[Inhibition of nuclear factor kappa B mediated by miRNA plasmid carried by novel polyethyleneimine nanogel particles in Kupffer cells of rats].
OBJECTIVE
To test a novel gene carrier, named polyethyleneimine nanogel particles (PEI-NP), for delivering the mircroRNA (miRNA) into Kupffer cells (KCs) for silencing the expression of nuclear transcription factor κB (NF-κB) P65.


METHODS
The capacity of PEI-NPs to carry miRNA was tested by gel electrophoresis. RAW264.7 cell line was transfected with the cationic polymers which mixed together PEI-NP and miRNA. Then, the cytotoxicity of the RAW264.7 cells was detected by cell counting kit-8 (CCK-8); the cell apoptosis and transfection efficiency were analyzed by flow cytometry combined with annexin V-FITC/PI staining; the gene and protein levels of NF-κB P65 were determined respectively by real-time quantitative PCR (qRT-PCR) and Western blotting. The distribution of the cationic polymers in RAW264.7 cells and liver KCs was observed by transmission electron microscope (TEM). The protein level of NF-κB P65 in KCs was detected by immunohistochemistry.


RESULTS
PEI-NPs were able to completely combine with miRNA at the least mass ratio of 20:1. The highest transfection efficiency of 48 hours was (33.63±1.94)% in vitro. The protein level of NF-κB P65 at 48 hours of PEI-NP/miRNA transfection into RAW264.7 cells was significantly inhibited. TEM showed the cationic polymers in RAW264.7 cytoplasm and only in KCs of liver tissue after transfection. Immunohistochemistry indicated that the protein expression of NF-κB P65 in KCs was significantly lower than that in control group.


CONCLUSIONS
Macrophages and KCs were successfully transfected with the cationic polymers in vitro and in vivo, respectively. The expression of NF-κB P65 was knocked down significantly after transfection.
Chongqing Key Laboratory of Hepatobiliary Surgery, Department of Hepatobiliary Surgery, Second Affiliated Hospital, Chongqing Medical University, Chongqing 400010, China.
Wenfeng
W
Zhang
Chongqing Key Laboratory of Hepatobiliary Surgery, Department of Hepatobiliary Surgery, Second Affiliated Hospital, Chongqing Medical University, Chongqing 400010, China.
Shipeng
S
Guo
Chongqing Key Laboratory of Hepatobiliary Surgery, Department of Hepatobiliary Surgery, Second Affiliated Hospital, Chongqing Medical University, Chongqing 400010, China.
Jianping
J
Gong
D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D007150:Immunohistochemistry; D007728:Kupffer Cells / Q000378:metabolism* / Q000648:ultrastructure; D008264:Macrophages / Q000378:metabolism / Q000648:ultrastructure; D051379:Mice; D035683:MicroRNAs / Q000235:genetics*; D046529:Microscopy, Electron, Transmission; D008856:Microscopy, Fluorescence; D000080385:Nanogels; D053758:Nanoparticles / Q000737:chemistry; D010957:Plasmids / Q000737:chemistry / Q000235:genetics*; D011092:Polyethylene Glycols / Q000737:chemistry; D011094:Polyethyleneimine / Q000737:chemistry; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction; D051996:Transcription Factor RelA / Q000235:genetics* / Q000378:metabolism
D035683:MicroRNAs; D000080385:Nanogels; D051996:Transcription Factor RelA; C413692:polyethylene glycol polyethyleneimine nanogel; D011092:Polyethylene Glycols; D011094:Polyethyleneimine