miR-4792 Inhibits Acute Myeloid Leukemia Cell Proliferation and Invasion and Promotes Cell Apoptosis by Targeting Kindlin-3

Abstract

It has been reported that kindlin-3 expression is closely associated with progression of many cancers and microRNA (miRNA) processing. However, the effects and precise mechanisms of kindlin-3 in acute myeloid leukemia (AML) have not been well clarified. Our study aimed to explore the interaction between kindlin-3 and miR-4792 in AML. In our study, we found that the expression of kindlin-3 was dramatically increased in AML samples and cell lines, and the miR-4792 level was significantly downregulated. Interestingly, the low miR-4792 level was closely associated with upregulated kindlin-3 expression in AML samples. Moreover, introduction of miR-4792 dramatically suppressed proliferation and invasion and induced apoptosis of AML cells. We demonstrated that miR-4792 could directly target kindlin-3 by using both bioinformatics analysis and luciferase reporter assay. In addition, kindlin-3 silencing had similar effects with miR-4792 overexpression on AML cells. Overexpression of kindlin-3 in AML cells partially reversed the inhibitory effects of miR-4792 mimic. miR-4792 inhibited cell proliferation and invasion and induced apoptosis of AML cells by directly downregulating kindlin-3 expression, and miR-4792 targeting kindlin-3 was responsible for the regulation of the proliferation, invasion, and apoptosis of AML cells.

Figure 1

The expressions of kindlin-3 and microRNA-4792 (miR-4792) in acute myeloid leukemia (AML) samples and cell lines. (A) Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis of kindlin-1, kindlin-2, and kindlin-3 expressions in AML samples and adjacent normal samples (n = 6). Transcript levels were normalized by glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression. (B) qRT-PCR analysis of kindlin-3 expressions in AML samples and adjacent normal samples (n = 30). Transcript levels were normalized by GAPDH expression. (C) Relative kindlin-3 expression analyzed by qRT-PCR in five AML cell lines were normalized with GAPDH (n = 6). (D) qRT-PCR analysis of miR-4792 level in AML samples and adjacent normal samples (n = 30). Transcript levels were normalized by U6 level. (E) Relative miR-4792 level analyzed by qRT-PCR in five AML cell lines were normalized with U6 (n = 6). (F) Pearson’s correlation analysis of the relative expression levels of miR-4792 and the relative kindlin-3 mRNA levels in AML samples (n = 30). All data are presented as mean ± standard error of the mean (SEM). *p < 0.05, **p < 0.01, ***p < 0.001 versus normal samples or HS-5.

Figure 2

Effects of miR-4792 on the proliferation and apoptosis in AML cells. HL-60 and Kg1a cells were transfected with miR-4792 mimic or miR-NC for 48 h. (A) The level of miR-4792 was determined by qRT-PCR assay. (B) Cell proliferation was assessed by BrdU-enzyme-linked immunosorbent assay (ELISA) assay. (C) The mRNA expressions of proliferating cell nuclear antigen (PCNA), CDK4, cyclin D1, and p27 were determined by qRT-PCR assay. (D, E) Cell apoptosis was measured by flow cytometric analysis of cells labeled with annexin V/propidium iodide (PI) double staining and nucleosomal degradation, respectively. (F) The activities of caspase 3 were determined by caspase 3 activity detection assay. All data are presented as mean ± SEM, n = 6. #p < 0.05, ##p < 0.01, ###p < 0.001 versus miR-NC.

Figure 3

The effects of miR-4792 on invasion and related molecules in AML cells. HL-60 and Kg1a cells were transfected with miR-4792 mimic or miR-NC for 48 h. (A) The invasion was assessed by Transwell assay. (B, C) Both mRNA and protein expressions of matrix metalloproteinase-2 (MMP-2), MMP-9, and tissue inhibitor of metalloproteinases 1 (TIMP-1) were determined by qRT-PCR and Western blot, respectively. All data are presented as mean ± SEM, n = 6. #p < 0.05, ##p < 0.01, ###p < 0.001 versus miR-NC.

Figure 4

Kindlin-3 was a direct target of miR-4792. HL-60 and Kg1a cells were transfected with miR-4792 mimic or miR-NC for 48 h. (A) Schematic representation of kindlin-3 3′-UTRs showing putative miRNA target site. (B) The analysis of the relative luciferase activities of kindlin-3-WT and kindlin-3-MUT. (C) The protein expression of kindlin-3 was determined by Western blot assay. All data are presented as mean ± SEM, n = 6. ##p < 0.01 versus miR-NC or anti-miR-NC.

Figure 5

The effects of kindlin-3 silencing on cell proliferation, invasion, and epithelial–mesenchymal transition (EMT) in AML cells. HL-60 and Kg1a cells were transfected with si-kindlin-3 or si-NC for 48 h. (A) The protein expressions of kindlin-3 were determined by Western blot assay. (B) Cell proliferation was assessed by BrdU-ELISA assay. (C) The mRNA expressions of PCNA, CDK4, cyclin D1, and p27 were determined by qRT-PCR. (D) The invasion was assessed by Transwell assay. (E) The protein expressions of MMP-2, MMP-9, and TIMP-1 were detected by Western blot assay. (F) Cell apoptosis was measured by nucleosomal degradation by using Roche’s cell death ELISA detection kit. (G) The activities of caspase 3 were determined by caspase 3 activity detection assay. All data are presented as mean ± SEM, n = 6. #p < 0.05, ##p < 0.01 versus si-NC.

Figure 6

Overexpression of kindlin-3 partially promoted cell proliferation, invasion, and EMT in miR-4792-overexpressing AML cells. HL-60 cells were cotransfected with miR-NC or miR-4792 mimic with pcDNA3.1 or pcDNA-kindlin-3 vector. (A) The protein expression of kindlin-3 was determined by Western blot assay. (B) Cell proliferation was assessed by BrdU-ELISA assay. (C) The mRNA expressions of PCNA, CDK4, cyclin D1, and p27 were determined by qRT-PCR. (D) The invasion was assessed by Transwell assay. (E) The expressions of MMP-2, MMP-9, and TIMP-1 were detected by Western blot assay. (F) Cell apoptosis was measured by nucleosomal degradation by using Roche’s cell death ELISA detection kit. (G) The activities of caspase 3 were determined by caspase 3 activity detection assay. All data are presented as mean ± SEM, n = 6. *p < 0.05, **p < 0.01 versus miR-NC; #p < 0.05, ##p < 0.01 versus miR-4792 mimic + pcDNA3.1.