Circular BANP knockdown inhibits the malignant progression of residual hepatocellular carcinoma after insufficient radiofrequency ablation

Abstract

Background: Circular 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).

Methods: Circ-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.

Results: Circ-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).

Conclusions: Knockdown 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.

Figure 1

Let-7f-5p is downregulated while circ-BANPand TLR4 are upregulated in HCC, especially in tumors after insufficient RFA. (A–C) circ-BANP, let-7f-5p andTLR4 mRNA expression in HCC and adjacent normal tissues detected using RT-qPCR; (D) protein expression of TLR4 in HCC and adjacent normal tissues detected using Western blotting analysis; (E) circ-BANP, let-7f-5p and TLR4 mRNA expression in Huh7 and Huh7-H cells assessed by RT-qPCR; (F) protein expression of TLR4 in Huh7 and Huh7-H assessed by Western blotting analysis; P < 0.01; para-tumor: n = 83, tumor without RFA: n = 44; residual tumor after RFA: n = 39; repetition = 3; the measurement data conforming to the normal distribution were expressed as mean ± standard deviation, unpaired t test was performed for comparisons between two groups, one-way ANOVA was used for comparisons among multiple groups and Tukey's multiple comparisons test was used for pairwise comparisons after one-way ANOVA. ANOVA: Analysis of variance; circ-BANP: Circular RNABTG3-associated nuclear protein; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; HCC: Hepatocellular carcinoma; RFA: Radiofrequency ablation; RT-qPCR: Reverse transcriptase-quantitative polymerase chain reaction; TLR4: Toll-like receptor 4.

Figure 2

Circ-BANP binds to let-7f-5p and let-7f-5p targets TLR4. (A) Binding sites between circ-BANP and let-7f-5p predicted at starbase; (B) the binding relationship between circ-BANP and let-7f-5p confirmed using dual luciferase reporter gene assay; (C) the binding relationship between circ-BANP and let-7f-5p detected by RNA pull-down assay; (D) binding sites between let-7f-5p and TLR4 predicted at starbase; (E) targeting relationship between let-7f-5p and TLR4 confirmed using dual luciferase reporter gene assay; ^∗P < 0.01; repetition = 3; the measurement data conforming to the normal distribution were expressed as mean ± standard deviation, one-way ANOVA was used for comparisons among multiple groups and Tukey's multiple comparisons test was used for pairwise comparisons after one-way ANOVA. ANOVA: Analysis of variance; circ-BANP: Circular RNA BTG3-associated nuclear protein; MUT: Mutant type; TLR4: Toll-like receptor 4; WT: Wild type.

Figure 3

Circ-BANP inhibition represses proliferation, migration, and EMT formation of residual HCC cells after insufficient RFA. (A) circ-BANP, let-7f-5p and TLR4 mRNA expression in cells after transfection assessed by RT-qPCR; (B) protein expression of TLR4, STAT3, and p-STAT3 in cells after transfection assessed by Western blotting analysis; (C) CCK-8 assay was used to evaluate cell proliferation after transfection; (D) EdU incorporation assay was employed to measure the proliferation rate of cells after transfection; (E) cell migration assessed by Transwell assay; (F) cell invasion assessed by Transwell assay; (G) protein expression of E-cadherin, N-cadherin, Vimentin, VEGFA, and VEGFR-2 in cells after transfection was determined using Western blotting analysis; P < 0.05, repetition = 3; the measurement data conforming to the normal distribution were expressed as mean ± standard deviation, one-way ANOVA was used for comparisons among multiple groups and Tukey's multiple comparisons test was used for pairwise comparisons after one-way ANOVA. ANOVA: Analysis of variance; CCK-8: Cell counting kit-8; circ-BANP: Circular RNA BTG3-associated nuclear protein; EMT: Epithelial-mesenchymal transformation; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; HCC: Hepatocellular carcinoma; RFA: Radiofrequency ablation; STAT3: Signal transducer and activator of transcription 3; TLR4: Toll-like receptor 4.

Figure 4

Circ-BANP knockdown inhibits proliferation, migration, and EMT formation of residual HCC cells after insufficient RFA via mediating let-7f-5p. (A) circ-BANP, let-7f-5p and TLR4 mRNA expression in cells after transfection assessed by RT-qPCR; (B) protein expression of TLR4, STAT3, and p-STAT3 in cells after transfection assessed by Western blotting analysis; (C) CCK-8 assay was used to evaluate cell proliferation after transfection; (D) EdU incorporation assay was employed to measure the proliferation rate of cells after transfection; (E) cell migration assessed by Transwell assay; (F) cell invasion assessed by Transwell assay; (G) protein expression of E-cadherin, N-cadherin, Vimentin, VEGFA, and VEGFR-2 in cells after transfection was determined using Western blotting analysis; ^∗P < 0.05, repetition = 3; the measurement data conforming to the normal distribution were expressed as mean ± standard deviation, one-way ANOVA was used for comparisons among multiple groups and Tukey's multiple comparisons test was used for pairwise comparisons after one-way ANOVA. ANOVA: Analysis of variance; CCK-8: Cell counting kit-8; circ-BANP: Circular RNA BTG3-associated nuclear protein; EMT: Epithelial-mesenchymal transformation; DAPI: 4’,6-diamidino-2-phenylindole; EDU: 5-ethynyl- 2’-deoxyuridine; HCC: Hepatocellular carcinoma; RFA: Radiofrequency ablation; RT-qPCR: Reverse transcriptase-quantitative polymerase chain reaction; STAT3: Signal transducer and activator of transcription 3; TLR4: Toll-like receptor 4; VEGFA: Vascular endothelial growth factor A; VEGFR-2: Vascular endothelial growth factor receptor 2.

