Effects of Smoothened gene targeting small interfering RNA lentiviral vector on biological characteristics of human pancreatic cancer cells

Abstract

Objective To observe the effect of lentiviral vector carrying Smoothened (SMO) gene targeting small interfering RNA (siRNA) on biological characteristics of pancreatic cancer cells in vitro. Methods Based on previous research methods, 3 lentiviral vectors with SMO siRNA (titer: 5×108, 6×108, 5×108 TU/ml) were synthesized and transfected into Patu-8988 cells. The expression of SMO gene in cells was detected by real-time quantitative polymerase chain reaction (Real-time PCR), and the inhibition rate of gene expression was calculated. Interference vectors with the highest inhibition rate were screened to transfect into Patu-8988 for 72 h. Cell growth was detected by methyl thiazol tetrazolium (MTT), cell apoptosis by flow cytometry, cell invasion and metastasis ability by Transwell invasion assay, and the expression of pathway target gene B cell lymphoma/leukemia-2 (bcl-2) by Western blotting, respectively. Results The inhibition rate of 3 lentiviral vectors of SMO siRNA on SMO gene expression in Patu-8988 cells was 82.4%, 93.1% and 89.1%, respectively. With the selection of interference vector with the highest inhibition rate, it was transfected into Patu-8988 cells for 72 h, and cells grew well. As compared with the negative control group, the growth inhibition rate of Patu-8988 cells in transfected group was (7.46±0.32)%, (25.41±0.28)%, (25.94±0.51)%, (26.01±0.94)% and (43.23±0.88)% from the first to the fifth day, respectively; the apoptotic rate was (1.35±0.11)%, which was lower than that of the control group in which no significant apoptosis was observed (t=-4.523, P=0.011). The number of cells observed in the Invasion chamber and Transwell chamber was (38.00±4.73)/HP and (78.00±3.71)/HP in the transfected group, respectively, significantly lower than those in the control group (128.00±10.91)/HP and (306.00±9.12)/HP, respectively (t=-22.808, P=0.000; t=-69.256, P=0.000); the relative expression of bcl-2 protein was down-regulated in the cells (t=-27.386, P=0.000). Conclusion SMO small interfering RNA lentiviral vector can effectively block the Hedgehog signaling pathway, and significantly inhibit the growth, invasion and metastasis of Patu-8988 cells. Key words: Pancreatic carcinoma; Hedgehog signal transduction pathway; Smoothened gene; Small interfering RNA