[Down-regulation of human intercellular adhesion molecule-1 expression in MCF-7 cells infected by lentiviral short hairpin RNA interference vectors].
OBJECTIVE
To construct lentiviral interference vectors of human intercellular adhesion molecule-1 (ICAM-1), then infect human breast cancer MCF-7 cells and identify the interference effects.


METHODS
Three short hairpin RNA (shRNA) interference sequences targeting human ICAM-1 gene (ICAM-1 shRNA1, ICAM-1 shRNA2 and ICAM-1 shRNA3) and a negative control sequence (NS) were designed, synthesized and cloned into the pLKO.1-SP6-PGK-GFP vector. After DNA sequencing, three plasmid-based lentiviral packaging system (vector plasmid-psPAX2-pMD2.G) was used to transfect HEK293T cells to package lentiviruses. The supernatants containing viruses were harvested to detect the viral titer. Human MCF-7 breast cancer cells were infected with the lentiviruses and the interference efficiency was detected by real-time quantitative PCR (qRT-PCR) and Western blotting.


RESULTS
PCR showed that the designed sequences were successfully inserted into the pLKO.1-SP6-PGK-GFP vector and DNA sequencing results were correct. The qRT-PCR and Western blotting showed that the mRNA and protein expression levels of ICAM-1 in the infected MCF-7 cells decreased significantly in the ICAM-1 shRNA3 group.


CONCLUSIONS
Lentiviral interference vectors of human ICAM-1 were constructed successfully and the expression of ICAM-1 in MCF-7 cells was down-regulated by ICAM-1 shRNA.
Department of Immunology, Weifang Medical University, Weifang 261053, China.
Dalin
D
Di
Department of Hematology, Hospital Affiliated to Weifang Medical University, Weifang 261031, China.
Lei
L
Chen
Department of Immunology, Weifang Medical University, Weifang 261053, China.
Lina
L
Wang
Prenatal Diagnosis Laboratory, Weifang People's Hospital, Weifang 261041, China.
Chengdong
C
Wang
Department of Immunology, Weifang Medical University, Weifang 261053, China.
Jiyu
J
Ju
D015153:Blotting, Western; D015536:Down-Regulation; D015972:Gene Expression Regulation, Neoplastic; D005822:Genetic Vectors / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D018799:Intercellular Adhesion Molecule-1 / Q000235:genetics* / Q000378:metabolism; D016086:Lentivirus / Q000235:genetics; D061986:MCF-7 Cells; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D034741:RNA, Small Interfering; D018799:Intercellular Adhesion Molecule-1