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Se-methylselenocysteine inhibits apoptosis induced by clusterin knockdown in neuroblastoma N2a and SH-SY5Y cell lines.

Wang C, Zeng Z, Liu Q, Zhang R, Ni J - Int J Mol Sci (2014)

Bottom Line: Its biological function is also associated with cell apoptosis.Se-methylselenocysteine (MSC) at an optimum concentration of 1 μM could reverse the alteration in antioxidative capacity, Bcl2/Bax ratio and caspase-8 activity caused by Clu-knockdown, thus inhibiting apoptosis and maintaining cell viability.The results hereby imply the potentiality of Clu and Se in neuroprotection.

View Article: PubMed Central - PubMed

Affiliation: Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, China. raulw2003@163.com.

ABSTRACT
Apoptosis, as a programmed cell death process, is essential for the maintenance of tissue function in organisms. Alteration of this process is linked to many diseases. Over-expression of clusterin (Clu) can antagonize apoptosis in various cells. Selenium (Se) is an essential trace element for human health. Its biological function is also associated with cell apoptosis. To explore the function of Clu and the impact of Se in the process of apoptosis, several short-hairpin RNAs (shRNA) were designed for the construction of two sets of recombinant plasmids: one set for plasmid-transfection of mouse neuroblastoma N2a cells (N2a cells); and the other set for lentiviral infection of human neuroblastoma SH-SY5Y cells (SH-SY5Y cells). These shRNAs specifically and efficiently interfered with the intracellular expression of Clu at both the mRNA and protein levels. The Clu-knockdown cells showed apoptosis-related features, including down-regulation of antioxidative capacity and the Bcl-2/Bax ratio and up-regulation of caspase-8 activity. Se-methylselenocysteine (MSC) at an optimum concentration of 1 μM could reverse the alteration in antioxidative capacity, Bcl2/Bax ratio and caspase-8 activity caused by Clu-knockdown, thus inhibiting apoptosis and maintaining cell viability. The results hereby imply the potentiality of Clu and Se in neuroprotection.

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Knockdown of Clu gene expression in SH-SY5Y cells. The cells were infected with lentivirus packaged from the plasmids pLKO–shRNAClu1 (Clu-1), pLKO–shRNAClu2 (Clu-2) and pLKO-shRNANc (Nc), respectively. The mRNA expression level was detected by the real-time PCR assay. Cells infected with the lentivirus containing the pLKO–shRNANc plasmid (Nc) were used as the control. ***p < 0.001 vs. the control.
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ijms-15-21331-f002: Knockdown of Clu gene expression in SH-SY5Y cells. The cells were infected with lentivirus packaged from the plasmids pLKO–shRNAClu1 (Clu-1), pLKO–shRNAClu2 (Clu-2) and pLKO-shRNANc (Nc), respectively. The mRNA expression level was detected by the real-time PCR assay. Cells infected with the lentivirus containing the pLKO–shRNANc plasmid (Nc) were used as the control. ***p < 0.001 vs. the control.

Mentions: The packaged lentivirus was used to interfere with Clu expression in SH-SY5Y cells. Three recombinant plasmids, Clu-1, Clu-2 and Nc, were successfully constructed, which are respectively co-transfected HEK293T cells with the packaged plasmids pCMV–VSV-G and pCMV-delta8.9. The packaged lentivirus were used to infect SH-SY5Y cells to knockdown Clu expression. Puromycin (0.5 μg/mL) was used to screen the infected cells for 10 days. The medium with puromycin was changed every 2–3 days. Total RNA of the infected cells was extracted for real-time PCR analysis to detect Clu expression. Results showed that lentivirus packaged from both Clu-1 and Clu-2 plasmids significantly decreased the expression of Clu (Figure 2). The expression level of Clu was significantly reduced by 88.99% (p < 0.001) by the lentivirus packaged from the Clu-1 plasmid. The inhibition efficiency of plasmids Clu-2 is 66.25% (p < 0.001). Thus, the Clu-1 plasmid was selected as the interfering plasmid for the following research.


