Long Noncoding RNA ZFAS1 Promotes Progression of Oral Squamous Cell Carcinoma Through Targeting miR-6499-3p/CCL5 Axis

Abstract

Background: Oral squamous cell carcinoma (OSCC) ranks sixth among malignancies in the world, and there are 200,000 new cases annually, rendering OSCC a significant global public health issue that has caused great burdens on patients, society, and the economy. Despite great progress in diagnosis and treatment methods, patient survival has not been greatly enhanced. Hence, there is an urgent need to identify novel targets that can serve as early diagnostic and therapeutic biomarkers for OSCC. Long non-coding RNAs (lncRNAs) participate in several cancer types, including OSCC. This work identified the competing endogenous RNA network related to lncRNA zinc finger nuclear transcription factor, X-box binding 1-type containing 1 antisense RNA 1 (ZFAS1) in OSCC, as well as the corresponding downstream targets.

Materials and methods: Firstly, we identified the lncRNA ZFAS1 levels in OSCC cells and tissues and confirmed its relationship to tumor progression. Secondly, we identified a lncRNA-miRNA-mRNA network, which was closely associated with OSCC development using bioinformatics methods. Next, our hypothesis that lncRNA ZFAS1 modulates OSCC progression was verified with in vitro and in vivo experiments.

Results: Firstly, we found lncRNA ZFAS1 expression increased within OSCC cells and tissues and was positively associated with tumor progression. Secondly, its lncRNA-miRNA-mRNA network was determined, and the target of ZFAS1 was identified as miR-6499-3p/C-C motif chemokine ligand 5 (CCL5). Mechanistically, we found that ZFAS1 up-regulated CCL5 by competitively sponging miR-6499-3p. Further studies demonstrated that ZFAS1 promoted tumor progression in vivo and in vitro.

Conclusion: Our results indicate that ZFAS1 serves as a crucial oncogenic factor in OSCC occurrence and development and may therefore serve as a possible therapeutic target for OSCC.

Keywords: CCL5; Oral squamous cell carcinoma; ZFAS1; ceRNA; lncRNA.

Figure 1. Long non-coding RNA (lncRNA) zinc finger nuclear transcription factor, X-box binding 1-type containing 1 antisense RNA 1 (ZFAS1) is up-regulated in oral squamous cell carcinoma (OSCC) tissues and cell lines. A: mRNA expression of lncRNA ZFAS1 was elevated in tumor tissues relative to adjacent non-carcinoma tissues from patients with OSCC. B: mRNA expression of ZFAS1 was elevated in OSCC cell lines compared to normal human oral keratinocytes (NHOK). Significantly different at: **p<0.01 and ***p<0.001.

Figure 2. A: In oral squamous cell carcinoma (OSCC) cells, small-interfering Iong non-coding RNA (si-lncRNA) zinc finger nuclear transcription factor, X-box binding 1-type containing 1 antisense RNA 1 (ZFAS1) down-regulated ZFAS1 expression in comparison with si-negative control (NC). B: si-lncRNA ZFAS1 markedly inhibited the proliferation of TSCCA and CAL-27 cells. C: ZFAS1 inhibits clone formation of OSCC cancer cells. D: si-lncRNA ZFAS1 suppressed migration of OSCC cells. E: si-lncRNA ZFAS1 suppressed invasion of OSCC cells. **Significantly different at p<0.01.

Figure 3. A: Predicted binding sites for long non-coding RNA (lncRNA) zinc finger nuclear transcription factor, X-box binding 1-type containing 1 antisense RNA 1 (ZFAS1), and miR-6499-3p using the DIANA bioinformatics tool (http://diana.imis.athena-innovation.gr/DianaTools/index.php). B: Relative luciferase activity in luciferase reporter plasmids, which included mutant-type (Mut) and wild-type (WT) pGL3-ZFAS1-3'UTR. Activity in pGL3-ZFAS1-Mut-transfected cells was not suppressed by miR-6499-3p. C: There was much more accumulated ZFAS1 binding to Argonaute 2 (AGO2) protein in the miR-6499-3p mimic group. D: Expression of miRNA-6499-3p was up-regulated in si-lncRNA ZFAS1 cells. E: The predicted binding site for C-C motif chemokine ligand 5 (CCL5) and miR-6499-3p by bioinformatics (http://www.targetscan.org). F: Relative luciferase activity in pGL3-CCL5-Mut-transfected cells was not suppressed by miRNA-6499-3p. G: and H: In cells transfected with miRNA-6499-3p mimic, expression of both CCL5 RNA and protein were reduced. Significantly different at: **p<0.01 and ***p<0.001.

Figure 4. A: miRNA-6499-3p inhibitor and C-C motif chemokine ligand 5 (CCL5) vector reduced the inhibition of proliferation under silencing of long non-coding RNA (lncRNA) zinc finger nuclear transcription factor, X-box binding 1-type containing 1 antisense RNA 1 (ZFAS1) with small interfering RNA (si-RNA). Effects of miRNA-6499-3p inhibitor and CCL5 vector on the inhibitory effect of lncRNA ZFAS1 silencing in colony formation (B), migration (C), and invasion (D) by cells. Significantly different at: *p<0.05, **/^##p<0.01 and ***p<0.001.

Figure 5. In vivo tumorigenicity assay in BALB/c mice injected in the right-back with CAL-27 cells with stable transfection of long non-coding RNA (lncRNA) zinc finger nuclear transcription factor, X-box binding 1-type containing 1 antisense RNA 1 (ZFAS1)-silencing (si) or control (NC) retroviral vectors. A: lncRNA ZFAS1-knockdown slowed tumor growth in vivo. B: LncRNA ZFAS1-knockdown reduced the terminal tumor volume. C: miR-6499-3p and C-C motif chemokine ligand 5 (CCL5) levels in tumor tissues. Significantly different at: *p<0.05 and **p<0.01.