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Knockdown of Mad2 induces osteosarcoma cell apoptosis-involved Rad21 cleavage
Ling Yu1, Weichun Guo, Shenghao Zhao
1Department of Orthopedics, Renmin Hospital, Wuhan University, Wuhan, Hubei, China.
Background:
Besides Mad2's role in carcinogenesis, recent study has shown that it is essential in cell survival. Here we found that knockdown of Mad2 causes osteosarcoma cell death through apoptosis, with the apoptotic signal resulting from Rad21 cleavage.
Methods:
U2OS and MG63 cells were divided into three groups: the Mad2 siRNA group, mock group and normal control group; the Mad2 siRNA group and mock group are transfected with Mad2 shRNA plasmid and mock plasmid, respectively. G418 was used to increase the transfection efficacy, which was evaluated by GFP fluorescence. Quantitative PCR and Western blotting analyses were used to detect the transcription and expression of Mad2, Rad21 and caspase-3, respectively. Flow cytometry assay using PE-labeled Annexin-V and PI, TUNEL assay and Hoechst 33258 staining were used to evaluate cell apoptosis.
Results:
We successfully achieved knockdown of Mad2 expression in cancer cells using RNA interference. We observed obvious apoptosis in the Mad2 siRNA group compared with the Mock and control group. We found that the apoptosis induced by Mad2 knockdown correlated with Rad21 cleavage.
Conclusion:
These results confirmed that knockdown of Mad2 causes osteosarcoma cell death through apoptosis and provides evidence that the apoptotic signal resulted from Rad21 cleavage. This study suggested that Mad2 has potential to be a novel target for cancer therapy.
Insights
Knockdown of Mad2 triggers osteosarcoma cell death via apoptosis, initiated by Rad21 cleavage. This finding highlights Mad2 as a potential therapeutic target for cancer treatment.
Area of Science:
- Molecular Biology
- Cell Biology
- Cancer Research
Background:
- Mad2 plays a role in carcinogenesis and is crucial for cell survival.
- Previous research indicates Mad2's involvement in cell survival pathways.
Purpose of the Study:
- To investigate the role of Mad2 in osteosarcoma cell death.
- To determine the mechanism by which Mad2 knockdown induces apoptosis.
Main Methods:
- Osteosarcoma cell lines (U2OS, MG63) were treated with Mad2 siRNA.
- Quantitative PCR and Western blotting assessed gene and protein expression.
- Apoptosis was evaluated using flow cytometry, TUNEL assay, and Hoechst staining.
Main Results:
- Mad2 knockdown was successfully achieved using RNA interference.
- Significant apoptosis was observed in Mad2-depleted cells compared to controls.
- Apoptosis induction correlated with Rad21 cleavage.
Conclusions:
- Mad2 knockdown induces osteosarcoma cell death through apoptosis.
- Rad21 cleavage is identified as the apoptotic signal pathway.
- Mad2 presents a potential novel therapeutic target for osteosarcoma.
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