEL assay was also performed to detect the apoptotic events in tumors. The representative images were shown (×200). White arrows indicated apoptotic cells. (D) Immunoblotting assay showed the expression of TRAIL and apoptotic pathway‐related proteins, including cleaved caspase 3 and PARP, in the tumor xenografts. GAPDH was used as loading control.
Figure 6
Figure 6
Ad‐TRAIL‐3MREs prevented liver tissue from cytotoxicity in mice. (A) TRAIL expression was investigated in liver tissues of the mice treated with indicated adenoviruses or PBS by immunohistological staining. The representative images were shown (×200). (B) Immunoblotting assay showed the expression of TRAIL and apoptotic pathway‐related proteins, including cleaved caspase 3 and PARP, in liver tissues of tumor‐bearing mice treated by adenoviruses. GAPDH was used as loading control. (C) 3 × 109 pfu of indicated adenoviruses or PBS was intravenously injected into 20 tumor‐free mice (4 group; n = 5). Subsequently, their blood was harvested to evaluate ALT levels. Means ± SD were shown.
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