CircRNA has_circ_0001806 promotes hepatocellular carcinoma progression via the miR-193a-5p/MMP16 pathway

Zhou, Hongmu; Chen, Ying

doi:10.1590/1414-431X2021e11459

Hongmu Zhou

Department of Geriatrics, General Hospital of The Yangtze River Shipping, Wuhan Brain Hospital, Wuhan, Hubei, China

https://orcid.org/0000-0001-9651-0459

Ying Chen Correspondence: Ying Chen: <yilf49254@163.com>

Department of Gastroenterology, Affiliated Hospital of Jianghan University, Wuhan Sixth Hospital, Wuhan, Hubei, China

https://orcid.org/0000-0002-5812-6277

Correspondence: Ying Chen: <yilf49254@163.com>

Figures (5)
Tables (2)

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Figure 1
circ_0001806 is up-modulated in hepatocellular carcinoma (HCC). A, 15 circRNAs were differentially expressed in two microarray datasets (GSE94508, GSE97332) containing the circRNA expression profiles of HCC tissues and normal liver tissues. Heat maps showing the up-modulated circRNAs in HCC tissues compared to the matched non-tumor tissues GSE94508 (B) and GSE97332 (C). D, The levels of circ_0001806 were examined by qRT-PCR after treatment with RNase R. E, The subcellular distribution of circ_0001806 was analyzed by qRT-PCR after cellular RNA fractionation. GAPDH was used as the control for cytoplasm and U6 was used as the control for nucleus. F, The expression levels of circ_0001806 in 49 paired HCC tissues and matched adjacent normal tissues were examined by qRT-PCR. G, The expression levels of circ_0001806 in human HCC cell lines (Huh-7, HepG2, SMMC-7721, Bel-7402, and HL-7702) and the normal hepatic cell line (L02) were examined by qRT-PCR. Data are reported as means±SD. ***P<0.001 (ANOVA).

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Figure 2
Knockdown of circ_0001806 inhibits multiplication, migration, and invasion of HCC cells. A, Three siRNAs targeting circ_0001806 (si-circ_0001806#1, si-circ_0001806#2, and si-circ_0001806#3) were transfected into Huh-7 and HepG2 cells and the expression level of circ_0001806 was detected by qRT-PCR. B, CCK-8 method was used to detect the multiplication of Huh-7 and HepG2 cells after transfection. C and D, Transwell experiment was employed to detect the migration and invasion of Huh-7 and HepG2 cells after transfection (scale bar 20 μm). Data are reported as means±SD. *P<0.05, **P<0.01, ***P<0.001; ns, not statistically significant (ANOVA and t-test).

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Figure 3
miR-193a-5p is the downstream target of circ_0001806. A, Bioinformatics analysis predicted that the sequence of miR-193a-5p matched the sequences of circ_0001806, and circ_0001806-WT (wild-type) and circ_0001806-MUT (mutant) luciferase reporter gene vectors were constructed. B, miR-193a-5p mimics (or miR control) were co-transfected with circ_0001806-WT (or circ_0001806-MUT) into HEK293T cells. After 48 h, the luciferase activity of the cells in each group was measured. C, The complex containing circ_0001806 and miR-193a-5p in Huh-7 and HepG2 cells was immunoprecipitated by anti-AGO2 using RIP assay. D, RNA pull-down assay was carried out to verify the interactions between circ_0001806 and miR-193a-5p. E, qRT-PCR was used to detect the expression of miR-193a-5p in Huh-7 and HepG2 cells after circ_0001806 knockdown. F, qRT-PCR was used to examine the expression levels of miR-193a-5p in 49 paired hepatocellular carcinoma (HCC) tissues and matched adjacent normal tissues. G, Pearson correlation analysis was utilized to evaluate the correlation between the expressions of circ_0001806 and miR-193a-5p in HCC tissues. Data are reported as means±SD. ***P<0.001 (t-test).

