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Combined transfection of Bcl-2 siRNA and miR-15a oligonucleotides enhanced methotrexate-induced apoptosis in Raji cells.

Ding L, Hu XM, Wu H, Liu GX, Gao YJ, He DM, Zhang Y - Cancer Biol Med (2013)

Bottom Line: CCK8 assay showed that cell proliferation was significantly decreased and was significantly lower in miR-15a ODN combined with Bcl-2 siRNA plus MTX group than in miR-15a ODN with methotrexate group, Bcl-2 siRNA with MTX group, and single MTX group (P<0.05).Among these groups, the apoptotic rate of miR-15a ODN combined with Bcl-2 siRNA plus MTX group was the highest; this apoptotic rate was also significantly different from that of miR-15a ODN or Bcl-2 siRNA plus MTX (P<0.05).Bcl-2 siRNA and miR-15a combined with MTX may be a useful approach to improve the treatment effects on lymphoma.

View Article: PubMed Central - PubMed

Affiliation: Institute of Hematology, Jinan University, Guangzhou 510632, China.

ABSTRACT

Objective: B-cell lymphoma 2 (Bcl-2) is an important member of the Bcl-2 family of proteins that regulate the induction of apoptosis. This study aims to investigate whether Bcl-2 small interfering RNA (siRNA) combined with miR-15a oligonucleotides (ODN) could enhance methotrexate (MTX)-induced apoptosis in Raji cells.

Methods: Chemically synthesized miR-15a ODN and Bcl-2 siRNA were transfected in Raji cells by using a HiPerFect Transfection Reagent and then combined with MTX. Expression levels of Bcl-2 protein were detected by Western blot. Cell proliferation was determined by CCK8 assay. The rate of cell apoptosis was determined by Annexin V/PI double staining. The morphology of apoptotic cells was observed by Hoechst-33 258 staining.

Results: After the cells were transfected with miR-15a ODN combined with Bcl-2 siRNA, Bcl-2 protein levels were evidently decreased. CCK8 assay showed that cell proliferation was significantly decreased and was significantly lower in miR-15a ODN combined with Bcl-2 siRNA plus MTX group than in miR-15a ODN with methotrexate group, Bcl-2 siRNA with MTX group, and single MTX group (P<0.05). Hoechst 33258 staining revealed numerous apoptotic cells. AnnexinV/PI double staining showed that the apoptotic rates were (13.13±1.60)%, (34.47±2.96)%, (32.87±3.48)%, and (45.47±2.16)% in MTX, Bcl-2 siRNA plus MTX, miR-15a ODN plus MTX, and miR-15a ODN combined with Bcl-2 siRNA plus MTX groups, respectively. Among these groups, the apoptotic rate of miR-15a ODN combined with Bcl-2 siRNA plus MTX group was the highest; this apoptotic rate was also significantly different from that of miR-15a ODN or Bcl-2 siRNA plus MTX (P<0.05).

Conclusions: Bcl-2 siRNA combined with miR-15a ODN could enhance MTX-induced apoptosis in Raji cells. Bcl-2 siRNA and miR-15a combined with MTX may be a useful approach to improve the treatment effects on lymphoma.

No MeSH data available.


Related in: MedlinePlus

Effect of combined transfection with miR-15a and Bcl-2 siRNA plus MTX on the morphology of Raji cells at 48 h. Nuclear morphology of cells stained with Hoechst-33 258 was analyzed by fluorescence microscopy (×200) at 48 h after transfection. Data are representative microscopic pictures. Untreated cells (A); negative siRNA (B); MTX (C); negative siRNA + MTX (D); scrambled ODN + MTX (E); miR-15a ODN + MTX (F); Bcl-2 siRNA + MTX (G); and miR-15a ODN + Bcl-2 siRNA + MTX (H).
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f3: Effect of combined transfection with miR-15a and Bcl-2 siRNA plus MTX on the morphology of Raji cells at 48 h. Nuclear morphology of cells stained with Hoechst-33 258 was analyzed by fluorescence microscopy (×200) at 48 h after transfection. Data are representative microscopic pictures. Untreated cells (A); negative siRNA (B); MTX (C); negative siRNA + MTX (D); scrambled ODN + MTX (E); miR-15a ODN + MTX (F); Bcl-2 siRNA + MTX (G); and miR-15a ODN + Bcl-2 siRNA + MTX (H).

Mentions: Two methods were used to assess apoptosis: (1) Hoechst staining and (2) Annexin V-FITC/PI double staining by FACS analysis. Figure 3 shows that the cells in the control group exhibited normal nuclear morphology. At 48 h after transfection with Bcl-2 siRNA and miR-15a ODN combined with MTX in Raji cells, significant nuclear condensation and morphological changes such as chromatin condensation and fragmentation were observed. AnnexinV-FITC and PI double staining with FACS were also used to detect cell apoptosis. Figure 4 shows that AnnexinV- FITC/PI double positive cells increased at 48 h after transfection with Bcl-2 siRNA and miR-15a combined with MTX in Raji cells. The apoptotic rate in Bcl-2 siRNA and miR-15a ODN combined with MTX group was (45.47±2.16)%. This apoptotic rate was significantly different compared with miR-15a ODN plus MTX group, Bcl-2 siRNA plus MTX group, or MTX alone group (P<0.05). The apoptotic rates in these three groups were (32.87±3.48)%, (34.47±2.96)%, and (13.13±1.60)%, respectively. These results also had a significant difference compared with the negative siRNA control plus MTX group [(13.33±0.72)%] and the scrambled ODN plus MTX group [(13.57±1.07)%].


