GRK2 蛋白介导长链非编码 RNA ANRIL 影响 Kasumi-1 细胞的增殖和凋亡。

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

机构信息

Center for Clinical Laboratories, The Affiliated Hospital of Guizhou Medical University, 550004 Guiyang, Guizhou, China.

Guizhou Center for Disease Control and Prevention, 550004 Guiyang, Guizhou, China.

出版信息

Front Biosci (Landmark Ed). 2024 Oct 21;29(10):362. doi: 10.31083/j.fbl2910362.

DOI:10.31083/j.fbl2910362

PMID:39473411

Abstract

BACKGROUND

A long non-coding RNAs (LncRNAs) called antisense noncoding RNA in the INK4 locus (), 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 in cell proliferation, apoptosis, and the underlying mechanism in AML cells.

METHODS

expression was quantified by real-time quantitative polymerase chain reaction (RT-qPCR). Kasumi-1 cells were transfected with LV- plasmid to upregulate expression, with or without co-transfection with a G Protein-Coupled Receptor Kinase 2 (GRK2) siRNA. Additionally, these cells were transfected with sh- plasmid to inhibit 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 and GRK2.

RESULTS

expression was increased in Kasumi-1 cells. 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- 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- 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 and GRK2.

CONCLUSION

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

摘要

背景

长非编码 RNA（lncRNAs）称为抗基因编码 RNA 在 INK4 基因座（），已成为急性髓系白血病（AML）中细胞存活的重要调节因子。然而，其在 AML 中的特异性和潜在机制尚不确定。在这项研究中，我们研究了在 AML 细胞中增殖、凋亡的作用及其潜在机制。

方法

实时定量聚合酶链反应（RT-qPCR）定量检测表达。用 LV-质粒转染 Kasumi-1 细胞上调表达，或与 G 蛋白偶联受体激酶 2（GRK2）siRNA 共转染。此外，用 sh-质粒转染这些细胞以抑制表达，或与 GRK2 过表达质粒共转染。用细胞计数试剂盒-8（CCK8）和流式细胞术测定细胞增殖和凋亡。用蛋白质印迹法检测磷脂酰肌醇 3-激酶（PI3K）、蛋白激酶 B（AKT）、磷酸化-Akt（p-AKT）、B 细胞淋巴瘤/白血病 2 蛋白（BCL-2）、增殖细胞核抗原（PCNA）、裂解的半胱天冬酶-3 和 GRK2 的蛋白表达水平。用 RNA 结合蛋白免疫沉淀（RIP）试验检测与 GRK2 的相互作用。

结果

在 Kasumi-1 细胞中表达增加。上调表达促进细胞增殖，抑制细胞凋亡。此外，其上调导致 PI3K、AKT、p-AKT、PCNA 和 BCL-2 的表达增加，而 BAX 在 Kasumi-1 细胞中的表达减少。此外，转染 GRK2 siRNA 可减弱 LV-对 Kasumi-1 细胞增殖和 PI3K/AKT 通路的促进作用，增加 BAX 和裂解的半胱天冬酶-3 的表达，减少 BCL-2 和 PCNA 的表达。GRK2 过表达逆转了 sh-对细胞增殖和 PI3K/AKT 通路的抑制作用。此外，它促进了 BCL-2 和 PCNA 的表达，抑制了 BAX 和裂解的半胱天冬酶-3 的表达。RIP 试验证实了与 GRK2 的物理相互作用。