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Published on: July 22, 2020
[Construction and identification of CerbB-2 siRNA expression plasmid and its transfer into human colon cancer cell
Dong-li Zhao1, Cheng-xue Dang, Yan-xia Sui
1Center of Oncology, First Affiliated Hospital of Medical College, Xi'an Jiaotong University, Xi'an, China. zhaodongli@tom.com
Objective:
To construct a plasmid carrying small interfering RNA (siRNA) targeting human C-erbB2 gene (pGenesil- erbB2) and test its effect on Her-2 expression at the post-transcriptional level in human colon cancer cell lines HT-29 cells that highly express erbB2.
Methods:
A HT-29 cell line that highly expressed CerbB-2 was selected using immunohistochemical method. The double-stranded siRNA targeting human CerbB-2 cDNA and the negative control fragment were cloned into pGenesil-1 vector, and after identification and sequence analysis, the constructed pGenesil-erbB2 plasmid was transfected into the selected HT-29 cell line.
Results:
The pGenesil-erbB2 plasmid was successfully constructed and stably transferred into HT-29 cells. The transfection resulted in significant inhibition of Her-2 protein expression in the HT-29 cells, as shown by Western blotting.
Conclusion:
The pGenesil-erbB plasmid we constructed can be stably transfected into HT-29 cells to inhibit the expression of Her-2 protein, and can be useful in further studies of increasing the radiosensitivity of HT-29 cell lines.
Insights
Researchers constructed a plasmid (pGenesil-erbB2) to silence the human C-erbB2 gene. This effectively reduced Her-2 protein expression in HT-29 colon cancer cells, showing potential for cancer therapy.
Area of Science:
- Molecular biology
- Cancer research
- Gene silencing
Context:
- Human colon cancer cell line HT-29 exhibits high C-erbB2 expression.
- C-erbB2 (Human Epidermal growth factor Receptor 2) is a key oncogene in various cancers.
- Post-transcriptional gene regulation is a critical mechanism in cancer progression.
Purpose:
- To construct a plasmid encoding small interfering RNA (siRNA) targeting the human C-erbB2 gene.
- To evaluate the efficacy of the constructed plasmid (pGenesil-erbB2) in inhibiting Her-2 expression.
- To assess the impact of Her-2 inhibition on HT-29 colon cancer cells.
Summary:
- A plasmid, pGenesil-erbB2, was successfully constructed by cloning siRNA targeting human C-erbB2 into the pGenesil-1 vector.
- The constructed plasmid was stably transfected into HT-29 cells, which highly express C-erbB2.
- Western blotting confirmed significant inhibition of Her-2 protein expression in HT-29 cells post-transfection.
Impact:
- The developed pGenesil-erbB2 plasmid effectively inhibits Her-2 protein expression at the post-transcriptional level.
- This study demonstrates the potential utility of pGenesil-erbB2 for further investigations into enhancing the radiosensitivity of HT-29 colon cancer cells.
- The findings contribute to the development of targeted therapies for C-erbB2-overexpressing cancers.
