Construction of a Bcl-2-shRNA expression vector and its effect on the mitochondrial apoptosis pathway in SW982 cells
- Weidong Zhang
- Xinjie Xue
- Teng Fu
Published online on: September 27, 2017
Apoptosis is considered to serve an important role in the pathogenesis of rheumatoid arthritis. The aim of the present study was to construct Bcl-2-short hairpin (sh)RNA expression vectors and transfect them into human synovial sarcoma SW982 cells, in order to screen for an effective interference sequence and analyze the effects of this interference on the expression levels of Bcl-2 and other molecules associated with the mitochondrial apoptosis pathway. Three different shRNAs (Bcl-2-sh1, 2 and 3) were designed according to the human Bcl-2 mRNA target sequence and were transformed into competent DH5α Escherichia coli cells following the construction of an expression vector, which was then transfected into SW982 cells. SW982 cells were grouped into a control group (transfected with a negative control shRNA), and Bcl-2-sh1, Bcl-2-sh2 and Bcl-2-sh3 groups (transfected with Bcl-2-sh1, 2 and 3, respectively). The expression levels of Bcl-2 mRNA were detected using reverse transcription-quantitative PCR (RT-qPCR). Bcl-2-sh1 was identified as the most effective shRNA sequence for interference, and was used for subsequent experiments. The mRNA and protein expression levels of Bcl-2, Bax, CytC and Caspase-3 were detected in SW982 cells by RT-qPCR and western blotting at various time-points (48 and 72 h) following transfection with Bcl-2-sh1, in order to observe the effectiveness of this interference. Compared with the control group, the expression levels of Bcl-2 were decreased, while those of Bax, CytC and Caspase-3 were increased in Bcl-2-sh1-transfected cells (P<0.01). The interference effect was greater at 48 h than at 72 h. In summary, an effective shRNA sequence (Bcl-2-sh1) targeting the Bcl-2 gene was identified from three candidates, and was demonstrated to significantly interfere with the expression of Bcl-2, Bax, CytC and Caspase-3 when transfected into SW982 cells. The interference effect of Bcl-2-sh1 was more pronounced at 48 h than at 72 h post-transfection.