Circular RNA circTRIM33-12 acts as the sponge of MicroRNA-191 to suppress hepatocellular carcinoma progression

Abstract

Background: Recently, the dysregulation of circular RNA (circRNA) have been shown to have important regulatory roles in cancer development and progression, including hepatocellular carcinoma (HCC). However, the roles of most circRNAs in HCC are still unknown.

Methods: The expression of circular tripartite motif containing 33-12 (circTRIM33-12) in HCC tissues and cell lines was detected by qRT-PCR. The role of circTRIM33-12 in HCC progression was assessed by western blotting, CCK-8, flow cytometry, transwell and a subcutaneous tumor mouse assays both in vitro and in vivo. In vivo circRNA precipitation, RNA immunoprecipitation, luciferase reporter assays were performed to evaluate the interaction between circTRIM33-12 and miR-191.

Results: Here, we found that circTRIM33-12, is downregulated in HCC tissues and cell lines. The downregulation of circTRIM33-12 in HCC was significantly correlated with malignant characteristics and served as an independent risk factor for the overall survival (OS) and recurrence-free survival (RFS) of patients with HCC after surgery. The reduced expression of circTRIM33-12 in HCC cells increases tumor proliferation, migration, invasion and immune evasion. Mechanistically, we demonstrated that circTRIM33-12 upregulated TET1 expression by sponging miR-191, resulting in significantly reduced 5-hydroxymethylcytosine (5hmC) levels in HCC cells.

Conclusions: These results reveal the important role of circTRIM33-12 in the proliferation, migration, invasion and immune evasion abilities of HCC cells and provide a new perspective on circRNAs in HCC progression.

Keywords: Circular RNA; Hepatocellular carcinoma; Histone methylation; TET1.

Fig. 1

Low circTRIM33–12 expression in HCC tissues and prognostic significance. a The heatmap shows circRNAs derived from the TRIM33 gene in HCC tissues compared with those in adjacent nontumor tissues analyzed by qRT-PCR. b The scheme illustrating the production of circTRIM33–12. c The differential expression of circTRIM33–12 in HCC tissues and adjacent nontumor tissues of 200 patients as indicated. d and e Prognostic analysis of circTRIM33–12 expression in 200 HCC patients

Fig. 2

High circTRIM33–12 expression inhibited the progression of HCC cells in vitro and in vivo. a Cell proliferation in HCC cells with the forced expression of circTRIM33–12 was assessed by a CCK-8 assay. b The cell cycle in HCC cells with the forced expression of circTRIM33–12 was detected by FCM. c The migration and invasion abilities of HCC cells with the forced expression of circTRIM33–12 was evaluated via a transwell assay. d Tumor growth in HCC cells with the forced expression of circTRIM33–12 was investigated by nude mice xenograft tumor models. e Serial sections from mouse lungs showed the metastatic ability of cancer cells expressing different levels of circTRIM33–12. The data are represented as the mean ± SD, n = 3. **P < 0.01

Fig. 3

circTRIM33–12 binds miR-191 in HCC cells. a RIP experiments were performed using an antibody against AGO2 on extracts from Huh 7 cells. b A schematic drawing showing the putative binding sites of miR-23a-3p, miR-191, and miR-224-5p with respect to circTRIM33–12. c The luciferase activity of luc-circTRIM33–12 or mutant luc-circTRIM33–12 in Huh 7 cells after cotransfection with miR-23a-3p, miR-191, or miR-224-5p. d circRIP was performed in Huh 7 cells using biotin-labeled miR-191 mimics and a negative control (NC). The enrichment of circTRIM33–12 was detected by qRT-PCR and was normalized to that of the NC. e The expression of circTRIM33–12 in Huh 7 cells after transfection with miR-191 or miR-191 siRNA. f The expression of circ miR-191 in SMMC-7721 and Huh 7 cells after transfection with circTRIM33–12 or circTRIM33–12 shRNA. The data are represented as the mean ± SD, n = 3. *P < 0.05; **P < 0.01; NS, no significant

Fig. 4

circTRIM33–12 upregulates TET1 expression in HCC cells. a-d The relative mRNA and protein levels of TET1 were detected in SMMC-7721 and Huh 7 cells after transfection with circTRIM33–12, circTRIM33–12 shRNA, or the control using qRT-PCR and western blotting, respectively. e The luciferase activity of luc-TET1 or mutant luc-TET1 in Huh 7 cells after cotransfection with circTRIM33–12 shRNA or circTRIM33–12 and miR-191 siRNA. f The luciferase activity of luc-TET1 or mutant luc-TET1 in SMMC-7721 cells after cotransfection with circTRIM33–12 or the mock control. g and h The expression of TET1 in 200 cases of HCC tissues was detected by qRT-PCR and IHC. A positive correlation between circTRIM33–12 and TET1 was observed in tumor tissues at the protein (R2 = 0.0441; P = 0.0028) and mRNA (R2 = 0.04751; P = 0.0019) levels. The data are represented as the mean ± SD, n = 3. *P < 0.05; NS, no significant

Fig. 5

High TET1 expression inhibited the progression of HCC cells in vitro. a The expression of 5hmC and 5mC was detected in SMMC-7721 cells after transfection with circTRIM33–12 or the control using IF. b The expression of 5hmC and 5mC was detected in SMMC-7721 cells after transfection with TET1 or the control using IF. c Cell proliferation in HCC cells with the forced expression of TET1 was assessed by a CCK-8 assay. d The cell cycle in HCC cells with the forced expression of TET1 was detected by FCM. e The migration and invasion abilities in HCC cells with the forced expression of TET1 were evaluated via a transwell assay. The data are represented as the mean ± SD, n = 3. *P < 0.05; **P < 0.01

Fig. 6

circTRIM33–12 and TET1 regulate the expression of several same genes in HCC cells. a The protein levels of WWC3, TP53INP1, ULBP1 and JHDM1D were detected in SMMC-7721 cells after transfection with circTRIM33–12, TET1, or the control using western blotting. b The protein levels of WWC3, TP53INP1, ULBP1 and JHDM1D were detected in Huh 7 cells after transfection with circTRIM33–12 shRNA, TET1 shRNA, or the control using western blotting. c The luciferase activity of luc-WWC3/TP53INP1/ULBP1/JHDM1D or mutant luc-WWC3/TP53INP1/ULBP1/JHDM1D in Huh 7 cells after cotransfection with miR-191. d Representative HCC cases in the tissue microarray were analyzed by immunohistochemical staining for NKG2D. e A positive correlation between circTRIM33–12 and the number of NKG2D-positive cells was observed in HCC tissues (R² = 0.1193; P < 0.001). The data are represented as the mean ± SD, n = 3. NS, no significant

Fig. 7

Silencing of miR-191 impairs the cell proliferation, migration and invasion abilities of HCC cells with reduced circTRIM33–12 expression. a miR-191 expression in HCC cells was modified by shRNA interference. b, c Reduced miR-191 expression enhanced TET1 protein expression but not TET1 mRNA in HCC cells. d, e The cell proliferation in HCC cells with the reduced expression of miR-191 was assessed by a CCK-8 assay. f, g The migration and invasion abilities in HCC cells with the reduced expression of miR-191 were evaluated via a transwell assay. The data are represented as the mean ± SD, n = 3. *P < 0.05; **P < 0.01; NS, no significant