Lentivirus-mediated SMO RNA interference inhibits SMO expression and cell proliferation, and affects the cell cycle in LNCaP and PC3 cancer cell lines.
Smoothened (SMO) is an important member of the Hedgehog signaling pathway. We constructed a specific recombinant lentiviral vector for RNA interference, targeting the SMO gene (NM_005631) to observe its effect on SMO expression, cell proliferation and the cell cycle in the human androgen-sensitive prostate cancer cell line, LNCaP, and in the androgen-independent prostate cancer cell line, PC3. Four siRNA sequences were designed and inserted into a lentiviral vector pGCSIL-GFP to construct four recombinant vectors. The vector with the highest interfering efficiency was co-transfected with packaging vectors (pHelper1.0 and pHelper2.0) in 293T cells to assemble lentivirus particles by liposome for infecting LNCaP and PC3 cell lines, respectively. The expression level of SMO mRNA, tumor cell proliferation and cell cycle were measured by quantitative realtime polymerase chain reaction (qRT-PCR), 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT) assay and flow cytometry, respectively. Sequence results showed that recombinant lentiviral vectors were constructed successfully. pGCSIL-GFP-723 had the highest interfering efficiency, named Lv-SIL-SMO723 after co-transfection, with which LNCaP and PC3 cell lines were infected. Compared with the control groups, results showed significantly decreased (P < 0.05) SMO mRNA expressions of LNCaP and PC3, lower mean percentage of S-phase cells and higher mean percentage of G(2)/M phase cells, as well as obviously slow proliferation (P < 0.01) of LNCaP in the infected group. Yet, the proliferation of PC3 was not altered (P > 0.05). In conclusion, the recombinant lentivirus particles were able to suppress SMO expression, regulate the cell cycle in the LNCaP and PC3 cell lines and markedly inhibit proliferation of LNCaP cells but not PC3 cells.
Yang
Bo
B
Department of Urology, Shanghai East Hospital, Tongji University, China. paulyang28@tom.com
Sun
Heng-Yun
HY
Chen
Wei-Hua
WH
Wen
Ji-Ling
JL
Shi
Xiang-Ting
XT
Wang
Yue-Min
YM
D001483:Base Sequence; D002453:Cell Cycle; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D017931:DNA Primers; D005434:Flow Cytometry; D005822:Genetic Vectors; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D008297:Male; D011471:Prostatic Neoplasms / Q000473:pathology*; D034622:RNA Interference; D043562:Receptors, G-Protein-Coupled / Q000235:genetics*; D000072086:Smoothened Receptor
D017931:DNA Primers; D043562:Receptors, G-Protein-Coupled; C491023:SMO protein, human; D000072086:Smoothened Receptor