Combined transfection of Bcl-2 siRNA and miR-15a oligonucleotides enhanced methotrexate-induced apoptosis in Raji cells.

Ding L, Hu XM, Wu H, Liu GX, Gao YJ, He DM, Zhang Y - Cancer Biol Med (2013)

Effect of combined transfection with miR-15a and Bcl-2 siRNA plus MTX on the morphology of Raji cells at 48 h. Nuclear morphology of cells stained with Hoechst-33 258 was analyzed by fluorescence microscopy (×200) at 48 h after transfection. Data are representative microscopic pictures. Untreated cells (A); negative siRNA (B); MTX (C); negative siRNA + MTX (D); scrambled ODN + MTX (E); miR-15a ODN + MTX (F); Bcl-2 siRNA + MTX (G); and miR-15a ODN + Bcl-2 siRNA + MTX (H).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3643686&req=5

f3: Effect of combined transfection with miR-15a and Bcl-2 siRNA plus MTX on the morphology of Raji cells at 48 h. Nuclear morphology of cells stained with Hoechst-33 258 was analyzed by fluorescence microscopy (×200) at 48 h after transfection. Data are representative microscopic pictures. Untreated cells (A); negative siRNA (B); MTX (C); negative siRNA + MTX (D); scrambled ODN + MTX (E); miR-15a ODN + MTX (F); Bcl-2 siRNA + MTX (G); and miR-15a ODN + Bcl-2 siRNA + MTX (H).
Mentions: Two methods were used to assess apoptosis: (1) Hoechst staining and (2) Annexin V-FITC/PI double staining by FACS analysis. Figure 3 shows that the cells in the control group exhibited normal nuclear morphology. At 48 h after transfection with Bcl-2 siRNA and miR-15a ODN combined with MTX in Raji cells, significant nuclear condensation and morphological changes such as chromatin condensation and fragmentation were observed. AnnexinV-FITC and PI double staining with FACS were also used to detect cell apoptosis. Figure 4 shows that AnnexinV- FITC/PI double positive cells increased at 48 h after transfection with Bcl-2 siRNA and miR-15a combined with MTX in Raji cells. The apoptotic rate in Bcl-2 siRNA and miR-15a ODN combined with MTX group was (45.47±2.16)%. This apoptotic rate was significantly different compared with miR-15a ODN plus MTX group, Bcl-2 siRNA plus MTX group, or MTX alone group (P<0.05). The apoptotic rates in these three groups were (32.87±3.48)%, (34.47±2.96)%, and (13.13±1.60)%, respectively. These results also had a significant difference compared with the negative siRNA control plus MTX group [(13.33±0.72)%] and the scrambled ODN plus MTX group [(13.57±1.07)%].

Bottom Line: CCK8 assay showed that cell proliferation was significantly decreased and was significantly lower in miR-15a ODN combined with Bcl-2 siRNA plus MTX group than in miR-15a ODN with methotrexate group, Bcl-2 siRNA with MTX group, and single MTX group (P<0.05).Among these groups, the apoptotic rate of miR-15a ODN combined with Bcl-2 siRNA plus MTX group was the highest; this apoptotic rate was also significantly different from that of miR-15a ODN or Bcl-2 siRNA plus MTX (P<0.05).Bcl-2 siRNA and miR-15a combined with MTX may be a useful approach to improve the treatment effects on lymphoma.

View Article: PubMed Central - PubMed

Affiliation: Institute of Hematology, Jinan University, Guangzhou 510632, China.

ABSTRACT

Objective: B-cell lymphoma 2 (Bcl-2) is an important member of the Bcl-2 family of proteins that regulate the induction of apoptosis. This study aims to investigate whether Bcl-2 small interfering RNA (siRNA) combined with miR-15a oligonucleotides (ODN) could enhance methotrexate (MTX)-induced apoptosis in Raji cells.

Methods: Chemically synthesized miR-15a ODN and Bcl-2 siRNA were transfected in Raji cells by using a HiPerFect Transfection Reagent and then combined with MTX. Expression levels of Bcl-2 protein were detected by Western blot. Cell proliferation was determined by CCK8 assay. The rate of cell apoptosis was determined by Annexin V/PI double staining. The morphology of apoptotic cells was observed by Hoechst-33 258 staining.

Results: After the cells were transfected with miR-15a ODN combined with Bcl-2 siRNA, Bcl-2 protein levels were evidently decreased. CCK8 assay showed that cell proliferation was significantly decreased and was significantly lower in miR-15a ODN combined with Bcl-2 siRNA plus MTX group than in miR-15a ODN with methotrexate group, Bcl-2 siRNA with MTX group, and single MTX group (P<0.05). Hoechst 33258 staining revealed numerous apoptotic cells. AnnexinV/PI double staining showed that the apoptotic rates were (13.13±1.60)%, (34.47±2.96)%, (32.87±3.48)%, and (45.47±2.16)% in MTX, Bcl-2 siRNA plus MTX, miR-15a ODN plus MTX, and miR-15a ODN combined with Bcl-2 siRNA plus MTX groups, respectively. Among these groups, the apoptotic rate of miR-15a ODN combined with Bcl-2 siRNA plus MTX group was the highest; this apoptotic rate was also significantly different from that of miR-15a ODN or Bcl-2 siRNA plus MTX (P<0.05).

Conclusions: Bcl-2 siRNA combined with miR-15a ODN could enhance MTX-induced apoptosis in Raji cells. Bcl-2 siRNA and miR-15a combined with MTX may be a useful approach to improve the treatment effects on lymphoma.

No MeSH data available.


Related in: MedlinePlus