ADORA2A promotes proliferation and inhibits apoptosis through PI3K/AKT pathway activation in colorectal carcinoma

Abstract

The third most often diagnosed disease globally and the second most prevalent cause of cancer-related death is colorectal cancer (CRC). Numerous human malignancies have been identified to have high expression of ADORA2A. However, it is still ambiguous about its function in CRC. RNA-seq with stable transfected SETDB1 knockdown cells was used to identify differentially expressed genes. Further, knockdown of ADORA2A in CRC cell lines SW620 and HCT116 was performed with siRNA and over expression of ADORA2A in SW480 cells was conducted with plasmids. CCK8, colony formation, wound healing, and transwell assay were used to detect the effects of cell proliferation, migration, and invasion after knockdown and over expression of ADORA2A. Also, apoptosis was analyzed by flow cytometry, apoptosis-related proteins and key PI3K/AKT pathway proteins were detected using Western blotting. ADORA2A was identified after RNA-seq analysis and played an important role in CRC prognosis. ADORA2A was relatively high in SW620 and HCT116 cell lines compared to SW480 cell lines. ADORA2A knockdown in SW620 and HCT116 inhibited cell proliferation, migration, and invasion, while ADORA2A overexpression had the opposite effect. In addition, ADORA2A also impacted the expression of apoptosis-related proteins, including Bcl-2, Bax, Cleaved caspase-3 and Cleaved caspase-9, and reduced apoptosis. Furthermore, this process may include the PI3K/AKT signaling pathway. ADORA2A promotes CRC progression and inhibits apoptosis by the PI3K/AKT signaling pathway. It may contribute to the management and treatment of CRC.

Figure 1

ADORA2A acts as an important progression factor in CRC. (A) DEGs of Stable transfected SETDB1 knockdown cells (DEG-UP: up-regulated genes with Stable transfected SETDB1 knockdown cells, DEG-DOWN: down-regulated genes with Stable transfected SETDB1 knockdown cells). (B) Biological process of GO and KEGG analysis with DEGs. (C) mRNA and protein expression of ADORA2A in colorectal cancer cells with stable knockdown of SETDB1. (D) Survival curve gained from HPA of CRC (low expression (n = 465); high expression (n = 132); p = 0.033).

Figure 2

The expression of ADORA2A in CRC cells. (A) The expression of ADORA2A in SW620, HCT116, SW480 CRC cell lines. (B) Transfection efficiency validation of ADORA2A overexpression in SW480. (C) Validation of ADORA2A knockdown by transfection effectiveness in SW620. (D) Validation of ADORA2A knockdown by transfection effectiveness in HCT116.

Figure 3

Knockdown of ADORA2A inhibits proliferation, migration, and invasion of CRC cells. (A–B) CCK8 assay of ADORA2A transfected with siRNA ADORA2A-2 and siRNA ADORA2A-3 in SW620 and HCT116. (C–D) Colony formation assay of ADORA2A transfected with siRNA ADORA2A-2 and siRNA ADORA2A-3 in SW620 and HCT116. (E–F) ADORA2A wound healing assay transfected with siRNA ADORA2A-2 and siRNA ADORA2A-3 in SW620 and HCT116. (G-H) Transwell assay of ADORA2A transfected with siRNA ADORA2A-2 and siRNA ADORA2A-3 in SW620 and HCT116. Bars:200 μm (E–F); 50 μm (G–H).

Figure 4

Overexpression of ADORA2A promotes malignant phenotypes in SW480 cells. (A–B) CCK8 assay and colony formation assay of SW480 cells transfected with ADORA2A overexpression plasmid. (C) Wound healing assay of SW480 cells transfected with ADORA2A overexpression plasmid. (D) Transwell assay of SW480 cells transfected with ADORA2A overexpression plasmid. Bars:200 μm (C); 50 μm (D).

Figure 5

ADORA2A inhibits apoptosis of CRC cells. (A–B) Cell apoptosis of ADORA2A transfected with siRNA ADORA2A-2 and siRNA ADORA2A-3 in SW620 and HCT116 determined by flow cytometry. Cell apoptosis was calculated as a sum of early and late apoptotic cells. (C) Cell apoptosis transfected with ADORA2A overexpression plasmid in SW480 determined by flow cytometry. Cell apoptosis was calculated as a sum of early and late apoptotic cells.

Figure 6

Expression of apoptosis-related proteins after ADORA2A knockdown or over expression. (A–B) Expression of Bcl-2, Bax, Cleaved caspase-3 and Cleaved caspase-9 in SW620 and HCT116. (C) Expression of Bcl-2, Bax, Cleaved caspase-3 and Cleaved caspase-9 proteins in SW480.

Figure 7

ADORA2A may be involved in the regulation of PI3K/AKT signaling. (A–B) Proteins expression of p-PI3K, PI3K, p-AKT and AKT in SW620 and HCT116. (C) Proteins expression of p-PI3K, PI3K, p-AKT and AKT in SW480.