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Updated: Jun 13, 2026
DNA Vector-based RNA Interference to Study Gene Function in Cancer
Published on: June 4, 2012
[Construction of lentiviral mediated CyPA siRNA and its functions in non-small cell lung cancer]
Yan-ming FENG1, Yi-ming WU, Xin-ming TU
1Institute of Medicine Henan University of Science and Tochnology, Luoyang 471003, China.
Objective:
To construct a lentiviral-vector-mediated CyPA small interference RNA (siRNA) and study its function in non-small cell lung cancer.
Methods:
First, four target sequences were selected according to CyPA mRNA sequence, the complementary DNA contained both sense and antisense oligonucleotides were designed, synthesized and cloned into the pGCL-GFP vector, which contained U6 promoter and green fluorescent protein (GFP). The resulting lentiviral vector containing CyPA shRNA was named Lv-shCyPA, and it was confirmed by PCR and sequencing. Next, it was cotransfected by Lipofectamine 2000 along with pHelper1.0 and pHelper 2.0 into 293T cells to package lentivirus particles. At the same time, the packed virus infected non-small cell lung cancer cell (A549), the level of CyPA protein at 5 d after infection was detected by Western Blot to screen the target of CyPA. A549 were infected with Lv-shCyPA and grown as xenografts in severe combined immunodeficient mice. Cell cycle and apoptosis were measured by FCM.
Results:
It was confirmed by PCR and DNA sequencing that lentiviral-vector-mediated CyPA siRNA (Lv-shCyPA) producing CyPA shRNA was constructed successfully. The titer of concentrated virus were 1 x 10(7) TU/ml. Flow cytometric analysis demonstrated G2-M phase (11.40% +/- 0.68%) was decreased relatively in A549/LvshCyPA compared with control groups (14.52% +/- 1.19%) (P<0.05). The apoptosis rate of A549/Lv-shCyPA (5.01% +/- 0.5%) was higher than control groups (0.35% +/- 0.17%) (P<0.05). Visible tumors were only detectable at 6th day after inoculated by A549/Lv-shCyPA. The xenograft tumors of A549/Lv-shCyPA remarkably delayed tumor growth and remained at a similarly small average size at 38th days after inoculation compared with the control group (P < 0.05).
Conclusion:
Lentiviral-vector-mediated siRNA technique effectively inhibits the expression of CyPA, induces the NSCLC cell apoptosis, inhibits the tumor growth. Elucidation of the precise role of CypA in these pathways may lead to new targeted therapies for non-small cell lung cancer.
Insights
This study successfully constructed lentiviral-vector-mediated CyPA small interference RNA (siRNA) to inhibit non-small cell lung cancer (NSCLC) growth. The siRNA effectively reduced CyPA expression, induced apoptosis, and suppressed tumor development in NSCLC models.
Area of Science:
- Molecular Biology
- Oncology
- Gene Therapy
Context:
- Non-small cell lung cancer (NSCLC) remains a leading cause of cancer-related mortality worldwide.
- Identifying novel therapeutic targets and strategies is crucial for improving NSCLC treatment outcomes.
- Cyclophilin A (CypA) has been implicated in cancer progression, but its specific role in NSCLC requires further elucidation.
Purpose:
- To construct a lentiviral vector expressing small interference RNA (siRNA) targeting Cyclophilin A (CyPA) for use in non-small cell lung cancer (NSCLC).
- To investigate the functional effects of CyPA knockdown on NSCLC cell proliferation, apoptosis, and tumor growth in vitro and in vivo.
Summary:
- A lentiviral vector (Lv-shCyPA) was successfully constructed and validated to express CyPA-specific shRNA.
- Lv-shCyPA infection of A549 NSCLC cells led to decreased CyPA protein levels, G2-M cell cycle arrest, and increased apoptosis.
- In vivo studies demonstrated that Lv-shCyPA significantly inhibited tumor growth in a xenograft mouse model, delaying tumor formation and reducing overall tumor size.
Impact:
- This study demonstrates the efficacy of lentiviral-vector-mediated siRNA delivery for targeting CyPA in NSCLC.
- The findings suggest that inhibiting CyPA expression can effectively suppress NSCLC progression and holds potential for developing new targeted therapies.
- Further research into the precise mechanisms by which CypA influences NSCLC pathways could pave the way for novel therapeutic strategies.
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