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Selective silencing of DNA topoisomerase IIβ in human mesenchymal stem cells by siRNAs (small interfering RNAs).

Kamaci N, Emnacar T, Karakas N, Arikan G, Tsutsui K, Isik S - Cell Biol Int Rep (2010) (2011)

Bottom Line: A high level of transfection efficiency (80%) was achieved by using unlabelled topo IIβ-specific siRNA oligos.Specifically, it was confirmed repeatedly that green labelled siRNAs interfere with the transfection of siRNAs.The reagent induced minimal cytotoxicity (3.5-4.5%), and cell viability of the transfected hMSCs decreased 20-30% compared with untreated cells, depending on the concentration of the reagent.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Fatih University, Stem Cell Research Laboratory, 34500 Buyukcekmece, Istanbul, Turkey.

ABSTRACT
hMSCs (human mesenchymal stem cells) express two isoforms of DNA topo II (topoisomerase II). Although both isoforms have the same catalytic activity, they are specialized for different functions in the cell: while topo IIα is essential for chromosome segregation in mitotic cells, topo IIβ is involved in more specific cellular functions. A number of inhibitors are available that inhibit the catalytic activity of both topo II isoforms. However, in order to investigate the isoform-specific inhibition of these two enzymes, it is necessary to use other techniques such as siRNA (small interfering RNA) interference to selectively silence either one of the isoforms individually. Depending on the lipid charge densities and protein varieties of the cell membrane, previous studies have demonstrated that transfection efficiencies of siRNAs to hMSCs are very low. In the study reported here, we demonstrate the use of Lipofectamine RNAiMAX as an efficient transfection reagent to introduce siRNAs into human mesenchymal stem cells with significantly great efficiency to silence topo IIβ selectively. A high level of transfection efficiency (80%) was achieved by using unlabelled topo IIβ-specific siRNA oligos. Specifically, it was confirmed repeatedly that green labelled siRNAs interfere with the transfection of siRNAs. The reagent induced minimal cytotoxicity (3.5-4.5%), and cell viability of the transfected hMSCs decreased 20-30% compared with untreated cells, depending on the concentration of the reagent.

No MeSH data available.


Related in: MedlinePlus

RT-PCR and Western blot analysis topo IIβ after siRNA transfections with Lipofectamine RNAiMAXTransfections were performed once at 0 h (A) and twice at 0 and 48 h (B, C). Although actin level did not change during transfection, topo IIβ disappeared at 24 h and reappeared at 72 h (A). While the level of topo IIα and actin did not change during 96 h of transfection, 80% of topo IIβ mRNA disappeared after 24 h, and this low level was observed even after 96 h (B). Protein level of topo IIβ decreased 85–90% during transfection after 24 h, but actin did not change.
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Figure 3: RT-PCR and Western blot analysis topo IIβ after siRNA transfections with Lipofectamine RNAiMAXTransfections were performed once at 0 h (A) and twice at 0 and 48 h (B, C). Although actin level did not change during transfection, topo IIβ disappeared at 24 h and reappeared at 72 h (A). While the level of topo IIα and actin did not change during 96 h of transfection, 80% of topo IIβ mRNA disappeared after 24 h, and this low level was observed even after 96 h (B). Protein level of topo IIβ decreased 85–90% during transfection after 24 h, but actin did not change.

Mentions: Two RNA duplexes of 21 nucleotides in length for topo IIβ (validated unlabelled siRNAs) were obtained from a commercial source, and hMSCs were transfected with a combination of these two siRNAs in Lipofectamine RNAiMAX reagent. Cells were assayed 24, 36, 48, 60 and 72 h after transfection by RT-PCR (Figure 3A). Specific human DNA primers were used for actin (as a control) and topo IIβ genes. Cells transfected with topo IIβ-specific siRNAs resulted in a significant reduction in topo IIβ at mRNA level. Most of the endogenous topo IIβ mRNA disappeared between 24 and 60 h after transfection. At 60 h of transfection, endogenous topo IIβ mRNA reappeared slightly, and after 72 h, almost more than half of the mRNA was retrieved (Figure 3A). A cytoskeleton protein, actin, was used to show that transfection is not non-specific. Actin mRNA was not affected by transfection. To extend the silencing period of the topo IIβ gene, a second transfection was performed after 48 h. Then, silencing of the topo IIβ was reanalysed between 24 and 96 h after transfection by RT-PCR (Figure 3B). At this time, almost 80% silencing was reached after 24 h of the transfection, and this low level of topo IIβ was observed even after 96 h after transfection. Also, since topo IIβ has its isoform, topo IIα, the mRNA level of topo IIα was also checked during 96 h of siRNA transfection to investigate whether topo IIβ is silenced selectively. The results clarified that topo IIα expression at mRNA level is not changed by the siRNA transfection (Figure 3B). Topo IIβ gene silencing was also confirmed by Western blotting at the protein level (Figure 3C). In a Western blot, monoclonal antibody raised against topo IIβ, clone 40, specifically recognized a 180-kDa polypeptide in hMSCs. On the first day after treatment, the topo IIβ protein level was approximately 10–15%, and no protein bands were observed in the later days of the transfection.


