[Proliferation inhibition and apoptosis promotion induced by deletion of TMPO in A549]
1Thoracic and Cardiac Surgery Department, Children's Hospital Affiliated to Zhengzhou University/Henan Children's Hospital/Zhengzhou Children's Hospital, Zhengzhou 450018, China.
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 G(0)/G(1) 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 G(2)/M phase were (20.33±1.43)%, (21.06±1.52)%, (31.84±2.76)%, respectively. The proportion of cells in G(0)/G(1) phase of transfection group was significantly lower than those of blank control and negative control group (P<0.05). The proportion of cells in G(2)/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.
Insights
Small interfering RNA (siRNA) targeting the thymopoietin (TMPO) gene significantly reduced lung cancer cell proliferation and induced apoptosis in A549 cells. This effect is linked to the inhibition of the Notch1/mTOR signaling pathway.
Area of Science:
- Oncology
- Molecular Biology
- Gene Regulation
Background:
- Lung cancer remains a leading cause of cancer-related mortality worldwide.
- Thymopoietin (TMPO) is a protein implicated in various cellular processes.
- Understanding the role of TMPO in lung cancer pathogenesis is crucial for developing novel therapeutic strategies.
Purpose of the Study:
- To investigate the impact of thymopoietin (TMPO) gene silencing using small interfering RNA (siRNA) on the proliferation and apoptosis of A549 lung cancer cells.
- To elucidate the underlying molecular mechanisms, particularly the involvement of the Notch1/mTOR signaling pathway.
Main Methods:
- A549 lung cancer cells were transfected with TMPO-specific siRNA.
- Gene expression was assessed using real-time quantitative PCR (RT-PCR) and Western blot.
- Cell proliferation was measured by MTT assay.
- Cell cycle distribution and apoptosis rates were analyzed via flow cytometry.
- Protein levels of PCNA, cleaved caspase-3, Notch1, and mTOR were quantified by Western blot.
Main Results:
- TMPO gene silencing significantly reduced TMPO mRNA and protein expression in A549 cells.
- TMPO knockdown markedly inhibited cell proliferation and induced significant apoptosis.
- Flow cytometry revealed a decrease in G0/G1 phase cells and an increase in G2/M phase cells, alongside elevated apoptosis.
- Western blot analysis indicated downregulation of PCNA, Notch1, and mTOR, and upregulation of cleaved caspase-3.
Conclusions:
- Inhibition of TMPO gene expression via siRNA effectively suppresses A549 lung cancer cell proliferation and promotes apoptosis.
- The observed effects are mediated through the downregulation of the Notch1/mTOR signaling pathway.
- Targeting TMPO presents a potential therapeutic approach for lung cancer treatment.
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