Blockage of Wnt1 signaling pathway by RNAi technology can inhibit proliferation of human neuroblasto-ma SH-SY5Y cells

Abstract

Objective To detect the expression of Wnt1 in human neuroblastoma cell line SH-SYSY, and to observe the effect of Wntl signaling pathway on the proliferation of the cells. Methods PCR, Western blotting and immunofluorescence technology were used to test the expression of Wnt1 in human neuroblastoma SH-SY5Y cells. RNAi technology was used to observe its inhibitory effect on the growth of the cells. SiRNA targeting Wnt1 was transfected into SH-SY5Y cells. The protein expression of Wntl and β-catenin were detected by Western blotting at 48 hours after transfec-tion. The quantity and the morphologic changes of the cells were observed by light microscope. The proliferative rate of SH-SY5Y cells after RNAi transfection was studied by MTT assay. Results Wnt1 expressed well in human neuroblastoma SH-SYSY cells. And the cells were successfully transfected with siRNA targeting Wntl mediated by Lipofectamine in vitro. The levels of the Wnt1 proteins ex-pression by western blotting in negative control, vector, nonsense-siRNA and Wnt-1 RNAi groups were 0. 341±0. 064,0. 401±0. 078,0. 328±0. 034,0. 041±0. 015, respectively. As the downstream molecular in the Wntl signaling pathway, β-catenin protein expression were 0. 275±0. 054, 0. 299± 0. 060, 0. 271±0. 034, 0. 037±0. 018, respectively. Quantification analysis revealed that Wnt-1 spe-cifie RNAi reduced Wnt-1 and β-catenin protein significantly(P 0. 05). The morphological changes of the SH-SYSY cells after 24h and 48h of transfection were observed under the light microscope. The density of the cells were obviously decreased in the specific Wnt1-siRNA group at 24 hours post-trans-fection, the formation of lamellipodium were blocked, and the time of focal adhesive clone were de-layed, apoptotic bodies were observed, especially at 48 hours post-transfection, accompanying with lots of floating, round and dead cells. However, in the other three groups the SH-SY5Y cells grew well and stretched out many lamellipodium and no obvious floating and dead cells were seen. Metabolic activity at 0, 12, 24, 36 and 48 hour after transfection was determined by MTT assay. The cell viability was reduced significantly after treatment with specific Wnt-1 siRNA as compared with the negative control or vector or non-specific siRNA group (P 0. 05). Conclusions Wnt1 can express in human neuroblastoma SH-SYSY cells. Wnt1 signaling pathway may play a very important role in the proliferation of Human neuroblastoma SH-SYSY cells. Key words: Neuroblastoma; SH-SY5Y; Wntl proteins siRNA; β-catenin