Construction of a Bcl-2-shRNA expression vector and its effect on the mitochondrial apoptosis pathway in SW982 cells

Abstract

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.

The pathogenic role of the EGFR/STAT5 signaling pathway in synovial fibroblasts of patients with rheumatoid arthritis

“…Studies have shown that STAT5 induces the expression of Bcl-2, Bcl-xL, IL-2 and IL-1 [17][18][19] . Anti-apoptotic factors Bcl-2 and Bcl-xL inhibit the apoptosis of RA-FLS by regulating the activities of Caspase-3 and other proteins related to cell proliferation and apoptosis [20][21][22] . Interleukin (IL) 2 is a soluble T cell growth factor that regulates the proliferation and differentiation of T cells and promotes the progression of systemic autoimmune diseases by binding to high-a nity receptor subunits.…”

Non-canonical roles of connexins

“…B-cell lymphoma-2 (Bcl-2) family proteins are key regulators of programmed cell death (Tsujimoto, 1998;Zhang et al, 2017). More specifically, Bcl-2 proteins control the intrinsic apoptotic pathway.…”

“…Apoptotic signals, like temperature variations, exposure to hydrogen peroxide and pH perturbations, induce mitochondrial outer-membrane permeabilization by triggering Bax. Consequently, cytochrome C is released leading to apoptotic cell death by activating cysteine proteases (Li et al, 1997;Zhang et al, 2017).…”

Rapeseed peptide inhibits HepG2 cell proliferation by regulating the mitochondrial and P53 signaling pathways

“…Therefore, they have attracted the attention of scholars at home and abroad 5 . At present, the antitumor mechanism of action of active peptides can be divided into two types: one is mediated by direct action on tumor cells, such as inhibiting tumor cell proliferation and inducing tumor cell apoptosis, and the second is mediated by indirect effects on tumor cells, such as by regulating the body's immune function and inhibiting tumor angiogenesis, among other ways 6‐8 …”

“…5 At present, the antitumor mechanism of action of active peptides can be divided into two types: one is mediated by direct action on tumor cells, such as inhibiting tumor cell proliferation and inducing tumor cell apoptosis, and the second is mediated by indirect effects on tumor cells, such as by regulating the body's immune function and inhibiting tumor angiogenesis, among other ways. [6][7][8] Rapeseed peptide is mainly the product of rapeseed protein obtained by acid hydrolysis, protease hydrolysis or microbial fermentation. Small peptides can be directly absorbed by the intestinal tract, which is faster and more bioactive than amino acids of the same content and composition.…”