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Background: Ablation is one of the main methods for local treatment of hepatocellular carcinoma (HCC). Different from radiofrequency ablation (RFA), microwave ablation (MWA) is not limited by tissue conductivity, and can use multiple electrodes at the same time to improve ablation efficiency. In addition, MWA can form a larger ablation area, which makes it possible to completely ablate large HCC. However, MWA may be incomplete due to factors such as larger tumors or tumors in high-risk areas. The mechanism by which the cellular and tumor immune microenvironment (TIME) is involved in the in vitro effects of incomplete microwave ablation (iMWA) needs to be further elucidated., Methods: H22 tumor-bearing C57BL/6 mice were treated with iMWA with several combinations of ablation power and time duration. The effects of iMWA on the genes of HCC cancer cells and the TIME were investigated by RNA sequencing, mass cytometry, immunohistochemistry, and immunofluorescence. The effect of iMWA in combination with anti-Gr-1 on HCC tumor growth was also evaluated., Results: Thermal stress generated by iMWA induced coagulative necrosis and apoptosis in the region of the ablation center of HCC. RNA sequencing analysis showed that iMWA can boost chemokine CXCL5, which was further confirmed by quantitative real time polymerase chain reaction (qRT-PCR). Mass cytometry results showed that relative to Ctrl group, iMWA-treated led to decreased CD4 + T, CD8 + T, Natural killer (NK), macrophages including both M1 and M2 types but increased monocytes and bone marrow-derived suppressor cells (MDSC). Therefore, inhibiting MDSC is the main target in the later stage of iMWA. In vivo results showed that the tumor volume and weight of iMWA+ anti-Gr-1 group were significantly reduced compared with iMWA+ anti-IgG group. In addition, the merged expressions of CD11b and Gr-1 proteins were found reduced in the iMWA+ anti-Gr-1 group compared with the iMWA+ anti-IgG group by immunofluorescence staining. Immunohistochemistry suggested that CD8 was enriched in the iMWA+ anti-Gr-1 group but not in the iMWA+ anti-IgG group., Conclusion: Our data suggests that iMWA and Gr-1 blocking combined therapy can further inhibit HCC growth and significantly improve the CD8 + T cells in the mouse subcutaneous tumor model, which brings good news to HCC patients., Competing Interests: Declarations. Ethics approval and consent to participate: All in vivo animal experiments were approved by the Committee on the Ethics of Animal Experiments of Nanjing Medical University. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Background: Circular RNAs (circRNAs) are enriched in exosomes and are extremely stable. Exosome-mediated intercellular transfer of specific biologically active circRNA molecules can drive the transformation of the tumor microenvironment and accelerate or inhibit the local spread and multifocal growth of hepatocellular carcinoma (HCC). In this study, we explored in depth about the biological roles of HCC cell-derived exosomes and exosome-transported circRNAs on HCC in vivo and in vitro., Methods: Exosomes extracted from HCC cells (Huh7 and HA22T) were characterized using transmission electron microscopy, nanoparticle size tracer analysis, and western blotting. Exosomes were observed for endocytosis using fluorescent labeling. The effects of HCC cell-derived exosomes and the circ_002136 they carried on cell growth, metastasis and apoptosis were determined by CCK-8 assay, transwell assay, flow cytometry analysis and TUNEL staining, respectively. The expressions of circ_002136, miR-19a-3p and RAB1A were detected by quantitative RT-PCR (qRT-PCR). Targeted binding between miR-19a-3p and circ_002136 or RAB1A was predicted and verified by bioinformatics analysis, dual-luciferase reporter and RNA pull-down experiments. The in vivo effect of circ_002136 was determined by constructing a xenograft tumor model., Results: The findings revealed that Huh7 and HA22T exosomes conferred enhanced viability as well as invasive ability to recipient HCC cells. Circ_002136 was shown for the first time to be differentially upregulated in HCC tissues and cells and transferred by HCC cell-derived exosomes. More importantly, selective silencing of circ_002136 depleted the malignant biological behaviors of HCC exosome-activated Huh7 and HA22T cells. Depletion of circ_002136 in vivo effectively retarded the growth of HCC xenograft tumors. Furthermore, a well-established circ_002136 ceRNA regulatory network was constructed, namely circ_002136 blocked miR-19a-3p expression, elevated RAB1A expression activity and stimulated HCC development. Finally, high levels of circ_002136 or RAB1A, as well as low levels of miR-19a-3p, negatively affected HCC patient survival., Conclusion: The study on circ_002136 provides good data to support our insight into the mechanism of to-be-silenced circRNA as a therapeutic agent in the progression of HCC., (© 2022. The Author(s).)

Background: Midazolam (MDZ) is an anaesthetic that is widely used for anxiolysis and sedation. More recently, MDZ has also been described to be related to the outcome of various types of carcinomas. However, how MDZ influences the progression of hepatocellular carcinoma (HCC) and its effects on the biological function and tumour immune microenvironment of this type of tumour remain unknown., Methods: The effects of MDZ on the proliferation, invasion, and migration of HCC cell lines were examined in vitro using the Cell Counting Kit 8 (CCK8), 5-ethynyl-2'-deoxyuridine (EdU), Transwell, and wound healing assays. Additionally, western blotting was employed to confirm that PD-L1 was expressed. Chromatin immunoprecipitation-seq (ChIP-seq) analysis was used to pinpoint the transcriptional regulation regions of NF-κB and programmed death-ligand 1 (PD-L1). A C57BL/6 mouse model was used to produce subcutaneous HCC tumors in order to evaluate the in vivo performance of MDZ. Mass spectrometry was also used to assess changes in the tumour immunological microenvironment following MDZ injection., Results: The HCC-LM3 and Hep-3B cell lines' proliferation, invasion, and migration were controlled by MDZ, according to the results of the CCK8, EdU, Transwell, and wound healing assays. PD-L1 expression was shown by ChIP-seq analysis to be boosted by NF-κB, and by Western blotting analysis, it was shown that MDZ downregulated the expression of NF-κB. Additionally, in vivo tests revealed that intraperitoneal MDZ injections reduced HCC tumor development and enhanced the effectiveness of anti-PD-1 therapy. The CD45 + immune cell proportions were higher in the MDZ group than in the PBS group, according to the mass spectrometry results. Injection of MDZ resulted in a decrease in the proportions of CD4 + T cells, CD8 + T cells, natural killer (NK) cells, monocytes, Tregs, and M2 macrophages and a rise in the proportion of dendritic cells. Additionally, the concentrations of the cytokines IFN-g and TNF-a were noticeably raised whereas the concentrations of the CD8 + T-cell fatigue markers ICOS, TIGIT, and TIM3 were noticeably lowered., Conclusion: According to this study, MDZ inhibited the progression of HCC by inhibiting the NF-κB pathway and reducing the exhaustion of CD8 + T cells. In clinical practice, MDZ combined with anti-PD-1 therapy might contribute to synergistically improving the antitumor efficacy of HCC treatment., (© 2022. The Author(s).)