Effect of RNA interference targeting brain-derived neurotrophic factor gene on HeLa cell proliferation and apoptosis / 中华病理学杂志

ABSTRACT

<p><b>OBJECTIVE</b>To screen effective sequences of short hairpin RNA on brain-derived neurotrophic factor (BDNF) gene and the effect of RNA interference on the proliferation and apoptosis of HeLa cells, a cervix carcinoma cell line with high expression of BDNF.</p><p><b>METHODS</b>Two recombinant eukaryotic human-BDNF siRNA expression vectors were designed and constructed. Sequences were confirmed by restrictive endonuclease digestion and DNA sequencing. The empty vector pGenesil-1 and two recombinant plasmids, pGenesil-shRNA-BDNF1 and pGenesil-shRNA-BDNF2, were transfected into HeLa cells using Lipofectamine 2000 (groups P(0), P(1) and P(2), respectively). The mRNA and protein levels of BDNF in HeLa cells were detected by RT-PCR and Western blot, respectively. The cellular proliferation rates were determined by MTT assay and the apoptotic rates were measured by flow cytometry and Hoechest 33258.</p><p><b>RESULTS</b>The recombinant eukaryotic BDNF siRNA expression vectors were successfully constructed. The expression of mRNA and protein of BDNF in P(1) group were significantly decreased, comparing with non-transfected group, P(0) and P(2) groups (F = 48.19, P < 0.01). P(2) group failed to meet the expected results (P > 0.05). In addition, the proliferation activity was reduced in P(1) group and the peak point of proliferation curve was prolonged. Moreover, the early cell apoptotic rates were statistically increased in P(1)[(53.4 +/- 4.2)%] VS. non-transfected [(0.8 +/- 0.4)%], P(0) [(5.1 +/- 1.8)%] and P(2)[(7.9 +/- 2.4)%] groups (F = 269.77, P < 0.01).</p><p><b>CONCLUSION</b>HeLa cells express a high level of BDNF. BDNF gene silencing by RNA interference increases the apoptosis of HeLa cells and inhibits cell proliferation, offering a possible target for efficient tumor therapy.</p>