CircSETD3 (Hsa_circ_0000567) acts as a sponge for microRNA-421 inhibiting hepatocellular carcinoma growth

Abstract

Background: Circular RNAs (circRNAs) play important roles in tumourigenesis and tumour progression. However, the expression profiles and functions of circRNAs in hepatocellular carcinoma (HCC) are largely unclear.

Methods: The expression profiles of circRNAs in HCC were identified through microarray analysis and were validated through quantitative reverse transcription polymerase chain reaction (qRT-PCR). Survival curves were plotted using the Kaplan-Meier method and compared using the log-rank test. The circular structure of candidate circRNA was confirmed through Sanger sequencing, divergent primer PCR, and RNase R treatments. Proliferation of HCC cells was evaluated in vitro and in vivo. The microRNA (miRNA) sponge mechanism of circRNAs was demonstrated using dual-luciferase reporter and RNA immunoprecipitation assays.

Results: CircSETD3 (hsa_circRNA_0000567/hsa_circRNA_101436) was significantly downregulated in HCC tissues and cell lines. Low expression of circSETD3 in HCC tissues significantly predicted an unfavourable prognosis and was correlated with larger tumour size and poor differentiation of HCC in patients. In vitro experiments showed that circSETD3 inhibited the proliferation of HCC cells and induced G1/S arrest in HCC cells. In vivo studies revealed that circSETD3 was stably overexpressed in a xenograft mouse model and inhibited the growth of HCC. Furthermore, we demonstrated that circSETD3 acts as a sponge for miR-421 and verified that mitogen-activated protein kinase (MAPK)14 is a novel target of miR-421.

Conclusion: CircSETD3 is a novel tumour suppressor of HCC and is a valuable prognostic biomarker. Moreover, circSETD3 inhibits the growth of HCC partly through the circSETD3/miR-421/MAPK14 pathway.

Keywords: Circular RNA; Growth; Hepatocellular carcinoma; SETD3; microRNA.

Fig. 1

Identification of hsa_circ_0000567 as a candidate biomarker of HCC. a Clustered heat map showed differentially expressed circRNAs in two paired HCC (T) and adjacent non-tumorous tissues (N). b-f The relative expression of three most upregulated circRNAs (hsa_circ_0005394, hsa_circ_0001741, hsa_circ_0006916) and two most downregulated circRNAs (hsa_circ_0000567, hsa_circ_0004058) were validated by qRT-PCR. g The relative expression of hsa_circ_0000567 in 132 HCC and 56 non-tumorous tissues. h and i Kaplan-Meier’s survival curves depicted the correlations between hsa_circ_0000567 and RFS or OS of HCC patients (the patients were stratified into two groups according to the median of hsa_circ_0000567). Log-rank test was used. For b-f, data are presented as means ± SD. HCC, hepatocellular carcinoma; T, tumor tissue; N, non-tumorous tissue; qRT-PCR, quantitative reverse transcription polymerase chain reaction; RFS, recurrence-free survival; OS, overall survival

Fig. 2

Confirmation of the circular structure of circSETD3. a Schematic illustration showed that circSETD3 is located at chromosome 14q32.2 and cyclized from exons 2–6 of SETD3, the PCR products of circSETD3 were confirmed by Sanger sequencing. b The existence of cricSETD3 was validated in HCC and paired non-tumorous tissues as well as Hep3B cells. Divergent primers detected circular RNAs in cDNA but not in gDNA. GAPDH was used as negative control. c PCR for detecting circSETD3 and SETD3 linear form RNA in HCC and paired non-tumorous tissues as well as HepG2 cells treated with or without RNase R digestion, circSETD3 was resistant to RNase R treatment. SETD3, SET domain-containing 3; cDNA, complementary DNA; gDNA, genomic DNA. PCR, polymerase chain reaction. ►◄, convergent primer; ◄►, divergent primer

