[Silencing effect study of Akt2-siRNA aimed at lung cancer cell NCI-H446.]
Tingting Cao1, Maolin Zhang, Guoqiang Zhao
1The 5th Department of Health Care, the Fifth Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan 450052, China.
Background:
Lung cancer is one of the most frequent malignant tumors and at the first place of mortality in today's world, It becomes more and more clear that the initiation and development of lung cancer is multistep processes involved in many genes. RNAi gets more and more attention in gene therapy because of its high efficiency and specificity. With RNAi technology, eukaryotic expression vectors aimed at AKT2 siRNA was designed and constructed, which is transfected into human small lung cancer cell line NCI-H446. The silencing effect was observed.
Methods:
Using the known sequence of AKT2's mRNA (M95936), we designed two oligonucleotides that have short hairpin configuration and one oligonucleotide as control. DNA double strands was annealed, and cloned into vectorpGEM-T. Then, blue-white selection and T7/SP6 PCR was used to screen the positive clones, the target DNA and the express vector pRNAT-U6.2 were cut down by BamHI and XhoI restriction enzyme, and linked with each other; use PCR test to filtrate the positive clones and sequenced the target DNA, then transfected it into human small lung cancer cell line (NCI-H446), cultured and screened by G418, RT-PCR was used to detect the expression level of AKT2 mRNA in NCI-H446.
Results:
validate the positive sequence by PCR and, transfect them to lung cancer cell line (NCI-H446), AKT2-mRNA's expression was evidently declined.
Conclusions:
The expression vector pRNAT-U6.2-AKT2 was constructed successfully and the expression of Akt2 mRNA in small lung cancer cell line (NCI-H446) was obviously silenced after transfection.
Insights
This study demonstrates successful silencing of AKT2 mRNA in small lung cancer cells using RNA interference (RNAi) technology. The developed expression vector effectively reduced AKT2 gene expression, offering potential for lung cancer gene therapy.
Area of Science:
- Molecular Biology
- Genetics
- Cancer Research
Context:
- Lung cancer is a leading cause of cancer mortality globally, driven by complex genetic alterations.
- RNA interference (RNAi) presents a promising therapeutic strategy due to its specificity and efficiency in gene silencing.
- Targeting specific genes involved in tumorigenesis is crucial for developing effective lung cancer treatments.
Purpose:
- To construct an eukaryotic expression vector for AKT2 small interfering RNA (siRNA).
- To evaluate the efficacy of the constructed vector in silencing AKT2 mRNA expression in the NCI-H446 small lung cancer cell line.
- To validate the potential of RNAi-mediated AKT2 silencing as a therapeutic approach for lung cancer.
Summary:
- Two distinct siRNA oligonucleotides targeting AKT2 mRNA were designed and cloned into the pRNAT-U6.2 vector.
- The resulting pRNAT-U6.2-AKT2 vector was successfully constructed and validated using PCR and sequencing.
- Transfection into NCI-H446 cells led to a significant reduction in AKT2 mRNA expression, confirmed by RT-PCR.
Impact:
- Successful construction of a functional RNAi vector targeting AKT2.
- Demonstration of effective AKT2 gene silencing in a human small lung cancer cell line.
- Provides a foundation for further investigation into RNAi-based therapies for lung cancer.
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