Se-methylselenocysteine inhibits apoptosis induced by clusterin knockdown in neuroblastoma N2a and SH-SY5Y cell lines.

Wang C, Zeng Z, Liu Q, Zhang R, Ni J - Int J Mol Sci (2014)

Knockdown of Clu gene expression in SH-SY5Y cells. The cells were infected with lentivirus packaged from the plasmids pLKO–shRNAClu1 (Clu-1), pLKO–shRNAClu2 (Clu-2) and pLKO-shRNANc (Nc), respectively. The mRNA expression level was detected by the real-time PCR assay. Cells infected with the lentivirus containing the pLKO–shRNANc plasmid (Nc) were used as the control. ***p < 0.001 vs. the control.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4264228&req=5

ijms-15-21331-f002: Knockdown of Clu gene expression in SH-SY5Y cells. The cells were infected with lentivirus packaged from the plasmids pLKO–shRNAClu1 (Clu-1), pLKO–shRNAClu2 (Clu-2) and pLKO-shRNANc (Nc), respectively. The mRNA expression level was detected by the real-time PCR assay. Cells infected with the lentivirus containing the pLKO–shRNANc plasmid (Nc) were used as the control. ***p < 0.001 vs. the control.
Mentions: The packaged lentivirus was used to interfere with Clu expression in SH-SY5Y cells. Three recombinant plasmids, Clu-1, Clu-2 and Nc, were successfully constructed, which are respectively co-transfected HEK293T cells with the packaged plasmids pCMV–VSV-G and pCMV-delta8.9. The packaged lentivirus were used to infect SH-SY5Y cells to knockdown Clu expression. Puromycin (0.5 μg/mL) was used to screen the infected cells for 10 days. The medium with puromycin was changed every 2–3 days. Total RNA of the infected cells was extracted for real-time PCR analysis to detect Clu expression. Results showed that lentivirus packaged from both Clu-1 and Clu-2 plasmids significantly decreased the expression of Clu (Figure 2). The expression level of Clu was significantly reduced by 88.99% (p < 0.001) by the lentivirus packaged from the Clu-1 plasmid. The inhibition efficiency of plasmids Clu-2 is 66.25% (p < 0.001). Thus, the Clu-1 plasmid was selected as the interfering plasmid for the following research.

Bottom Line: Its biological function is also associated with cell apoptosis.Se-methylselenocysteine (MSC) at an optimum concentration of 1 μM could reverse the alteration in antioxidative capacity, Bcl2/Bax ratio and caspase-8 activity caused by Clu-knockdown, thus inhibiting apoptosis and maintaining cell viability.The results hereby imply the potentiality of Clu and Se in neuroprotection.

View Article: PubMed Central - PubMed

Affiliation: Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, China. raulw2003@163.com.

ABSTRACT
Apoptosis, as a programmed cell death process, is essential for the maintenance of tissue function in organisms. Alteration of this process is linked to many diseases. Over-expression of clusterin (Clu) can antagonize apoptosis in various cells. Selenium (Se) is an essential trace element for human health. Its biological function is also associated with cell apoptosis. To explore the function of Clu and the impact of Se in the process of apoptosis, several short-hairpin RNAs (shRNA) were designed for the construction of two sets of recombinant plasmids: one set for plasmid-transfection of mouse neuroblastoma N2a cells (N2a cells); and the other set for lentiviral infection of human neuroblastoma SH-SY5Y cells (SH-SY5Y cells). These shRNAs specifically and efficiently interfered with the intracellular expression of Clu at both the mRNA and protein levels. The Clu-knockdown cells showed apoptosis-related features, including down-regulation of antioxidative capacity and the Bcl-2/Bax ratio and up-regulation of caspase-8 activity. Se-methylselenocysteine (MSC) at an optimum concentration of 1 μM could reverse the alteration in antioxidative capacity, Bcl2/Bax ratio and caspase-8 activity caused by Clu-knockdown, thus inhibiting apoptosis and maintaining cell viability. The results hereby imply the potentiality of Clu and Se in neuroprotection.

Show MeSH
Related in: MedlinePlus