Downregulation of SMIM3 inhibits growth of leukemia via PI3K-AKT signaling pathway and correlates with prognosis of adult acute myeloid leukemia with normal karyotype

Abstract

Background: Acute myeloid leukemia (AML) patients with normal karyotype (NK-AML) have significant variabilities in outcomes. The European Leukemia Net stratification system and some prognostic models have been used to evaluate risk stratification. However, these common standards still have some limitations. The biological functions and mechanisms of Small Integral Membrane Protein 3 (SMIM3) have seldomly been investigated. To this date, the prognostic value of SMIM3 in AML has not been reported. This study aimed to explore the clinical significance, biological effects and molecular mechanisms of SMIM3 in AML.

Methods: RT-qPCR was applied to detect the expression level of SMIM3 in bone marrow specimens from 236 newly diagnosed adult AML patients and 23 healthy volunteers. AML cell lines, Kasumi-1 and THP-1, were used for lentiviral transfection. CCK8 and colony formation assays were used to detect cell proliferation. Cell cycle and apoptosis were analyzed by flow cytometry. Western blot was performed to explore relevant signaling pathways. The biological functions of SMIM3 in vivo were validated by xenograft tumor mouse model. Survival rate was evaluated by Log-Rank test and Kaplan-Meier. Cox regression model was used to analyze multivariate analysis. The correlations between SMIM3 and drug resistance were also explored.

Results: Through multiple datasets and our clinical group, SMIM3 was shown to be significantly upregulated in adult AML compared to healthy subjects. SMIM3 overexpression conferred a worse prognosis and was identified as an independent prognostic factor in 95 adult NK-AML patients. Knockdown of SMIM3 inhibited cell proliferation and cell cycle progression, and induced cell apoptosis in AML cells. The reduced SMIM3 expression significantly suppressed tumor growth in the xenograft mouse model. Western blot analysis showed downregulation of p-PI3K and p-AKT in SMIM3-knockdown AML cell lines. SMIM3 may also be associated with some PI3K-AKT and first-line targeted drugs.

Conclusions: SMIM3 was highly expressed in adult AML, and such high-level expression of SMIM3 was associated with a poor prognosis in adult AML. Knockdown of SMIM3 inhibited the proliferation of AML through regulation of the PI3K-AKT signaling pathway. SMIM3 may serve as a potential prognostic marker and a therapeutic target for AML in the future.

Keywords: Acute myeloid leukemia; Cell cycle; Cell proliferation; PI3K-AKT; Prognosis; SMIM3.

Fig. 1

AML samples showed a higher expression of SMIM3 compared to normal samples. A Expression levels of SMIM3 in paired samples of normal and tumor patients in different cancers. B In the TCGA-GTEx database, SMIM3 showed significantly higher expression in AML patients (n = 173) than in normal samples (n = 70). C In the ImmuCo database, the gene expression level of SMIM3 was higher in AML bone marrow mononuclear cells (BMMC, n = 814) than in hematopoietic stem cells (HSC, n = 113). D AML samples (n = 236) from the ZZU cohort showed a significant increase in SMIM3 expression compared to normal bone marrow samples (n = 22). E SMIM3 expression in different cell lines. *, P < 0.05; **, P < 0.01; ***, P < 0.001

Fig. 2

Overall survival (OS) of adult subjects with AML according to SMIM3. A OS of 151 AML subjects in TCGA. B OS of 69 AML subjects with normal karyotype in TCGA. C OS of 163 AML subjects in GSE12417-GPL97. D OS of 79 AML subjects in GSE12417-GPL570. G OS of 95 AML subjects with normal karyotype in ZZU cohort. H Differences in OS between the transplant and non-transplant groups in ZZU NK-AML cohort. Overall survival (OS) of adult subjects with AML according to risk-stratification system. E OS of 236 AML subjects in ZZU cohort. F OS of 95 AML subjects with normal karyotype in ZZU cohort

Fig. 3

Downregulation of SMIM3 inhibited cell proliferation and colony formation in AML cells. The efficiency of SMIM3 knockdown in the Kasumi-1 and THP-1 cell lines was verified by A, B reverse transcription-quantitative polymerase chain reaction and E, F immune fluorescence, respectively. C, D Cell proliferation was detected by the CCK-8 assay in Kasumi-1 and THP-1. G, H The colony formation of SMIM3-CTRL and SMIM3-KD in Kasumi-1 and THP-1. CTRL, control; KD, knockdown; *P < 0.05 compared with CTRL cells; **P < 0.01 compared with CTRL cells; ***P < 0.001 compared with CTRL cells; Error bars indicate the standard deviation

Fig. 4

Apoptosis and cell cycle analysis in AML cell lines. A, B The flow cytometry was applied to analyze apoptosis in SMIM3 knockdown cells compared with control. C–E Propidium iodide (PI) staining was applied to analyze the cell cycle of SMIM3 knockdown cells compared with control. F Alterations in apoptosis and cell cycle-related protein assay in SMIM3 knockdown cells compared with control. G, H Densitometric analysis of the WB signals. CTRL, control; KD, knockdown; *P < 0.05 compared with CTRL cells; **P < 0.01 compared with CTRL cells; ***P < 0.001 compared with CTRL cells

Fig. 5

GO/KEGG enrichment analysis of subjects with high or low SMIM3 expression in the TCGA-LAML dataset. A Volcano map of the DEGs, including 231up-regulated genes and 1889 down-regulated genes. B Heat map showing the top ten up-regulated and the top ten down-regulated genes. The samples are shown on the X-axis, and the DEGs are shown on the Y-axis. D Venn diagram of the overlap among the DEGs and correlated genes. C GO and KEGG enrichment analysis of the co-expressed genes of DEGs and correlated genes. MF, molecular function. CC, cellular component. BP, biological process. Different categories were shown on the Y-axis, and the X-axis reflected the percentage of DEGs. PI3K-AKT signaling pathway is critical for SMIM3-mediated changes. G Western blot analysis of PI3K-AKT signaling pathway in SMIM3-KD cells and controls. H, I Densitometric analysis of the WB signals. E SC79 (10 mM) reversed the inhibitory effect of SMIM3 knockdown on the proliferation of Kasumi-1, F at 24 h. CTRL, control; KD, knockdown; ***P < 0.001 compared with CTRL cells; Error bars indicate the standard deviation

Fig. 6

SMIM3 knockdown reduced the proliferation of AML cells in nude mice by inhibiting PI3K-AKT signaling pathway. A–D, F–I SMIM3-KD inhibits tumor growth in Kasumi-1 and THP-1 cells. E, J The expression of PI3K-AKT signaling pathway proteins in tumor tissues by Western blot. CTRL, control; KD, knockdown; ***P < 0.001 compared with CTRL cells

Fig. 7

The relationship between drugs sensitivity and the expression level of SMIM3