CREB3 Transactivates lncRNA ZFAS1 to Promote Papillary Thyroid Carcinoma Metastasis by Modulating miR-373-3 p/MMP3 Regulatory Axis

Abstract

The incidence rate of thyroid carcinoma ranks ninth among human malignancies, and it accounts for the most frequent malignancy in endocrine-related tumors. This study aimed to investigate the role of long noncoding RNA (lncRNA) ZFAS1 in the metastasis of papillary thyroid carcinoma (PTC) and the potential molecular mechanisms. Both ZFAS1 and MMP3 were highly expressed in thyroid carcinoma and PTC cell, as measured by the q-PCR and TCGA database. In addition, ZFAS1 induced TPC-1 metastasis through inducing the epithelial-mesenchymal transition (EMT) process. Besides, ZFAS1 knockdown by siRNA induced miR-373-3p expression and reduced MMP3 expression, as quantified by q-PCR and Western blotting. According to the luciferase assay, both ZFAS1 and MMP3 were classified as the direct targets of miR-373-3p. However, MMP3 itself did not affect ZFAS1. Using the online prediction tool, CREB3 was predicted as the transcription factor (TF) of ZFAS1 that contained two binding sites on its promoter region, and CREB3 was positively correlated with ZFAS1 in thyroid carcinoma cohorts. Results from the dual-luciferase assay and ChIP-qPCR indicated that both the two binding sites were essential for the transcription of ZFAS1. In conclusion, CREB3 activated lncRNA ZFAS1 at the transcriptional level to promote PTC metastasis by modulating miR-373-3p/MMP3.

Figure 1

ZFAS1 expression in thyroid carcinoma. (a) ZFAS1 expression profile in primary tumor and normal tissues obtained from TCGA-THCA datasets (^∗∗∗p < 0.001). (b) ZFAS1 expression profile in paired primary tumor and normal tissues obtained from TCGA-THCA datasets (^∗∗∗p < 0.001). (c) ZFAS1 expression profile in different cancer stages in thyroid carcinoma obtained from TCGA-THCA datasets (^∗p < 0.05). (d) ZFAS1 expression profile in different nodal metastasis status of thyroid carcinoma obtained from TCGA-THCA datasets (^∗∗∗p < 0.001). (e) The relations between ZFAS1 expression and survival rate of THCA patients. The cut-off value is 50%–50% (high-low). (f) ZFAS1 expression was measured by q-PCR in TPC-1 cells and Nthy-ori- 3-1 cells (^∗∗p < 0.01).

Figure 2

The effect of ZFAS1 on TPC-1 metastasis. (a) EMT markers were tested by q-PCR in TPC-1 cells and Nthy-ori 3–1 cells (^∗∗p < 0.01). (b) ZFAS1 was knocked down by siRNA (^∗∗p < 0.01). (c) The effect of ZFAS1 siRNA (siZFAS1) on EMT markers in TPC-1 cells tested by q-PCR (^∗∗p < 0.01). (d) Invasion ability of TPC-1 cells were measured after transfected with siZFAS1 and quantified with cell numbers (^∗∗p < 0.01).

Figure 3

Target confirmed between ZFAS1 and miR-373-3p. (a) ZFAS1 was predicted as a direct target of miR-373-3p. (b) The effect of miR-373-3p on ZFAS1 expression in TPC-1 cell was tested by q-PCR (^∗∗p < 0.01). (c) Target between ZFAS1 and miR-373-3p was confirmed by luciferase assay (^∗∗p < 0.01). (d) The effect of miR-373-3p and miR-373-3p/pcD-ZFAS1 cotransfection on EMT markers in TPC-1 cells tested by q-PCR (^∗∗p < 0.01). (e) The effect of ZFAS1 overexpression on miR-373-3p expression level tested by q-PCR (^∗∗p < 0.01).

Figure 4

ZFAS1 modulated miR-373-3p/MMP3 axis. (a) MMP3 was predicted as a direct target of miR-373-3p. (b) Target between MMP3 and miR-373-3p was confirmed by luciferase assay (^∗∗p < 0.01). (c) The effect of miR-373-3p on MMP3 expression in TPC-1 cell was tested by q-PCR (^∗∗p < 0.01). (d) MMP3 protein level was decreased by miR-373-3p mimics (^∗∗p < 0.01). (e) miR-373-3p and MMP3 expression were tested by q-PCR after ZFAS1 knocking down in TPC-1 cells (^∗∗p < 0.01, ^∗∗∗p < 0.001). (f) MMP3 expression profile in primary tumor and normal tissues obtained from TCGA-THCA datasets (^∗∗∗p < 0.001). (g) The effect of MMP3 knocking down on ZFAS1 expression was tested by q-PCR. (h) ZFAS1 and MMP3 enrichment with AGO2 antibody analyzed by RNA immunoprecipitation followed q-PCR test after ectopically expressed miR-373-3p (^∗∗p < 0.01).

Figure 5

CREB3 transactivated ZFAS1 expression. (a) and (b) JASPAR predicted the positions of the putative CREB3 binding motif at −2,000 bp in the human ZFAS1 promoter. (c) CREB3 was found to be positively related to ZFAS1 expression in thyroid carcinoma. (d) CREB3 expression profile in primary tumor and normal tissues obtained from TCGA-THCA datasets (^∗∗∗p < 0.001). (e) Ectopically expressed CREB3 induced ZFAS1 level tested by q-PCR (^∗∗p < 0.01). (f) Luciferase reporter assay performed following cotransfection of the full-length ZFAS1 promoter or deleted ZFAS1 promoter fragments with the CREB3 expression plasmid or blank vector in TPC1 cells. (g) Luciferase activities were expressed as relative to that of the pGL3 vector (^∗∗p < 0.01). (h) The binding between CREB3 and ZFAS1 was confirmed by q-ChIP assay; AchR was served as a negative control (^∗∗p < 0.01). (i) Regulation model of CREB3/ZFAS1/miR-373-3p/MMP3 in EMT process.