Selective silencing of DNA topoisomerase IIβ in human mesenchymal stem cells by siRNAs (small interfering RNAs).

Kamaci N, Emnacar T, Karakas N, Arikan G, Tsutsui K, Isik S - Cell Biol Int Rep (2010) (2011)

RT-PCR and Western blot analysis topo IIβ after siRNA transfections with Lipofectamine RNAiMAXTransfections were performed once at 0 h (A) and twice at 0 and 48 h (B, C). Although actin level did not change during transfection, topo IIβ disappeared at 24 h and reappeared at 72 h (A). While the level of topo IIα and actin did not change during 96 h of transfection, 80% of topo IIβ mRNA disappeared after 24 h, and this low level was observed even after 96 h (B). Protein level of topo IIβ decreased 85–90% during transfection after 24 h, but actin did not change.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3475440&req=5

Figure 3: RT-PCR and Western blot analysis topo IIβ after siRNA transfections with Lipofectamine RNAiMAXTransfections were performed once at 0 h (A) and twice at 0 and 48 h (B, C). Although actin level did not change during transfection, topo IIβ disappeared at 24 h and reappeared at 72 h (A). While the level of topo IIα and actin did not change during 96 h of transfection, 80% of topo IIβ mRNA disappeared after 24 h, and this low level was observed even after 96 h (B). Protein level of topo IIβ decreased 85–90% during transfection after 24 h, but actin did not change.
Mentions: Two RNA duplexes of 21 nucleotides in length for topo IIβ (validated unlabelled siRNAs) were obtained from a commercial source, and hMSCs were transfected with a combination of these two siRNAs in Lipofectamine RNAiMAX reagent. Cells were assayed 24, 36, 48, 60 and 72 h after transfection by RT-PCR (Figure 3A). Specific human DNA primers were used for actin (as a control) and topo IIβ genes. Cells transfected with topo IIβ-specific siRNAs resulted in a significant reduction in topo IIβ at mRNA level. Most of the endogenous topo IIβ mRNA disappeared between 24 and 60 h after transfection. At 60 h of transfection, endogenous topo IIβ mRNA reappeared slightly, and after 72 h, almost more than half of the mRNA was retrieved (Figure 3A). A cytoskeleton protein, actin, was used to show that transfection is not non-specific. Actin mRNA was not affected by transfection. To extend the silencing period of the topo IIβ gene, a second transfection was performed after 48 h. Then, silencing of the topo IIβ was reanalysed between 24 and 96 h after transfection by RT-PCR (Figure 3B). At this time, almost 80% silencing was reached after 24 h of the transfection, and this low level of topo IIβ was observed even after 96 h after transfection. Also, since topo IIβ has its isoform, topo IIα, the mRNA level of topo IIα was also checked during 96 h of siRNA transfection to investigate whether topo IIβ is silenced selectively. The results clarified that topo IIα expression at mRNA level is not changed by the siRNA transfection (Figure 3B). Topo IIβ gene silencing was also confirmed by Western blotting at the protein level (Figure 3C). In a Western blot, monoclonal antibody raised against topo IIβ, clone 40, specifically recognized a 180-kDa polypeptide in hMSCs. On the first day after treatment, the topo IIβ protein level was approximately 10–15%, and no protein bands were observed in the later days of the transfection.

Bottom Line: A high level of transfection efficiency (80%) was achieved by using unlabelled topo IIβ-specific siRNA oligos.Specifically, it was confirmed repeatedly that green labelled siRNAs interfere with the transfection of siRNAs.The reagent induced minimal cytotoxicity (3.5-4.5%), and cell viability of the transfected hMSCs decreased 20-30% compared with untreated cells, depending on the concentration of the reagent.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Fatih University, Stem Cell Research Laboratory, 34500 Buyukcekmece, Istanbul, Turkey.

ABSTRACT
hMSCs (human mesenchymal stem cells) express two isoforms of DNA topo II (topoisomerase II). Although both isoforms have the same catalytic activity, they are specialized for different functions in the cell: while topo IIα is essential for chromosome segregation in mitotic cells, topo IIβ is involved in more specific cellular functions. A number of inhibitors are available that inhibit the catalytic activity of both topo II isoforms. However, in order to investigate the isoform-specific inhibition of these two enzymes, it is necessary to use other techniques such as siRNA (small interfering RNA) interference to selectively silence either one of the isoforms individually. Depending on the lipid charge densities and protein varieties of the cell membrane, previous studies have demonstrated that transfection efficiencies of siRNAs to hMSCs are very low. In the study reported here, we demonstrate the use of Lipofectamine RNAiMAX as an efficient transfection reagent to introduce siRNAs into human mesenchymal stem cells with significantly great efficiency to silence topo IIβ selectively. A high level of transfection efficiency (80%) was achieved by using unlabelled topo IIβ-specific siRNA oligos. Specifically, it was confirmed repeatedly that green labelled siRNAs interfere with the transfection of siRNAs. The reagent induced minimal cytotoxicity (3.5-4.5%), and cell viability of the transfected hMSCs decreased 20-30% compared with untreated cells, depending on the concentration of the reagent.

No MeSH data available.


Related in: MedlinePlus