Leucine-Rich Alpha-2-Glycoprotein1 Gene Interferes with Regulation of Apoptosis in Leukemia KASUMI-1 Cells

Abstract

BACKGROUND Leukemia cells have strong proliferation and anti-apoptosis capabilities. The purpose of this study was to investigate the effect of silencing the leucine-rich alpha-2-glycoprotein1 (LRG1) gene, which was found to regulate tumor proliferation and apoptosis in acute myeloid leukemia (AML) cell lines. MATERIAL AND METHODS Plasmid interference technique was used to silence the LRG1 gene in the KASUMI-1 cell line. The cell counting kit-8 (CCK-8) assay was used to test the effect of transduction on cell viability. Cell cycle and apoptosis were detected by flow cytometry. Western blot and quantitative real-time polymerase chain reaction (RT-qPCR) were applied to detect the expression levels of proteins and mRNA, respectively. RESULTS KASUMI-1 cells with the CD34⁺CD38⁻ phenotype were sorted by flow cytometry. After transfection of the siLRG1 plasmid, the level of LRG1 expression was downregulated and cell viability was reduced. Silencing of LRG1 gene blocked KASUMI-1 cells in G0/G1 phase and promoted apoptosis. Further experiments found that LRG1 gene silencing significantly downregulated cell cycle-associated proteins and anti-apoptotic proteins, while upregulating pro-apoptotic proteins. Downregulation of LRG1 gene expression also inhibits signal transduction of the JAK-STAT pathway. CONCLUSIONS LRG1 gene silencing regulates the expression of cyclin and apoptosis-related proteins to reduce cell viability and promote apoptosis, probably through inhibition of the JAK-STAT pathway.

Figure 1

Flow CD34⁺CD38⁻ KASUMI-1 cells were sorted by flow cytometry. (A) Only 63.4% of KASUMI-1 cells were CD34⁺CD38⁻ phenotype before sorting. (B) After selection, 90.3% of KASUMI-1 cells had CD34⁺CD38⁻ phenotype.

Figure 2

Effects of siLRG1 plasmid on the expression of LRG1 and cell viability in KASUMI-1 cells. KASUMI-1 cells were transfected with PBS (control group), negative-siRNA plasmid (NC group), siLRG1 plasmid (siLRG1 group). (A) Western blot was used to assess the protein level of LRG1 in different gruops. (B) RT-qPCR was performed to test the mRNA level of LRG1 in different gruops. (C) The cell viability was analyzed by CCK-8 analysis. * P<0.05, ** P<0.01, versus control group.

Figure 3

Effect of siLRG1 plasmid transfection on cell cycle. (A–D) Flow cytometry was applied to detect the cell cycle of each group after transfection. (E, F) Western blot was used to assess the protein level of Cyclin D1 and PCNA in different gruops. (G) RT-qPCR was used to detect mRNA of M Cyclin D1 and PCNA. * P<0.05, ** P<0.01, versus control group.

Figure 4

Effect of siLRG1 plasmid transfection on cell apoptosis. (A–D) Flow cytometry was applied to detect the cell apoptosis of each group after transfection. (E, F) Western blot was used to assess the protein level of Bcl-2, Bax, pro-Caspase-3 and cleaved-Caspase-3 in different gruops. (G) RT-qPCR was used to detect mRNA of Bcl-2, Bax, pro-Caspase-3 and cleaved-Caspase-3. * P<0.05, ** P<0.01, versus control group.

Figure 5

Effect of siLRG1 plasmid transfection on JAK-STAT pathway. (A, B) Western blot was used to assess the protein level of JAK-2, p-JAK-2, STAT3 and p-STAT3 in different gruops. * P<0.05, ** P<0.01, versus control group.