[Inhibitory effects of RNA interference on MyD88 expression and biological activity in murine myeloid dendritic cells].
OBJECTIVE
To investigate inhibitory effects of RNA interference on MyD88 expression in murine myeloid dendritic cells(DCs) and detect the biological activity of DCs.


METHODS
Three pairs of myeloid differentiation factor 88(MyD88) siRNA were synthesized and transfected into DCs by RNAi-mate. The mRNA and protein expression of MyD88 were analyzed by semi-quantified RT-PCR and Western blot. Mouse DCs were divided into control group and RNA interference group. One of the highest effective siRNA was transfected into RNA interference group. 12 hours later, LPS of the final concentrations of 1.0 mg/L was added in two groups and continued to culture for 3 days. The phenotype and functional properties of DCs were detected by flow cytometry and mixed lymphocyte reaction (MLR). The concentration of TNF-alpha, IFN-gamma and IL-12 in the supernatant was detected by ELISA, and the concentration of NF-kappaB was detected by immunochemistry.


RESULTS
mRNA and protein expression were reduced 90% and 85% in sequence2 siRNA, 92% and 88% in sequence3 siRNA respectively but no change was found in other groups. LPS stimulation increased the expression of CD80, CD86 and MHC-II in the cytomembrane of DCs, the concentration of TNF-alpha, IFN-gamma and IL-12 in the supernatant in control group. Besides, LPS stimulation promoted the shift of NF-kappaB to karyon and the proliferation of allogeneic T cells in control group. RNA interference inhibited these effects induced by LPS.


CONCLUSIONS
RNA interference can reduce MyD88 expression in murine myeloid dendritic cells and inhibit the maturation of DCs, which may provide a new strategy of gene therapy for related diseases.
Chen
Jia-Jun
JJ
Department of Cardiovascular Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China. cjj329@163.com
Sun
Zong-Quan
ZQ
Dong
Nian-Guo
NG
Su
Gang
G
Liu
Chao
C
Liu
Jin-Ping
JP
Deng
Yong-Zhi
YZ
D000818:Animals; D018122:B7-1 Antigen / Q000378:metabolism; D051940:B7-2 Antigen / Q000378:metabolism; D015153:Blotting, Western; D002460:Cell Line; D003713:Dendritic Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D005786:Gene Expression Regulation; D007120:Immunochemistry; D007371:Interferon-gamma / Q000378:metabolism; D018664:Interleukin-12 / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology; D007959:Lymphocyte Culture Test, Mixed; D051379:Mice; D008807:Mice, Inbred BALB C; D022423:Myeloid Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D053594:Myeloid Differentiation Factor 88 / Q000235:genetics* / Q000378:metabolism*; D016328:NF-kappa B / Q000378:metabolism; D034622:RNA Interference / Q000502:physiology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D018122:B7-1 Antigen; D051940:B7-2 Antigen; D008070:Lipopolysaccharides; C507780:Myd88 protein, mouse; D053594:Myeloid Differentiation Factor 88; D016328:NF-kappa B; D014409:Tumor Necrosis Factor-alpha; D018664:Interleukin-12; D007371:Interferon-gamma