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High-throughput Quantitative Real-time RT-PCR Assay for Determining Expression Profiles of Types I and III Interferon Subtypes
Published on: March 24, 2015
Interferon gamma-induced hub genes and key pathways: A study based on biological network analysis and experimental
Reza Heidari-Japelaghi1, Mostafa Valizadeh2, Raheem Haddad1
1Department of Biotechnology Engineering, Faculty of Agriculture and Natural Resources, Imam Khomeini International University, Qazvin, Iran.
Abstract:
By performing a biological network analysis, we identified some hub genes, which were up- or down-regulated in the breast cancer (BC) cell line after treatment with IFN-γ. Moreover, several pathways including cytokine-cytokine receptor interaction, TNF signaling pathway, NOD-like receptor signaling pathway, and NF-κB signaling pathway were detected that their activation leads to the antiproliferation, proapoptosis, and antiviral activities. To validate in silico results, the bioactivity of recombinant human IFN-γ (hIFN-γ) produced in different hosts was analyzed by antiviral and anticancer assays. The antiviral role of the hIFN-γ preparations was evaluated by inhibition of Vesicular Stomatitis Virus (VSV)-mediated cytopathic effects on Vero cells. A dose-dependent increase in cell viability was observed at different concentrations of recombinant proteins. The maximum amount of the cell viability detected for the hIFN-γ preparations was determined at a concentration of 32.00 pg/mL. To analyze the cytotoxic efficacy of the hIFN-γ preparations on the growth and development of tumor cells, a BC cell line (MCF-7) was treated with both recombinant protein forms in a time- and dose-dependent way. The highest level of inhibiting cell proliferation was detected at a concentration of 32.00 pg/mL hIFN-γ after 72 h incubation. Anticancer and antiviral functions of IFN-γ were confirmed via the expression analysis of hub genes cd74, cxcl10, il6, and stat1 using RT-PCR. Furthermore, the hIFN-γ preparations were significantly able to up-regulate the expression of proapoptotic Bax and p53 and to down-regulate Bcl-2 as an antiapoptotic gene, showing the cytotoxic effect of hIFN-γ toward MCF-7 cells via apoptosis induction.
Insights
Interferon-gamma (IFN-γ) exhibits anticancer and antiviral properties by regulating key genes and pathways. Recombinant human IFN-γ (hIFN-γ) effectively inhibits breast cancer cell proliferation and induces apoptosis.
Area of Science:
- Molecular Biology
- Immunology
- Oncology
Background:
- Interferon-gamma (IFN-γ) is a crucial cytokine involved in immune responses.
- Its role in cancer and viral infections is well-established, but the precise molecular mechanisms require further elucidation.
- Biological network analysis can identify key genes and pathways affected by IFN-γ.
Purpose of the Study:
- To identify hub genes and signaling pathways modulated by IFN-γ in breast cancer (BC).
- To validate the in silico findings by assessing the antiviral and anticancer bioactivity of recombinant human IFN-γ (hIFN-γ).
- To confirm the anticancer effects through apoptosis induction and gene expression analysis.
Main Methods:
- Biological network analysis to identify hub genes and pathways.
- Antiviral assays using Vesicular Stomatitis Virus (VSV) on Vero cells.
- Anticancer assays on MCF-7 breast cancer cell line with recombinant hIFN-γ.
- RT-PCR analysis of hub genes (cd74, cxcl10, il6, stat1) and apoptosis-related genes (Bax, p53, Bcl-2).
Main Results:
- Network analysis identified hub genes and pathways (cytokine-cytokine receptor interaction, TNF, NOD-like receptor, NF-κB signaling) linked to antiproliferation, proapoptosis, and antiviral activities.
- Recombinant hIFN-γ demonstrated dose-dependent antiviral activity against VSV and significant inhibition of MCF-7 cell proliferation, with maximum efficacy at 32.00 pg/mL.
- hIFN-γ treatment upregulated proapoptotic genes (Bax, p53) and downregulated antiapoptotic gene (Bcl-2), confirming apoptosis induction in MCF-7 cells.
Conclusions:
- IFN-γ plays a significant role in antiproliferation, proapoptosis, and antiviral activities through specific signaling pathways.
- Recombinant hIFN-γ possesses potent anticancer and antiviral bioactivity, validating its therapeutic potential.
- The study confirms IFN-γ's mechanism of action involves modulating key gene expression and inducing apoptosis in breast cancer cells.
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