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

Li Ding, Xiao-mao Hu, Hong Wu, Ge-Xiu Liu, Yang-Jun Gao, Dong-Mei He, Yuan Zhang

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.

Keywords


B-cell lymphoma 2 (Bcl-2); small interfering RNA; oligonucleotide; methotrexate; Raji cell; miR-15a; apoptosis

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