ZFPM2-AS1 transcriptionally mediated by STAT1 regulates thyroid cancer cell growth, migration and invasion via miR-515-5p/TUSC3

Abstract

Objective: Our purpose was to study the roles and molecular mechanisms of long non-coding RNA (lncRNA) ZFPM2 Antisense RNA 1 (ZFPM2-AS1) in thyroid cancer. Methods: Firstly, the expression of ZFPM2-AS1, miR-515-5p and TUSC3 was detected in thyroid cancer tissues and cells. Secondary, their biological functions (proliferation, apoptosis, migration and invasion) were analyzed by a serious of functional experiments including cell counting kit-8 (CCK-8), clone formation, 5-Ethynyl-2'-deoxyuridine (EdU), enzyme-linked immunosorbent assay (ELISA), wound healing and Transwell assays. Thirdly, the mechanisms of STAT1/ZFPM2-AS1 and ZFPM2-AS1/miR-515-5p/TUSC were validated using chromatin immunoprecipitation (CHIP), pull-down and luciferase reporter assays. Results: ZFPM2-AS1 and TUSC were both highly expressed and miR-515-5p was down-regulated in thyroid cancer tissues as well as cells. Their knockdown weakened thyroid cancer cell growth, migration, and invasion. ZFPM2-AS1 was mainly distributed in the nucleus and cytoplasm of thyroid cancer cells. Mechanistically, up-regulation of ZFPM2-AS1 was induced by transcription factor STAT1 in line with CHIP and luciferase reporter assays. Furthermore, as a sponge of miR-515-5p, ZFPM2-AS1 decreased the ability of miR-515-5p to inhibit TUSC3 expression by pull-down, luciferase reporter and gain-and-loss assays, thereby promoting malignant progression of thyroid cancer. Conclusion: ZFPM2-AS1 acted as an oncogene in thyroid cancer, which was transcriptionally mediated by STAT1. Furthermore, ZFPM2-AS1 weakened the inhibitory effect of miR-515-5p on TUSC3. Thus, ZFPM2-AS1 could be an underlying biomarker for thyroid cancer.

Keywords: TUSC3; ZFPM2-AS1; miR-515-5p; thyroid cancer; transcription factor..

Figure 1

Expression and clinical features of ZFPM2-AS1 in thyroid cancer. (A) High ZFPM2-AS1 expression was detected in thyroid cancer tissues via qRT-PCR. (B) The difference in ZFPM2-AS1 expression between I-II and III-IV thyroid cancer patients. (C) qRT-PCR was utilized to monitor ZFPM2-AS1 expression in thyroid cancer and normal cells. (D) ROC was depicted to determine the efficiency of ZFPM2-AS1 expression as a diagnosed marker of thyroid cancer. **p<0.01.

Figure 2

ZFPM2-AS1 is transcriptionally regulated by STAT1 in thyroid cancer. (A) Schematic diagram showing the binding sites in the promoter region of ZFPM2-AS1 for STAT1. (B, C) qRT-PCR examining STAT1 expression in thyroid cancer tissues as well as cells. (D, E) qRT-PCR verifying the transfected efficiencies of si-STAT1 and pcDNA3.1-STAT1 in thyroid cancer SW579 and 8505C cells. (F, G) ZFPM2-AS1 expression was tested in SW579 and 8505C cells with si-STAT1 and pcDNA3.1-STAT1. (H) ChIP assay and (I) Dual luciferase reporter assay validating the binding sites of STAT1 and ZFPM2-AS1 promoter region. **p<0.01.

Figure 3

ZFPM2-AS1 knockdown weakens proliferation and facilitates apoptosis for thyroid cancer cells. (A) siRNAs were used to interfere with ZFPM2-AS1 expression in SW579 and 8505C cells. (B) CCK-8 experiment detected that the viability of SW579 and 8505C cells with si-ZFPM2-AS1 was suppressed. (C) Clone formation experiment showed that the number of clone formations was weakened in SW579 and 8505C cells with si-ZFPM2-AS1. (D, E) EdU experiment detecting the changes in cell proliferation after transfection of si-ZFPM2-AS1 as well as its corresponding negative controls. Scale bar: 20 μm. Magnification: 200×. (F) ELISA showed lower Caspase 3 and 9 levels in cells with si-ZFPM2-AS1. **p<0.01.

