Lentiviral vector-mediated siRNA knockdown of SR-PSOX inhibits foam cell formation in vitro.
OBJECTIVE
To investigate the expression of scavenger receptor that binds phosphatidylserine and oxidized lipoprotein (SR-PSOX)/CXC chemokine ligand 16 (CXCL16) in the human monocyte-derived cell line THP-1, and the effect of lentiviral vectors for the stable delivery of SR-PSOX/CXCL16 short hairpin RNA on foam cell formation.


METHODS
A lentiviral expression vector containing enhanced green fluorescence protein (GFP) and SR-PSOX small interfering RNA (siRNA) (Lenti-SR-PSOXsi), or the control siRNA (Lenti-NC) gene was constructed. A human monocyte-derived cell line THP-1 was transfected with a different multiplicity of infection (MOI) of Lenti-SR-PSOXsi or Lenti-NC, and cultured to obtain stably-transfected THP- 1KD and THP-1NC cells. After incubation with oxidatively-modified, low-density lipoprotein (Ox-LDL), the expression of SR-PSOX/CXCL16 mRNA was determined by real-time PCR. The expression of the SR-PSOX/CXCL16 protein was detected by flow cytometry analysis. The effect of Lenti-SR-PSOXsi on foam cell formation was assessed by Oil red O-stain analysis.


RESULTS
Ox-LDL increased the expression of SR-PSOX/CXCL16 mRNA in a time- and dose-dependent manner in THP-1 cells. Four days after transfection with Lenti-SR-PSOXsi (MOI: 100), the percentage of GFP expression cells was over 89.3%. The expression of the SR-PSOX/ CXCL16 mRNA and protein in THP-1KD cells significantly decreased compared with the parent cells, even the THP-1KD cells stimulated with 40 mg/L Ox-LDL. Ox-LDL uptake experiments in THP-1- and THP-1KD-derived macrophages indicated that SR-PSOX/CXCL16 deficiency decreased the development of macrophage- derived foam cell formation.


CONCLUSIONS
The above data showed that SRPSOX siRNA delivered by using lentiviral vectors in THP-1 cells was a powerful tool for studying the effect of SR-PSOX, and decreased the expression of the SRPSOX gene by inhibiting macrophage-derived foam cell formation.
Zhang
Li
L
Tumor Immunology and Gene Therapy Center, Second Military Medical University, Shanghai 200438, China.
Liu
Hou-jia
HJ
Li
Tie-jun
TJ
Yang
Yang
Y
Guo
Xian-ling
XL
Wu
Meng-chao
MC
Rui
Yao-cheng
YC
Wei
Li-xin
LX
D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D000075743:Chemokine CXCL16; D019743:Chemokines, CXC / Q000235:genetics* / Q000502:physiology*; D008078:Cholesterol, LDL / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005487:Foam Cells / Q000187:drug effects*; D005822:Genetic Vectors; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D009000:Monocytes / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics*; D051116:Receptors, Scavenger / Q000235:genetics* / Q000502:physiology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014161:Transduction, Genetic
C423727:CXCL16 protein, human; D000075743:Chemokine CXCL16; D019743:Chemokines, CXC; D008078:Cholesterol, LDL; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D051116:Receptors, Scavenger