The Novel Tumor Suppressor Gene ZNF24 Induces THCA Cells Senescence by Regulating Wnt Signaling Pathway, Resulting in Inhibition of THCA Tumorigenesis and Invasion

Abstract

Object: Clinically, the effective treatment options available to thyroid cancer (THCA) patients are very limited. Elucidating the features of tumor suppressor genes (TSGs) and the corresponding signal transduction cascade may provide clues for the development of new strategies for targeted therapy of THCA. Therefore, this paper aims to explore the mechanism of ZNF24 underlying promoting THCA cell senescence at molecular level.

Methods: We performed RT-PCR and Western Blotting for evaluating associated RNA and protein expression. CCK8, colony forming, wound healing and Transwell chamber assays were conducted to examine THCA cell proliferation, invasion and migration. β-galactosidase staining assay was performed to detect THCA cells senescence. The size and volume of xenotransplanted tumors in nude mice are calculated to asses ZNF24 effect in vivo.

Results: Ectopic expression of ZNF24 significantly inhibited the cell viability, colony forming, migration and invasion abilities of THCA cell lines (K1/GLAG-66i and BCPAPi) (P < 0.05). ZNF24 induced BCPAPi cells senescence through regulating Wnt signaling pathway. ZNF24 inhibited Wnt signaling pathway activition by competitively binding β-catenin from LEF1/TCF1-β-catenin complex. In nude mice, both Ectopic expression of ZNF24 and 2,4-Da (the strong β-catenin/Tcf-4 inhibitor) treatment significantly decreased both the size and weight of xenotransplanted tumors when compared with control mice (P < 0.05).

Conclusion: Results obtained in vivo and in vitro reveal the role of ZNF24 in significantly suppressing THCA tumorigenesis and invasion by regulating Wnt signaling pathway.

Keywords: Wnt signaling pathway; ZNF24; senescence; thyroid cancer; tumor suppressor genes.

Figure 1

ZNF24 serves as a new suppressor gene of THCA with clinical relevance (A, B) mRNA levels of ZNF24 within the GTEX-derived thyroid samples and TCGA-derived THCA samples; (C): mRNA expression of ZNF24 in para-tumoral tissues and in tumor tissues; (D): Western blotting on the ZNF24 level within specific lung tissue samples, T: tumor, N: para-tumor; (E): Western blotting on the ZNF24 level within specific THCA cells (F) Impacts of DOX-mediated ZNF24 expression within stable BCPAPi and K1/GLAG-66i cells. Immunoblots that contained specific antibodies were used to analyze specific cells-derived whole-cell lysates; (G, H) ZNF24 ectopic expression inhibited K1/GLAG-66i and BCPAPi cells cell viability. The K1/GLAG-66i (F) and BCPAPi cells (G) (1 × 10³) were exposed to 48 h of 1 µg/ml DOX treatment or not, and CCK-8 assay was conducted to detect cell viability at specific time points; (I) ZNF24 suppressed the ability of form colonies in BCPAPi and K1/GLAG-66i cells. K1/GLAG-66i and BCPAPi cells were exposed to 10 days of DOX treatment prior to colony formation assay; (J) ZNF24 suppressed the ability to form spheres in BCPAPi and K1/GLAG-66i cells. Typical sphere formation image for BCPAPi and K1/GLAG-66i cells (upper), as well as sphere formation statistic image (lower). Values are obtained from three independent assays, while unpaired t-test was used for analysis. Error bars = SD. *P < 0.05, **P < 0.01.

Figure 2

ZNF24 inhibited K1/GLAG-66i and BCPAPi cells migration and invasion. (A–C) Cell migration and invasion detection by Wound Healing test (A) and transwell chamber assay (B, C) of K1/GLAG-66i and BCPAPi cells treated without or with DOX (1 µg/ml); (D) Cell migration and invasion protein makers expression level detection by Western Blot assay of K1/GLAG-66i and BCPAPi cells treated without or with DOX (1 µg/ml), indicated antibodies were added during Western Blot assay; (E, F) Statistical analysis of cell migration and invasion protein makers expression level based on western blot assay result Values are obtained from 3 independent assays, while unpaired t-test was used for analysis. Error bars= SD. *P < 0.05; **P < 0.01.

