Circular RNA circ_0008274 upregulates granulin to promote the progression of hepatocellular carcinoma via sponging microRNA -140-3p

Figures & data

Figure 1. Circ_0008274 expression is significantly up-regulated in HCC tissues and cells

A. Circ_0008274 was generated from the transcript of UGGT2 gene.B. Volcanic map was used to show the changes of circRNAs’ expression in HCC tissues compared with normal tissues adjacent to cancer. CircRNAs whose expression was significantly up-regulated were marked with orange, while those with significantly down-regulated expression were marked with blue (log₁₀|fold change| > 1, P < 0.05).C. The differentially expressed circRNAs in HCC tissues and adjacent normal tissues were shown in the heatmap.D. The expression of circ_0008274 in HCC tissues and adjacent tissues was detected by qRT-PCR.E. The expression of circ_0008274 in HCC cell lines and THLE-3 cell was detected by qRT-PCR.F. The subcellular localization of circ_0008274 in Huh-7 and HepG2 cells was detected by qRT-PCR.** P < 0.01, and *** P < 0.001.

Table 1. Primers used for qRT-PCR

	Forward	Reverse
circ_0008273	5ʹ-CCAGATCTCCCTGTTTCACC CCTGTCCTCCTAAACCTCCAAG-3’	5ʹ-CTTGCCCTCTTTGGCTCTCT-3’
miR-140-3p	5ʹ-TACCACAGGGTAGAACCACGG-3ʹ	5ʹ-CCACAGGGTAGAACCACGG-3ʹ
GRN	5ʹ-ATCTTTACCGTCTCAGGGACTT-3’	5ʹ-CCATCGACCATAACACAGCAC-3’
U6	5ʹ-TGCGGGTGCTCGCTTCGGCAGC-3’	5ʹ-CCAGTGCAGGGTCCGAGGT-3’
GAPDH	5ʹ-GCACCGTCAAGGCTGAGAAC-3’	5ʹ-TGGTGAAGACGCCAGTGGA-3’

Table 2. Correlation between clinicopathological features and expression of circ_0008274 in HCC tissues

Pathological Parameters	Number (n = 55)	Circ_0008274 expression High(n = 28) Low(n = 27)		P-Value
Gender				0.3341
Male	38	18	20
Female	17	10	7
Age (years)				0.1103
<50	40	23	17
≥50	15	5	10
Tumor size (cm)				0.0329*
>5	38	23	15
<5	17	5	12
TNM stage				0.0425*
I+ II	23	8	15
III+IV	32	20	12
Degree of differentiation				0.0780
Low, medium	33	20	13
High	22	8	14

*P < 0.05.

Figure 2. Circ_0008274 promotes the proliferation, migration and invasion of HCC cells

A&B. pcDNA-NC or pcDNA-circ_0008274 was transfected into Huh-7 cells, and si-NC, si-circ_0008274-1 and si-circ_0008274-2 were transfected into HepG2 cells. Then the expression of circ_0008274 was detected by qRT-PCR.C&D. CCK-8 and EdU assays were used to detect the effects of overexpression or knockdown of circ_0008274 on the proliferation of Huh-7 or HepG2 cells.E. Transwell assay was used to measure the effects of overexpression or knockdown of circ_0008274 on the migration and invasion of Huh-7 or HepG2 cells.* P < 0.05, ** P < 0.01, and *** P < 0.001.

Figure 3. Circ_0008274 adsorbs miR-140-3p

(a).The potential binding site between circ_0008274 and miR-140-3p was predicted by Circinteractome database.(b). The effects of miR-140-3p mimics or miR-140-3p inhibitors on the relative luciferase activity of the reporters carrying the sequence of circ_0008274, were detected by dual-luciferase reporter gene assay.(c). RIP assay was used to detect the enrichment degree of circ_0008274 and miR-140-3p, to indicate their interaction.(d). The expression of miR-140-3p in HCC cells was detected by qRT-PCR after overexpression or knockdown of circ_0008274.(e).The expression of miR-140-3p in HCC tissues and adjacent tissues was detected by qRT-PCR. (f). The expression of miR-140-3p in HCC cell lines and THLE-3 cells was detected by qRT-PCR. (g). Pearson correlation analysis showed the correlation between circ_0008274 and miR-140-3p expressions in HCC tissues.* P < 0.05, ** P < 0.01, and *** P < 0.001.

Figure 4. Circ_0008274 can promote the malignant behaviors of HCC cells by targeting miR-140-3p

(a).pcDNA-NC, pcDNA-circ_0008274 and pcDNA-circ_0008274+ miR-140-3p mimics were transfected into Huh-7 cells; and si-NC, si-circ_0008274-1 and si-circ_0008274-1+ miR-140-3p inhibitors were transfected into HepG2 cells. MiR-140-3p expression was detected by qRT-PCR.B & C The proliferation of Huh-7 or HepG2 cells were detected by CCK-8 and EdU experiments.D. Transwell assay was used to detect the migration and invasion of Huh-7 or HepG2 cells.* P < 0.05, ** P < 0.01, and *** P < 0.001.

Figure 5. Circ_0008274 regulates miR-140-3p/GRN axis

A.The potential binding sites between GRN mRNA 3’-UTR and miR-140-3p were predicted by TargetScan database.B.The relative luciferase activity was detected by dual-luciferase reporter gene assay.C&D. The effects of circ_0008274 and miR-140-3p on the expression of GRN protein and mRNA were detected by western blot and qRT-PCR.E. The expression of GRN mRNA in HCC tissues and adjacent tissues was detected by qRT-PCR.F&G. Pearson correlation analysis was used to detect correlations between miR-140-3p expression and GRN mRNA expression, and miR-140-3p expression and GRN mRNA expression in HCC samples.* P < 0.05, ** P < 0.01, and *** P < 0.001.

Data availability statement

The data used to support the findings of this study are available from the corresponding author upon request.