[IL-1beta antisense RNA enhanced sensitivity of HepG2 cells to NK cell mediated cytotoxicity].
OBJECTIVE
To investigate the inhibitory effect of IL-1beta antisense RNA on the sensitivity of HepG2 cells to the NK cell mediated cytotoxicity.


METHODS
Two gene segments of IL-1beta [IL-1beta1(17-331, 315 bp) and IL-1beta2(246-505, 260 bp)] were selected for antisense RNA. Total RNA was extracted from PBMC of a healthy donor treated with LPS. IL-1beta1 and IL-1beta2 were prepared by RT-PCR. PCR products were cloned into pMD18-T-simple vector and then sub-cloned to construct the pcDNA3.0-antiIL-1beta1 and pcDNA3.0-antiIL-1beta2 antisense RNA expression vectors. HepG2 cells were transfected by jetPEI, the expression of antisense RNA in HepG2 cells was assayed by RT-PCR, level of IL-1beta was analyzed by intracellular staining. The response of HepG2 cells to NK-92 cells was assessed by MTT assay.


RESULTS
Two gene fragments of 260 bp and 315 bp products were obtained by RT-PCR. The purified gene fragments were cloned to construct pMD18 T-IL-1beta1 and pMD18 T-IL-1beta2 which were verified by PCR, restriction enzyme assay (Xho I) and DNA sequencing. The PCR products using Pfu DNA polymerase from cloning vectors were sub-cloned to create the antisense RNA expression vectors of pcDNA3.0-antiIL-1beta1 and pcDNA3.0-antiIL-1beta2 which were confirmed by PCR, restriction enzyme assay (Pst I) and DNA sequencing. When transfected into HepG2 cells, HepG2 cells expressed high level of antisense RNA, and simultaneously expression of IL-1beta was markedly suppressed which rendered HepG2 cells to be more sensitive to NK-92 cell mediated cytotoxicity compared with the cells transfected by pcDNA3.0. The cytolytic activity of NK-92 cells to HepG2 cells increase about 20% at the effector to target ratio of 10:1.


CONCLUSIONS
Inhibiting of proinflammatory cytokine IL-1beta can reduce the sensitivity of hepatoma cells to the NK cell mediated cytolysis which provide an useful way of rendering NK cell activity against hepatoma.
Liang
Shu-juan
SJ
Key Laboratory of Molecular Immunology, Weifang Medical College, Weifang 261042, China.
Xiao
Wei-ling
WL
Mu
Dong-zhen
DZ
Wu
Hui-na
HN
Wang
Xue-jing
XJ
D006528:Carcinoma, Hepatocellular; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D006801:Humans; D053583:Interleukin-1beta / Q000235:genetics* / Q000502:physiology; D007694:Killer Cells, Natural / Q000502:physiology*; D016372:RNA, Antisense / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D053583:Interleukin-1beta; D016372:RNA, Antisense