Analysis of the expression of PHTF1 and related genes in acute lymphoblastic leukemia

Abstract

Background: Previous study showed that downregulated BCL11B expression in T cell acute lymphoblastic leukemia (T-ALL) cell line Molt-4 inhibited cell proliferation and induce apoptosis, which may be related to PHTF1 gene overexpression. The objective of this study was to investigate the expression of PHTF1 and related genes in ALL and further explore its function in T-ALL cell lines.

Methods: Real-time PCR was used to determine the gene expression level of PHTF1 in hematologic malignancies. The PHTF1, BCL11B, FEM1B and Apaf-1 gene expression levels and correlations were analyzed in patients with primary ALL (including T-ALL and B-ALL) and healthy individuals (HIs). Inhibition and overexpression of PHTF1 by lentiviral transduction were performed using the Molt-4 and Jurkat cell lines. Cell growth and apoptosis were measured by the Cell Counting Kit-8 assay and flow cytometry, respectively. Upon PHTF1 overexpression, the BCL11B, FEM1B and Apaf-1 gene expression levels were determined by real-time PCR.

Results: PHTF1 overexpression was found in both T-ALL (p = 0.004) and B-ALL (p < 0.001) groups compared with HIs group. A trend toward a negative correlation between the PHTF1 and BCL11B genes was detected for the T-ALL group, while positively correlated expression was found for the PHTF1 and BCL11B genes in HIs (P = 0.001). FEM1b and Apaf-1 overexpression was found in recently diagnosed ALL patients compared with HIs (p < 0.05). Positively correlated expression was found for the PHTF1, FEM1b and Apaf-1 genes in patients with ALL (p < 0.05) and HIs (p < 0.05). Direct up-regulation of PHTF1 expression inhibited the proliferation of Jurkat and Molt-4 cells and effectively induced apoptosis in Molt-4 cells. Direct inhibition of PHTF1 expression had no significant effect on the proliferation or apoptosis of Jurkat and Molt-4 cells. FEM1b and Apaf-1 overexpression, which did not obviously alter the BCL11B expression level, was detected in PHTF1-transduced T-ALL cell lines.

Conclusions: PHTF1 overexpression is responsible for regulating cell proliferation and apoptosis in T-ALL cell lines. PHTF1 may be a tumor-suppressor like gene and a therapeutic target for triggering the PHTF1-FEM1b-Apaf-1 apoptosis pathway.

Keywords: ALL; Apaf-1; Apoptosis; BCL11B; FEM1b; Overexpression; PHTF1; Proliferation.

Fig. 1

PHTF1 and BCL11B expression level in the different ALL subtypes and healthy individuals. a PHTF1 expression level in the different ALL subtypes and healthy individuals. PHTF1 overexpression was detected in recently diagnosed T-ALL and B-ALL patients. P = 0.004 for T-ALL versus HIs, P < 0.001 for B-ALL versus HIs. b BCL11B expression level in the different ALL subtypes and healthy individuals. BCL11B overexpression was detected in patients with T-ALL. P < 0.001 for T-ALL versus B-ALL

Fig. 2

Correlation analyses of the PHTF1 and BCL11B expression levels. a No significant correlation was found for the PHTF1 and BCL11B genes in B-ALL patients. b A negative correlation trend was detected for the PHTF1 and BCL11B genes in the T-ALL patients. c A positively correlated expression level for the PHTF1 and BCL11B genes was found in the HIs

Fig. 3

FEM1b and Apaf-1 expression levels in the ALL patients and healthy individuals. FEM1b and Apaf-1 overexpression was found in recently diagnosed ALL patients versus HIs. P = 0.014 for FEM1b, P = 0.001 for Apaf-1

Fig. 4

Correlation analysis of the PHTF1, FEM1b and Apaf-1 expression levels. Positively correlated expression for the PHTF1, FEM1b and Apaf-1 genes was found in ALL patients (a PHTF1 vs. FEM1b, b FEM1b vs. Apaf-1) and HIs (c PHTF1 vs. FEM1b, d FEM1b vs. Apaf-1)

