THAP7 promotes cell proliferation by regulating the G1/S phase transition via epigenetically silencing p21 in lung adenocarcinoma

Abstract

Purpose: Lung adenocarcinoma (LUAD) is one of the most common cancers worldwide. The THanatos-Associated Proteins (THAP) family plays an essential role in multiple cancers. However, the role of THAP7 in cancers has remained elusive.

Methods: THAP7 expression status in LUAD tissues was analysed by using the Oncomine database and qRT-PCR, and its expression level in LUAD cell lines was detected by qRT-PCR and Western blotting. The role of THAP7 in LUAD cells was determined by proliferation, colony formation, and cell cycle analyses. In vivo role of THAP7 was studied on xenograft models. Luciferase reporter assays and chromatin immunoprecipitation (ChIP) were used to determine the activity and acetylation of the p21 promoter.

Results: THAP7 expression was increased in LUAD tissues and cell lines. Moreover, the high expression of THAP7 was correlated with poor prognosis. The overexpression of THAP7 accelerated the G1/S phase transition and promoted tumour growth both in vitro and in vivo. A mechanistic study revealed that THAP7 reduced the acetylation of histone H3 on the p21 promoter to suppress p21 transcription.

Conclusion: For the first time, we demonstrated the function of THAP7 in LUAD, and our findings suggested that THAP7 may be a potential molecular therapy target in LUAD.

Keywords: THAP7; cell cycle; lung adenocarcinoma; p21; proliferation.

Figure 1

THAP7 is upregulated in lung adenocarcinoma samples and is associated with shorter overall survival. Notes: (A) Meta-analysis of THAP7 mRNA levels in lung cancer samples from the Oncomine database (probe ID: 218492_s_at, normal, n=20, LUAD, n=226). (B) qRT-PCR evaluation of THAP7 expression in LUAD samples compared with adjacent normal tissues (n=14). (C, D) The expression of THAP7 was determined in human bronchial epithelial cells (BEAS-2B) and LUAD cell lines, including H1975, A549 and H1299 cells, through qRT-PCR (C) and Western blotting (D). GAPDH was used as an internal control. The bars correspond to the mean ± standard error. (E) Patients with high THAP7 expression exhibited relatively shorter overall survival (OS) according to the KMplot database. ***P<0.001. Abbreviations: LUAD, lung adenocarcinoma; qRT-PCR, quantitative real-time PCR; THAP7, Thanatos-associated protein-7.

Figure 2

THAP7 promotes cell proliferation and colony formation in vitro and tumour growth in vivo in lung adenocarcinoma. Notes: (A) Western blotting of THAP7 expression in THAP7-overexpressing A549 cells and THAP7-knockdown H1299 cells. (B) Cell proliferation was assessed in THAP7-overexpressing A549 cells (left) and THAP-knockdown H1299 cells (right). (C, D) The colony formation ability of THAP7-overexpressing A549 cells (C) and THAP7-knockdown H1299 cells (D) was evaluated. (E) Nude mice were transplanted with A549 cells overexpressing THAP7 (n = 6) or empty vector for control (n = 6) for 18 days. Representative image of tumours excised from the mice (upper). Mean tumour weights were calculated (down). *P<0.05, **P<0.01, ***P<0.001. Abbreviations: THAP7, Thanatos-associated protein-7.

Figure 3

THAP7 promotes the G1 to S phase transition. Notes: The cell cycles were analysed via flow cytometry in THAP7-overexpressing A549 cells (A) and THAP7-knockdown H1299 cells (B). *P<0.05, **P<0.01. Abbreviations: THAP7, Thanatos-associated protein-7.

Figure 4

THAP7 reduces the acetylation of histone H3 on the p21 promoter to inhibit p21 expression. Notes: The expression of p21 was determined by qRT-PCR (A) and Western blotting (B) in THAP7-overexpressing A549 cells and THAP7-knockdown H1299 cells. A luciferase reporter assay revealed that overexpressing THAP7 in A549 cells or knocking down THAP7 in H1299 cells suppressed (C) or increased (D) the promoter activity of p21, respectively. (E) The promoter activity of p21 in THAP7-overexpressing A549 cells in the presence or absence of HDAC3 knocking down. The acetylation of histone H3, located in the region of the p21 methylation promoter, was examined by ChIP assays in THAP7-overexpressing A549 cells (F) and THAP7-knocked down H1299 cells (G). (H) A negative correlation between THAP7 and p21 mRNA expression in lung cancer tissues. The correlation was analysed with a Pearson’s correlation analysis. *P<0.05, **P<0.01. Abbreviations: ChIP, chromatin immunoprecipitation; qRT-PCR, quantitative real-time PCR; THAP7, Thanatos-associated protein-7.