Fig. 3

CircSETD3 inhibits the proliferation of HCC cells in vitro. a The expression levels of circSETD3 in multiple HCC cell lines. b The expression level of circSETD3 in Huh7 cells were successfully overexpressed by circSETD3 letivirus. c The expression level of circSETD3 in Hep3B cells were knocked down by circSETD3 siRNA. d-g CCK-8 (d and e), colony formation (f), and EdU (g) assays showed the overexpression of circSETD3 inhibited the growth of Huh7 cells while knockdown of circSETD3 promoted the growth of Hep3B cells. h Cell cycle was analysed using flow cytometry after transfection with circSETD3 letivirus or siRNA. Overexpression of circSETD3 induced G1/S arrest in Huh7 cells. Knockdown of circSETD3 relieved the G1/S arrest in Hep3B cells. HCC, hepatocellular carcinoma; NC, negative control. **P < 0.01, ***P < 0.001. Error bars indicate SD

Fig. 4

SETD3 is not the target of circSETD3 whereas circSETD3 acts as a sponge for miR-421 in HCC. a The expression level of SETD3 mRNA in 56 pairs of HCC and non-tumorous tissues. b CircSETD3 positively correlated with SETD3 mRNA in HCC tissues. c and d The mRNA and protein levels of SETD3 were not changed after artificially changed the expression of circSETD3 in HCC cells. e 10 most possible miRNAs that could be sequestered by circSETD3 were identified. Among them, miR-421 is a well-demonstrated tumor promoter. f miR-421 was significantly upregulated in HCC tissues compared with matched non-tumorous tissues. g The expression of miR-421 was negatively associated with circSETD3 in HCC tissues. h and i Kaplan-Meier’s survival curves depicted the correlations between miR-421 and RFS or OS of HCC patients (the patients were stratified into two groups according to median of Mr-421). Log-rank test was used. j Schematic of circSETD3 wild-type (WT) and mutant (Mut) luciferase reporter vectors. k The relative luciferase activities were analyzed in 293 T cells co-transfected with miR-421 mimics, miR-mimics-NC, miR-421 inhibitors or miR-inhibitors-NC and WT or Mut luciferase reporter vectors. l The Ago2 RIP showed that Ago2 significantly enriched circSETD3 and miR-421. m Expression change of circSETD3 did not affect the expression of miR-421. n Expression change of miR-421 did not affect the expression of circSETD3. HCC, hepatocellular carcinoma; NS, not significant; RIP, RNA immunoprecipitation. **P < 0.01, ***P < 0.001. Error bars indicate SD

Fig. 5

CircSETD3 inhibits the growth of HCC through thecircSETD3/miR-421/MAPK14 pathway. a and b Both qRT-PCR and IHC showed MAPK14 was significantly downregulated in HCC tissues compared with matched non-tumorous tissues. c and d MAPK14 negatively correlated with miR-421 whereas positively correlated with circSETD3 in HCC tissues. e Schematic of MAPK14 wild-type (WT) and mutant (Mut) luciferase reporter vectors. f The relative luciferase activities were analyzed in 293 T cells co-transfected with miR-421 mimics or miR-mimics-NC and WT or Mut luciferase reporter vectors. g MiR-421 inhibitor up-regulated MAPK14 and down-regulated cyclinD1 and PCNA in Hep3B cells. MiR-421 mimics down-regulated MAPK14 and up-regulated cyclinD1 and PCNA in Huh7 cells. h CircSETD3 letivirus up-regulated MAPK14 and down-regulated cyclinD1 and PCNA in Huh7 cells, this effect could be reversed by co-transfected with miR-421 mimics. i circSETD3 siRNA down-regulated MAPK14 and up-regulated cyclinD1 and PCNA in Hep3B cells, this effect can be reversed by co-transfected with miR-421 inhibitors. HCC, hepatocellular carcinoma; qRT-PCR, quantitative reverse transcription polymerase chain reaction; in, inhibitors; mi, mimics; IHC, immunohistochemistry. ***P < 0.001. Error bars indicate SD

Fig. 6

CircSETD3 stably maintained in xenograft tumor models and inhibit tumor growth by targeting MAPK14. a and b Smaller tumor size and lower tumor weight were observed in circSETD3-overexpressing group. c Over-expressed circSETD3 could stably maintained in xenograft tumor models. d The expression of MAPK14 was increased, Ki-67, PCNA and Cyclin D1 were decreased in circSETD3-overexpressing group when compared with control group. **P < 0.01, ***P < 0.001. Error bars indicate SD