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Updated: Jul 13, 2026
Measurement of Natural Killer Cell-Mediated Cytotoxicity and Migration in the Context of Hepatic Tumor Cells
Published on: February 22, 2020
[IL-1beta antisense RNA enhanced sensitivity of HepG2 cells to NK cell mediated cytotoxicity]
Shu-juan Liang1, Wei-ling Xiao, Dong-zhen Mu
1Key Laboratory of Molecular Immunology, Weifang Medical College, Weifang 261042, China.
Aim:
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.
Conclusion:
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.
Insights
Inhibiting interleukin-1 beta (IL-1beta) with antisense RNA makes hepatoma cells more sensitive to natural killer (NK) cell attacks. This approach enhances NK cell activity against liver cancer cells.
Area of Science:
- Immunology
- Molecular Biology
- Hepatology
Context:
- Hepatocellular carcinoma (HCC) is a major global health concern.
- Natural killer (NK) cells are crucial in innate immunity and cancer surveillance.
- Interleukin-1 beta (IL-1beta) is a pro-inflammatory cytokine implicated in cancer progression.
Purpose:
- To investigate the efficacy of IL-1beta antisense RNA in sensitizing HepG2 hepatoma cells to NK cell-mediated cytotoxicity.
- To explore the potential of targeting IL-1beta to enhance anti-cancer immunity.
Summary:
- Antisense RNA expression vectors targeting two IL-1beta gene segments were constructed and verified.
- HepG2 cells transfected with these vectors showed suppressed IL-1beta expression.
- Transfected HepG2 cells exhibited significantly increased sensitivity to NK-92 cell-mediated lysis, with a ~20% increase in cytolytic activity at a 10:1 E:T ratio.
Impact:
- Inhibiting IL-1beta can overcome resistance of hepatoma cells to NK cell-mediated killing.
- This strategy offers a novel therapeutic approach to augment NK cell-based immunotherapy for liver cancer.
- Findings suggest a potential role for IL-1beta modulation in cancer treatment.
