GRK2 Protein Mediates the ANRIL, a lncRNA, to Affect the Proliferation and Apoptosis of Kasumi-1 Cells

Abstract

Background: A long non-coding RNAs (LncRNAs) called antisense noncoding RNA in the INK4 locus (ANRIL), has emerged as substantial regulators of cell survival in acute myeloid leukemia (AML). However, its speciffc and potential mechanism is uncertain in AML. In this research, we investigated the role of ANRIL in cell proliferation, apoptosis, and the underlying mechanism in AML cells.

Methods: ANRIL expression was quantified by real-time quantitative polymerase chain reaction (RT-qPCR). Kasumi-1 cells were transfected with LV-ANRIL plasmid to upregulate ANRIL expression, with or without co-transfection with a G Protein-Coupled Receptor Kinase 2 (GRK2) siRNA. Additionally, these cells were transfected with sh-ANRIL plasmid to inhibit ANRIL expression, with or without co-transfection with a GRK2 overexpression plasmid. Cell proliferation and apoptosis were determined using the cell counting kit-8 (CCK8) and flow cytometry. Protein expression levels of phosphatidylinositide 3-kinases (PI3K), protein kinase B (AKT), phosphorylated-Akt (p-AKT), Bcl-2-associated protein x (BAX), B-cell leukemia/lymphoma 2 protein (BCL-2), proliferating cell nuclear antigen (PCNA), cleaved caspase-3, and GRK2 were detected by western blot. The RNA-binding protein immunoprecipitation (RIP) assay was conducted to investigate the interaction between ANRIL and GRK2.

Results: ANRIL expression was increased in Kasumi-1 cells. ANRIL upregulation expression promoted cell proliferation and inhibited apoptosis. Furthermore, its upregulation led to increased expressions of PI3K, AKT, p-AKT, PCNA, and BCL-2, and decreased expression of BAX in Kasumi-1 cells. Additionally, transfection with GRK2 siRNA attenuated the promoting effect of LV-ANRIL on Kasumi-1 cells proliferation and the PI3K/AKT pathway, increased BAX and cleaved caspase-3 expressions, and decreased BCL-2 and PCNA expressions. GRK2 overexpression reversed the inhibitory effect of sh-ANRIL on cell proliferation and the PI3K/AKT pathway. Furthermore, it promoted BCL-2 and PCNA expressions, and inhibited BAX and cleaved caspase-3 expressions. RIP assay confirmed the physical interaction between ANRIL and GRK2.

Conclusion: The GRK2 protein-mediated ANRIL, increasing Kasumi-1 cell proliferation and inhibiting apoptosis by activating the PI3K/AKT/BCL-2 pathway.

Keywords: ANRIL; GRK2; PI3K/AKT/BCL-2 pathway; acute myeloid leukemia; apoptosis; proliferation.