title: Combined transfection of Bcl-2 siRNA and miR-15a oligonucleotides enhanced methotrexate-induced apoptosis in Raji cells.
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.
authors.affiliation: Institute of Hematology, Jinan University, Guangzhou 510632, China.
authors.forename: Li
authors.initials: L
authors.lastname: Ding
authors.forename: Xiao-Mao
authors.initials: XM
authors.lastname: Hu
authors.forename: Hong
authors.initials: H
authors.lastname: Wu
authors.forename: Ge-Xiu
authors.initials: GX
authors.lastname: Liu
authors.forename: Yang-Jun
authors.initials: YJ
authors.lastname: Gao
authors.forename: Dong-Mei
authors.initials: DM
authors.lastname: He
authors.forename: Yuan
authors.initials: Y
authors.lastname: Zhang
keywords: B-cell lymphoma 2 (Bcl-2); Raji cell; apoptosis; methotrexate; miR-15a; oligonucleotide; small interfering RNA