Figure 4

ZFPM2-AS1 knockdown inhibits migrated and invasive capacities of thyroid cancer cells. (A, B) Lower migratory rate was detected in SW579 and 8505C cells with si-ZFPM2-AS1. Magnification: 200×. (C, D) The number of cells that penetrated Matrigel to reach the lower layer of the chamber was reduced for cells with si-ZFPM2-AS1. Magnification: 200×. (E, F) Western blot was utilized to examine EMT-related markers (E-cadherin, N-cadherin and Vimentin) in SW579 and 8505C cells interfered with si-ZFPM2-AS1. **p<0.01.

Figure 5

ZFPM2-AS1 may sponge miR-515-5p in thyroid cancer cells. (A) Subcellular localization of ZFPM2-AS1. (B) Putative binding sites between ZFPM2-AS1 and miR-515-5p. (C) Potential targeted mRNAs of miR-515-5p. (D) KEGG enrichment analyses of target mRNAs. (E, F) qRT-PCR was utilized to examine miR-515-5p expression in thyroid cancer tissues as well as cells. (G, H) Cell viability and invasion were assessed in cells with miR-515-5p mimics using CCK-8 and Transwell. (I, J) Pull-down and luciferase reporter assays confirmed the direct binding between ZFPM2-AS1 and miR-515-5p. (K, L) qRT-PCR examined the expressions of miR-515-5p or ZFPM2-AS1 in cells with si-ZFPM2-AS1 or miR-515-5p mimics. **p<0.01.

Figure 6

TUSC3 is a targeted mRNA of miR-515-5p in thyroid cancer. (A) Overlapping target mRNAs of miR-515-5p and overexpressed mRNAs in thyroid cancer. (B, C) Molecular functions and KEGG pathway analysis of target mRNAs. (D) Expression patterns of TUSC3 across different cancers using TCGA database. (E) Differences in expression patterns of TUSC3 in thyroid cancer and normal tissues. (F) Differences in expression patterns of TUSC3 in different clinical stages. (G) Differences in expression patterns of TUSC3 in nodal non-metastasis and metastasis status. (H) qRT-PCR was utilized to verify TUSC3 expression in thyroid cancer tissues as well as cells. (I, J) Dual luciferase report validated that miR-515-5p bound to the two binding sites in the 3'UTR region of TUSC3. **p<0.01.

Figure 7

miR-515-5p may suppress proliferation and invasion for thyroid cancer cells by inhibiting TUSC3 expression. (A) Western blot detecting the expression of TUSC3 protein after transfection with miR-515-5p mimics and / or pcDNA3.1-TUSC3 in SW579 and 8505C cells. (B, C) CCK-8 assay was presented to examine the cell viability of SW579 and 8505C cells following transfection with miR-515-5p mimics and / or pcDNA3.1-TUSC3. (D) Clone formation experiment of transfected two thyroid cancer cells. (E) Edu assay results for transfected two thyroid cancer cells. (F) Transwell assay results showing the invasive ability of SW579 and 8505C cells transfected with miR-515-5p mimics and / or pcDNA3.1-TUSC3. **p<0.01.

Figure 8

ZFPM2-AS1 mediates TUSC3 expression via miR-515-5p in thyroid cancer cells. (A) Transfection efficiencies of si-TUSC3 in SW579 and 8505C cells were verified using western blot. (B-D) Cell proliferation was detected in cells transfected with si-TUSC3 using CCK-8, clone formation and EdU assays. (E) Invasive capacity was assessed when transfected with si-TUSC3. (F, G) ZFPM2-AS1 and TUSC3 expression was validated after transfection with miR-515-5p mimics / inhibitors using qRT-PCR. (H) TUSC3 expression was determined when transfection with pcDNA3.1-ZFPM2-AS1 or si-ZFPM2-AS1 via qRT-PCR. (I) qRT-PCR was utilized to assess TUSC3 expression in cells transfected with pcDNA3.1-ZFPM2-AS1 and / or miR-515-5p mimics. **p<0.01.

Figure 9

A mechanism diagram of STAT1/ZFPM2-AS1/miR-515-5p/TUSC3 axis in thyroid cancer.