Figure 3

Ectopic ZNF24 expression led to THCA cell aging. (A) ZNF24 induces G0/G1 cell cycle arrest in K1/GLAG-66i and BCPAPi cells. Cell proportions at G0/G1, S, G2/M phases. BCPAPi and K1/GLAG-66i cell lines were exposed to 3 days of 1 µg/ml DOX treatment or not, the distribution of cell cycle (left) and the related statistics (right) were analyzed by FACS analysis. (B) ZNF24 ectopic expression induced senescence of THCA cells. BCPAPi and K1/GLAG-66i cell lines were exposed to 48 h of 1 µg/ml DOX treatment or not, the activity of aging-related β-galactosidase (left) and the related statistics (right) were analyzed to determine the aging cells. Values are obtained from three independent assays, while unpaired t-test was used for analysis. Error bars=SD. *P < 0.05.

Figure 4

ZNF24 inhibits the activation of Wnt signaling pathway in BCPAPi cells by competitively binding β-catenin. (A) ZNF24 inhibits Wnt signaling pathway traget genes mRNA expression. BCPAPi cells (2 × 10⁵) were subjected to 48 h of 1 µg/ml DOX treatment or not, then qPCR was conducted to extract total RNA; (B) Wnt signaling pathway traget genes protein expression level detection by Western Blot assay of BCPAPi cells treated without or with DOX (1 µg/ml), indicated antibodies were added during Western Blot assay. (C) Endogenous ZNF24 is associated with β-catenin within the BCPAPi cell line. CoIP assays were carried out using anti-β-catenin or anti-ZNF24, then immunoblots that contained specific antibodies were used to analyze whole-cell lysate and immunoprecipitates (input). (D) ZNF24 diminish the interaction between β-catenin and TCF1 or LEF1. 2.5 g plasmids were used to transfect 293T cells (2 × 10⁶). CoIP and immunoblot assays were carried out using specific antibodies. Values are obtained from 3 independent assays, while unpaired t-test was used for analysis. Error bars=SD. *P < 0.05, **P < 0.01.

Figure 5

β-catenin reverse the ZNF24 effect of BCPAPi cells. (A, B) Cell viability and colony forming dectection of BCPAPi cells without or with DOX (1 µg/ml) or ICG001 (0.1 μg/ml) treatment. (C) ICG001 induces senescence of BCPAPi cells. BCPAPi cells without or with DOX (1 µg/ml) or ICG001 (0.1 μg/ml) treatment for 48 h, later the aging cells were measured through activity of aging-related β-galactosidase (left) and the corresponding statistics (right). (D) β-catenin expression level detection by Western Blot assay. 0.1 μg β-catenin expression plasmids were transfected into BCPAPi cells (1 × 10⁴), followed by DOX (1 µg/ml of DOX treatment or not, indicated antibodies were added during Western Blot assay. (E, F) Cell viability and colony forming dectection of BCPAPi cells. 0.1 μg β-catenin expression plasmids were transfected into BCPAPi cells (1 × 10⁴), followed by 1 µg/ml of DOX treatment or not. (G) BCPAPi cells (1 × 10⁴) were transfected with β-catenin expression plasmid (0.1 μg), followed by 1 µg/ml of DOX treatment or not. The aging cells were measured through the activity of aging-related β-galactosidase (left) and the corresponding statistics (right). Values are obtained from three independent assays, while unpaired t-test was used for analysis. Error bars=SD. *P < 0.05, ***P < 0.001.

Figure 6

ZNF24 and 2,4-Da inhibited THCA xenografted tumors growth in nude mice. (A, B, D) The growth and weight of DOX or 2,4-Da exposed tumor samples was significantly inhibited relative to controls. We measured tumor growth at intervals of 2 days by measuring its diameter with Vernier caliper. (C) The Real Time PCR analysis of ZNF24 on ex vivo tumor extracts. (E) 2,4 Dia or DOX made no difference to the mouse body weight measured at intervals of 5 days. (F) Protein expression level of ZNF24 of THCA xenografted tumors of nude mice treated with DOX (DOX+) or without DOX (DOX-). (G, H) IHC staining of THCA xenografted tumors of nude mice treated with DOX (DOX+), DOX+2,4-Da or without DOX (DOX-), indicated antibodies were added during IHC staining assay. Values are obtained from three independent assays, while unpaired t-test was used for analysis. Error bars=SD. *P < 0.05; **P < 0.01; ***P < 0.001.