Fig. 5

Knockdown and overexpression of PHTF1 in T-ALL cell lines. a, b PHTF1 knockdown with PHTF1 shRNA lentivirus in Jurkat (a) and Molt-4 (b) cells. PHTF1 was down-regulated approximately 3-fold in both Jurkat and Molt4 cells. P < 0.01 for bar 1 versus bar 2 and bar 3 versus bar 4. c, d PHTF1 overexpression by PHTF1 lentivirus in Jurkat (c) and Molt-4 (d) cells. PHTF1 was up-regulated approximately 2291-fold in Jurkat cells and approximately 1100-fold in Molt4 cells. P < 0.05 for bar 1 versus bar 2 and bar 3 versus bar 4. The results represent the mean ± SEM (n = 3)

Fig. 6

PHTF1 overexpression inhibits the proliferation of Jurkat and Molt-4 cells. a Jurkat cells infected with PHTF1 (PHTF1) or control (CON 2) lentivirus were seeded in a 96-well plate and incubated for 72 h. The CCK8 assay demonstrated that cells infected with PHTF1 have lower viability compared with control. The results shown are the mean ± SEM (n = 3). P = 0.004 for bar 1 versus bar 2. b Molt-4 cells infected with PHTF1 (PHTF1) or control (CON 2) lentivirus were seeded in a 96-well plate and incubated for 72 h. The CCK8 assay demonstrated that cells infected with PHTF1 have lower viability compared with controls. The results represent the mean ± SEM (n = 3). P = 0.007 for bar 1 versus bar 2. c Jurkat cells infected with PHTF1 shRNA (RNAi) or control (CON 1) lentivirus were seeded in a 96-well plate and incubated for 72 h. The CCK8 assay demonstrated that cells infected with PHTF1 shRNA have no significant difference compared with controls. The results represent the mean ± SEM (n = 3). NS no significance. d Molt4 cells infected with PHTF1 shRNA (RNAi) or control (CON 1) lentivirus were seeded in a 96-well plate and incubated for 72 h. The CCK8 assay demonstrated that cells infected with PHTF1-shRNA have no significant difference compared with controls. The results represent the mean ± SEM (n = 3). NS no significance

Fig. 7

PHTF1 overexpression induces apoptosis in Molt-4 cells. a Representative FACS plots and summary (right) of apoptotic Molt 4 cells after transduction with PHTF1 (PHTF1, left) or control (CON 2, middle) lentivirus. Data represent the mean ± SEM (n = 3). P = 0.021 for bar 1 versus bar 2. b Representative FACS plots and summary (right) of apoptotic Jurkat cells after transduction with PHTF1 (PHTF1, left) or control (CON 2, middle) lentivirus. Data represent the mean ± SEM (n = 3). NS no significance. c, d Summary of apoptotic Jurkat (c) or Molt-4 (d) cells after transduction with PHTF1 shRNA (RNAi) or control (CON 1) lentivirus. Data represent the mean ± SEM (n = 3). NS no significance

Fig. 8

Genes activated with PHTF1 overexpression. a Expression level of BCL11B, FEM1b and Apaf-1 in Jurkat cells after PHTF1 overexpression by transduction with PHTF1 lentivirus (PHTF1) or in the presence of control lentivirus (CON 2). The BCL11B expression level was unchanged versus control; FEM1b and Apaf-1 were up-regulated 2.4- and 4.4-fold, respectively, versus control. Data shown are the mean ± SEM (n = 3), P = 0.007 for bar 3 versus bar 4; P = 0.009 for bar 5 versus bar 6. b Expression levels of BCL11B, FEM1b and Apaf-1 in Molt-4 cells after PHTF1 overexpression by PHTF1 lentivirus (PHTF1) or control lentivirus (CON 2). The BCL11B expression level was unchanged versus control; FEM1b and Apaf-1 were up-regulated 6.3- and 29.4-fold, respectively, versus control. Data shown are the mean ± SEM (n = 3), P = 0.020 for bar 3 versus bar 4; P = 0.005 for bar 5 versus bar 6