Yonsei Med J.  2019 Feb;60(2):182-190. 10.3349/ymj.2019.60.2.182.

Silencing the PIK3CA Gene Enhances the Sensitivity of Childhood Leukemia Cells to Chemotherapy Drugs by Suppressing the Phosphorylation of Akt

• Liang X
• Xin X
• Qi D
• Fu C
• Ding M

• KMID: 2431635
• DOI: http://doi.org/10.3349/ymj.2019.60.2.182

Abstract

PURPOSE
This study aimed to investigate the effects of PIK3CA on the sensitivity of acute B lymphocytic leukemia cells (Nalm-6 cells) to chemotherapy drugs.
MATERIALS AND METHODS
Children's normal B lymphocytes and Nalm-6 cells were cultured. Nalm-6 cells were transfected with PIK3CA siRNA (siPIK3CA group) or its negative control (PIK3CA-Control group). Normal Nalm-6 cells were named Mock group. Nalm-6 cells transfected by PIK3CA siRNA were treated with Akt inhibitor (siPIK3CA+Akti-1/2 group). mRNA and protein expression was detected by qRT-PCR and Western blot. Proliferation and sensitivity to chemotherapeutic drugs was detected by MTT assay. Cell cycle and apoptosis was explored by low cytometry. Transwell assay was performed to test invasion.
RESULTS
PIK3CA mRNA (p=0.008) and protein (p=0.006) expression was higher in Nalm-6 cells than that in normal B lymphocytes. Compared with the Mock group and PIK3CA-Control group, Nalm-6 cells of the siPIK3CA group had lower OD495 values (all p < 0.05) and invasion cell numbers (p=0.03 and p=0.025), as well as a higher proportion of G0/G1 phase cells (p=0.020 and p=0.022), percentage of apoptosis (p=0.016 and p=0.022), and inhibition rate (all p < 0.05). pAkt expression in the siPIK3CA group (p=0.026 and p=0.031) and siPIK3CA+Akti-1/2 group (p=0.019 and p=0.023) was lower than that in the Mock group.
CONCLUSION
PIK3CA silencing inhibited Nalm-6 cell proliferation and invasion, and promoted their apoptosis and sensitivity to chemotherapeutic drugs, potentially through regulation of the PI3K/AKT signaling pathway.

Keyword

PIK3CA; childhood leukemia cells; proliferation; apoptosis; invasion

MeSH Terms

Apoptosis
B-Lymphocytes
Blotting, Western
Cell Count
Cell Cycle
Cell Proliferation
Drug Therapy*
Leukemia*
Leukemia, B-Cell
Phosphorylation*
RNA, Messenger
RNA, Small Interfering
RNA, Messenger
RNA, Small Interfering

Figure

• 

  Fig. 1 PIK3CA up-regulated in Nalm-6 cells. (A) PIK3CA mRNA was up-regulated in Nalm-6 cells. (B) PIK3CA protein was increased in Nalm-6 cells. *p<0.01.

• 

  Fig. 2 PIK3CA silenced in Nalm-6 cells by siPIK3CA transfection. (A) PIK3CA siRNA transfection inhibited PIK3CA mRNA expression in Nalm-6 cells. (B) PIK3CA siRNA transfection suppressed PIK3CA protein expression in Nalm-6 cells. *p<0.01 when compared with Mock group, †p<0.01 when compared with PIK3CA-Control group.

• 

  Fig. 3 PIK3CA silencing inhibits Nalm-6 cell proliferation. *p<0.05 when compared with Mock group at the same time, †p<0.05 when compared with PIK3CA-Control group at the same time.

• 

  Fig. 4 PIK3CA silencing arrests Nalm-6 cells at G0/G1 phase. (A) Flow cytometry was used to detect cell cycle distributions of cells in each group. The process in this figure included G0/G1, S, and G2/M stage. (B) Statistics on the proportion of cells at different cell cycles of each group. *p<0.05 when compared with Mock group, †p<0.05 when compared with PIK3CA-Control group.

• 

  Fig. 5 PIK3CA silencing promotes Nalm-6 cell apoptosis. (A) Flow cytometry was used to detect apoptosis in each group. (B) Statistics on the percentage of apoptotic cells in each group. *p<0.05 when compared with Mock group, †p<0.05 when compared with PIK3CA-Control group.

• 

  Fig. 6 PIK3CA silencing suppresses Nalm-6 cell invasion. (A) Crystal violet staining (×300) of invasive cells in each group. (B) Statistics on the number of invasive cells in each group. *p<0.05 when compared with Mock group, †p<0.05 when compared with PIK3CA-Control group.

• 

  Fig. 7 PIK3CA silencing enhances Nalm-6 cell sensitivity to chemotherapeutic drugs. (A) Inhibition rate of cells in each group after being treated by vincristine. (B) Inhibition rate of cells in each group after daunorubicin treatment. *p<0.05 when compared with Mock group at the same time, †p<0.05 when compared with PIK3CA-Control group.

• 

  Fig. 8 PIK3CA silencing inhibits the phosphorylation of Akt. (A) Western blot of proteins in each group. (B) Statistical graph of proteins relative expression in each group. *p<0.05 when compared with Mock group, †p<0.05 when compared with PIK3CA-Control group.

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