Proliferation inhibition and apoptosis promotion induced by deletion of TMPO in A549
10.3760/cma.j.issn.0253-3766.2019.10.004
- VernacularTitle: 小干扰RNA抑制胸腺生成素基因的表达对肺癌A549细胞增殖和凋亡的影响
- Author:
Xiangyang DONG
1
;
Wenjing LI
2
;
Bo ZHAI
1
Author Information
1. Thoracic and Cardiac Surgery Department, Children′s Hospital Affiliated to Zhengzhou University/Henan Children′s Hospital/Zhengzhou Children′s Hospital, Zhengzhou 450018, China
2. Endocrinology and Hereditary Metabolites Department, Children′s Hospital Affiliated to Zhengzhou University/Henan Children′s Hospital/Zhengzhou Children′s Hospital, Zhengzhou 450018, China
- Publication Type:Journal Article
- Keywords:
Lung neoplasms;
Thymopoietin;
Cell proliferation;
Apoptosis;
Notch1/mTOR signaling pathway
- From:
Chinese Journal of Oncology
2019;41(10):742-747
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To investigate the effect of thymopoietin (TMPO) gene deleted by small interfering RNA (RNAi) on the proliferation and apoptosis of lung cancer cell A549 and its mechanism.
Methods:TMPO siRNA was transfected into A549 cells by lipofection. The transfected siRNA control was used as a negative control, and the parent cells were used as blank control. Forty-eight hours later, the expression of TMPO in the transfected cells was detected by real-time fluorescent quantitative polymerase chain reaction (RT-PCR) and Western blot. Cell proliferation was detected by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H tetrazolium bromide (MTT) assay, cell cycle and apoptosis were detected by flow cytometry, the protein levels of proliferating cell nuclear antigen (PCNA), cleaved caspase-3, notch receptor 1 (Notch1) and mammalian rapamycin target protein (mTOR) were detected by Western blot analysis.
Results:The results of RT-PCR and Western blot showed that the expression levels of TMPO mRNA in the blank control group, the negative control group and TMPO siRNA transfected group were (1.01±0.11), (0.99±0.10), (0.36±0.04), respectively, the protein levels were (0.27±0.02), (0.29±0.03), (0.08±0.10), respectively. The expression levels of TMPO mRNA and protein in the transfected group were significantly lower than those in the blank control and negative control group (P<0.05). The results of MTT assay showed that the OD values of the blank control group, the negative control group and the transfected group were (0.35±0.04), (0.37±0.04) and (0.34±0.03) at 24 h of transfection, respectively. The OD values at 48 h were (0.47±0.06), (0.46±0.08), (0.37±0.04), the OD values at 72 h were (0.75±0.08), (0.73±0.07), (0.49±0.05), respectively, and the OD values at 96 h were (1.09±0.07), (1.06±0.08), (0.56±0.06). The proliferation abilities of the transfected cells at 48, 72, 96 h were significantly lower than those of the blank control and the negative control group (P<0.05). The results of flow cytometry showed that the proportion of G0/G1 phase cells in blank control group, negative control group and transfection group were (62.55±2.03)%, (61.24±3.15)%, (47.35±2.44)%, respectively. The proportion of cells in S phase were (17.12±1.31)%, (17.70±2.01)%, and (20.81±2.06)%, respectively. The proportion of cells in G2/M phase were (20.33±1.43)%, (21.06±1.52)%, (31.84±2.76)%, respectively. The proportion of cells in G0/G1 phase of transfection group was significantly lower than those of blank control and negative control group (P<0.05). The proportion of cells in G2/M phase of transfection group was significantly higher than those of blank control and negative control group (P<0.05). The apoptosis ratio of the transfection group was (34.10±2.69)%, significantly higher than (2.96±0.03)% of the blank control and (3.01±0.04)% of the negative control group (P<0.05). Western blot analysis showed that PCNA, Notch1 and mTOR proteins were down-regulated while cleaved caspase-3 protein was up-regulated in A549 cells after deletion of TMPO.
Conclusion:The inhibition of TMPO gene expression induced by small interfering RNA can significantly inhibit the proliferation and induce apoptosis of A549 cells, and the mechanism is associated with the inhibition of the activation of Notch1/mTOR signaling pathway.
