• Guoming Li, Jian Kong, Shuying Dong, Haigang Niu, Shilun Wu, and Wenbing Sun.
• Department of Hepatobiliary Surgery, Beijing ChaoYang Hospital Affiliated to Capital Medical University, Beijing 100043, China.
• Chin. Med. J. 2022 Aug 8.

BackgroundCircular RNAs (circRNAs) are endogenous non-coding RNAs, some of which have pathological roles. The current study aimed to explore the role of circRNA BTG3-associated nuclear protein (circ-BANP) binding with let-7f-5p and its regulation of the toll-like receptor 4 (TLR4)/signal transducer and activator of transcription 3 (STAT3) signaling pathway in residual hepatocellular carcinoma (HCC) after insufficient radiofrequency ablation (RFA).MethodsCirc-BANP, let-7f-5p, and TLR4 expressions in HCC samples were assessed using reverse transcription- quantitative polymerase chain reaction (RT-qPCR) and Western blotting. Bioinformatics prediction, RNA pull-down assay, and dual luciferase reporter gene assay were used to analyze the relationships among circ-BANP, let-7f-5p, and TLR4. Huh7 cells were used to generate an in vitro model of residual HCC, defined as Huh7-H cells, which were transfected with either a plasmid or the sequence of circ-BANP, let-7f-5p, or TLR4. Expression of circ-BANP, let-7f-5p, and TLR4 mRNA was determined by RT-qPCR. TLR4, STAT3, p-STAT3, vascular endothelial growth factor A, vascular endothelial growth factor receptor-2, and epithelial-mesenchymal transformation (EMT)-related factors proteins were determined by Western blotting. Cell proliferation was determined by cell counting kit-8 and 5-Ethynyl-2'-deoxyuridine (EdU) assay and cell migration and invasion by Transwell assay. Animal studies were performed by inducing xenograft tumors in nude mice.ResultsCirc-BANP and TLR4 mRNAs were upregulated in HCC tissues (the fold change for circ-BANP was 1.958 and that for TLR4 was 1.736 relative to para-tumors) and expression further increased following insufficient RFA (fold change for circ- BANP was 2.407 and that of TLR4 was 2.224 relative to para-tumors). Expression of let-7f-5p showed an opposite tendency (fold change for let-7f-5p in HCC tissues was 0.491 and that in tumors after insufficient RFA was 0.300 relative to para-tumors). Competitive binding of circ-BANP to let-7f-5p was demonstrated and TLR4 was identified as a target of let-7f-5p (P < 0.01). Knockdown of circ-BANP or elevation of let-7f-5p expression inhibited the TLR4/STAT3 signaling pathway, proliferation, invasion, migration, angiogenesis, and EMT in Huh7 and Huh7-H cells (P < 0.01). The effects induced by circ-BANP knockdown were reversed by let-7f-5p inhibition. Overexpression of TLR4 reversed the impact of let-7f-5p upregulation on the cells (P < 0.01). Silencing of circ-BANP inhibited the in vivo growth of residual HCC cells after insufficient RFA (P < 0.01).ConclusionsKnockdown of circ-BANP upregulated let-7f-5p to inhibit proliferation, migration, and EMT formation in residual HCC remaining after insufficient RFA. Effects occur via regulation of the TLR4/STAT3 signaling pathway.Copyright © 2022 The Chinese Medical Association, produced by Wolters Kluwer, Inc. under the CC-BY-NC-ND license.

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