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Published on: December 13, 2018
[Effect of Interfering the ADAM10 by Lentivirul Vector-Mediated shRNA on Multiple Myeloma MM.1S Cell Proliferation]
Li-Fan Xu1, Jian-Ping Luo1, Hu-Jun Li1
1Department of Hematology,The Affiliated Hospital of Xuzhou Medical College, Xuzhou 221002, Jiangsu Province, China.
Objective:
To explore the effect of interfering ADAM10 on proliferation and apoptosis of multiple myeloma MM.1S cells, and its possible mechanism.
Methods:
Four pairs of shRNA-coding sequences directed against different sites of ADAM10 mRNA were designed and inserted into lentiviral vector plasimd pLVshRNA-EGFP(2A) Puro for constructing the sh/ADAM10-1, sh/ADAM10-2, sh/ADAM10-3, sh/ADAM10-4 and sh/Con. These plasmids and lentiviral packaging plasmids were co-transfected into the packaging cells 293FT, then the virus particles were collected and the viral titer was assayed after concentration, and these viral particles were transfected to MM.1S cells. The flow cytometry was used to sort GFP+ cells. Real-time quantitative PCR, and Western blot were used to detect the effect of interfering the ADAM10 gene by lentiviral vector mediated shRNA. The proliferation-inhibition curve was plotted by CCK-8 method, the cell viability and apoptosis were detected by flow cytometry with Annexin V and 7-AAD staining, the transcripts of pro-apoptosis gene BAD, BAK, BIK, anti-apoptotic genes BCL-2, c-Myc and Notch1 target gene Hes-1 were detected by real-time PCR.
Results:
Lentivirus vector was successfully constructed, that could specifically interfere ADAM10 expression. Interfering ADAM10 gene could inhibit the MM.1S cell proliferation and induce apoptosis. After the interferencing ADAM10 gene the mRNA levels of pro-apoptosis gene BAD, BAK and BIK were increased, and the mRNA levels of anti-apoptotic genes BCL-2 and c-Myc were reduced. Q-PCR results showed that the mRNA level of Notch1 were increased, but that of Hes-1 were reduced.
Conclusion:
Down-regulated ADAM10 expression can significantly inhibit multiple myeloma MM.1S cell proliferation and promote the apotosis. Its mechanism may be related to Notch1 signaling pathways.
Insights
Down-regulating ADAM10 in multiple myeloma cells inhibits proliferation and promotes apoptosis. This effect is linked to the Notch1 signaling pathway, offering potential therapeutic insights.
Area of Science:
- Molecular Biology
- Cancer Research
- Cell Biology
Background:
- Multiple myeloma (MM) is a hematologic malignancy characterized by uncontrolled proliferation of plasma cells.
- ADAM10 (a disintegrin and metalloproteinase domain-10) is implicated in various cellular processes, including cell growth and survival.
- Understanding the role of ADAM10 in MM pathogenesis is crucial for developing targeted therapies.
Purpose of the Study:
- To investigate the impact of interfering with ADAM10 expression on the proliferation and apoptosis of multiple myeloma MM.1S cells.
- To elucidate the underlying molecular mechanisms, particularly the involvement of the Notch1 signaling pathway.
Main Methods:
- Lentiviral vectors expressing short hairpin RNA (shRNA) targeting ADAM10 were constructed and introduced into MM.1S cells.
- Cell proliferation was assessed using the CCK-8 assay.
- Apoptosis, cell viability, and gene expression (including BAD, BAK, BIK, BCL-2, c-Myc, Notch1, and Hes-1) were analyzed by flow cytometry and real-time quantitative PCR.
Main Results:
- Successful construction of lentiviral vectors specifically interfering with ADAM10 expression.
- Interference with ADAM10 significantly inhibited MM.1S cell proliferation and induced apoptosis.
- Upregulation of pro-apoptotic genes (BAD, BAK, BIK) and downregulation of anti-apoptotic genes (BCL-2, c-Myc) were observed.
- Notch1 mRNA levels increased, while its target gene Hes-1 mRNA levels decreased.
Conclusions:
- Down-regulation of ADAM10 effectively inhibits multiple myeloma cell proliferation and promotes apoptosis.
- The observed effects are potentially mediated through the Notch1 signaling pathway.
- Targeting ADAM10 represents a promising therapeutic strategy for multiple myeloma.