Figure 5

Elevated let-7f-5p reduces proliferation, migration, and EMT formation of residual HCC cells after insufficient RFA. (A) circ-BANP, let-7f-5p and TLR4 mRNA expression in cells after transfection assessed by RT-qPCR; (B) protein expression of TLR4, STAT3, and p-STAT3 in cells after transfection assessed by Western blotting analysis; (C) CCK-8 assay was used to evaluate cell proliferation after transfection; (D) EdU incorporation assay was employed to measure the proliferation rate of cells after transfection; (E) cell migration assessed by Transwell assay; (F) cell invasion assessed by Transwell assay; (G) protein expression of E-cadherin, N-cadherin, Vimentin, VEGFA, and VEGFR-2 in cells after transfection was determined using Western blotting analysis; ^∗P < 0.01; repetition = 3; the measurement data conforming to the normal distribution were expressed as mean ± standard deviation, one-way ANOVA was used for comparisons among multiple groups and Tukey's multiple comparisons test was used for pairwise comparisons after one-way ANOVA. ANOVA: Analysis of variance; CCK-8: Cell counting kit-8; circ-BANP: Circular RNA BTG3-associated nuclear protein; EMT: Epithelial-mesenchymal transformation; HCC: Hepatocellular carcinoma; RFA: Radiofrequency ablation; RT-qPCR: Reverse transcriptase-quantitative polymerase chain reaction; STAT3: Signal transducer and activator of transcription 3; TLR4: Toll-like receptor 4.

Figure 6

Let-7f-5p elevation targets TLR4 to restrain proliferation, migration, and EMT formation of residual HCC cells after insufficient RFA. (A) circ-BANP, let-7f-5p and TLR4 mRNA expression in cells after transfection assessed by RT-qPCR; (B) protein expression of TLR4, STAT3, and p-STAT3 in cells after transfection assessed by Western blotting analysis; (C) CCK-8 assay was used to evaluate cell proliferation after transfection; (D) EdU incorporation assay was employed to measure the proliferation rate of cells after transfection; (E) cell migration assessed by Transwell assay; (F) cell invasion assessed by Transwell assay; (G) protein expression of E-cadherin, N-cadherin, Vimentin, VEGFA, and VEGFR-2 in cells after transfection was determined using Western blotting analysis; ^∗P < 0.01; repetition = 3; the measurement data conforming to the normal distribution were expressed as mean ± standard deviation, unpaired t test was performed for comparisons between two groups, one-way ANOVA was used for comparisons among multiple groups and Tukey's multiple comparisons test was used for pairwise comparisons after one-way ANOVA. ANOVA: Analysis of variance; CCK-8: Cell counting kit-8; circ-BANP: Circular RNA BTG3-associated nuclear protein; EMT: Epithelial-mesenchymal transformation; HCC: Hepatocellular carcinoma; RFA: Radiofrequency ablation; RT-qPCR: Reverse transcriptase-quantitative polymerase chain reaction; STAT3: Signal transducer and activator of transcription 3; STAT3: Signal transducer and activator of transcription 3; TLR4: Toll-like receptor 4.

Figure 7

Silencing circ-BANP inhibits in vivo growth of residual HCC cells after insufficient RFA. (A) circ-BANP expression in Huh7 and Huh7-H cells after transfection of sh-circ-BANP or shNC was determined using RT-qPCR; (B) Huh7 and Huh7-H cells that had been transfected with sh-circ-BANP or sh-NC were subcutaneously injected into nude mice and 4 weeks later, the mice were euthanized. Images of tumor volume were exhibited; (C, D) tumor size was measured with a caliper rule every 4 days and tumor weight was measured at the end of the experiment; ^∗P < 0.01; n = 5; the measurement data conforming to the normal distribution were expressed as mean ± standard deviation, one-way ANOVA was used for comparisons among multiple groups and Tukey's multiple comparisons test was used for pairwise comparisons after one-way ANOVA. ANOVA: Analysis of variance; circ-BANP: Circular RNA BTG3- associated nuclear protein; HCC: Hepatocellular carcinoma; RFA: Radiofrequency ablation; RT-qPCR: Reverse transcriptase-quantitative polymerase chain reaction.