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Figure 4
MMP16 is the direct target of miR-193a-5p. A, Bioinformatics analysis was performed with starBase, TargetScan, and miRDB to predict the candidate mRNAs, which could be targeted by miR-193a-5p. B, Bioinformatics analysis predicted that the sequence of MMP16 3UTR matched the sequences of miR-193a-5p, and MMP16-WT (wild-type) and MMP16-MUT (mutant) luciferase reporter gene vectors were constructed. C, The miR-193a-5p mimics (or miR control) were co-transfected with MMP16-WT (or MMP16-MUT) into HEK293T cells, and the luciferase activity of the cells in each group was measured. D, Huh-7 and HepG2 cells were transfected with anti-miR-193a-5p and negative control (NC), and the expression level of miR-193a-5p was detected by qRT-PCR. E, qRT-PCR was used to detect the expression of MMP16 mRNA in Huh-7 and HepG2 cells after circ_0001806 knockdown. F, qRT-PCR was used to detect the expression of MMP16 mRNA in Huh-7 and HepG2 cells transfected with anti-miR-193a-5p. G, Western blot was used to detect the expression of MMP16 protein in Huh-7 and HepG2 cells after circ_0001806 knockdown (G) and in Huh-7 and HepG2 cells transfected with anti-miR-193a-5p (H). Data are reported as means±SD. **P<0.01, ***P<0.001 (t-test).

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Figure 5
Circ_0001806 enhances the multiplication and invasion of hepatocellular carcinoma (HCC) cells through miR-193a-5p/MMP16 axis. Huh-7 and HepG2 cells were transfected with si-NC + anti-miR-Con, si-circ_0001806#1 + anti-miR-Con, or si-circ_0001806#1 + anti-miR-193a-5p. A, qRT-PCR was used to detect the expression of miR-193a-5p in Huh-7 and HepG2 cells. B, qRT-PCR was used to detect the expression of MMP16 mRNA in Huh-7 and HepG2 cells. C, Western blot was used to detect the expression of MMP16, MMP2, and MMP9 protein in Huh-7 and HepG2 cells. D, CCK-8 method was used to detect the multiplication of Huh-7 and HepG2 cells after transfection. The transwell experiment was employed to detect the migration and invasion of Huh-7 cells (E) and HepG2 cells (F) after transfection (scale bar 20 μm). Data are reported as means±SD. *P<0.05, **P<0.01, and ***P<0.001 (ANOVA).

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Table 1
Primer sequences used in this study.

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Table 2
The intersected circRNAs form differentially expressed circRNAs in 2 gene sets.

Gene	Sequence
circ_0001806	Forward: 5-GTGATCTGAAAGGGCCAGAG-3
	Reverse: 5-TCCACATCACCCTTCACCTT-3
MMP16	Forward: 5-TAGAGCGTGCAGATAATGACAAGGA-3
	Reverse: 5-TGAACTGCTAGCCTCTGGATTTGA-3
miR-193a-5p	Forward: 5-TATATGGGTCTTTGCGGGCG-3
	Reverse: 5-GTGCAGGGTCCGAGGT-3
GAPDH	Forward: 5-GGAGCGAGATCCCTCCAAAAT-3
	Reverse: 5-GGCTGTTGTCATACTTCTCATGG-3
U6	Forward: 5-ATTGGAACGATACAGAGAAGATT-3
	Reverse: 5-GGAACGCTTCACGAATTTG-3

Gene symbol	GSE94508		GSE97332
Gene symbol	LogFC	P value	LogFC	P value
hsa_circ_0067934	1.831013	0.0223	2.6748404	0.00315
hsa_circ_0043438	1.554496	7.38E-09	2.0000682	0.0397
hsa_circ_0014879	1.548521	7.18E-09	1.5874528	0.0302
hsa_circ_0000981	1.541864	1.13E-08	1.6756029	0.0152
hsa_circ_0001806	1.539452	2.25E-09	2.3077778	0.000246
hsa_circ_0079480	1.524299	2.47E-06	-1.714286	4.57E-06
hsa_circ_0009581	-1.547747	0.000224	2.5098621	0.000521
hsa_circ_0084429	-1.566135	0.00311	1.7969988	0.000559
hsa_circ_0027774	-1.595124	0.000121	1.6627344	0.000397
hsa_circ_0056548	-1.759187	0.0000021	-1.66516	0.000954
hsa_circ_0007762	-1.951053	0.0000012	-1.553882	0.00238
hsa_circ_0078279	-2.060631	0.0000212	-1.501581	0.00155
hsa_circ_0059859	-2.064442	0.0000086	1.5601697	0.000159
hsa_circ_0055033	-2.843088	2.48E-06	2.6107158	0.0000107
hsa_circ_0004913	-6.093206	8.96E-13	-3.585576	0.0000169

[1] Correspondence: Ying Chen: <yilf49254@163.com>

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CircRNA has_circ_0001806 promotes hepatocellular carcinoma progression via the miR-193a-5p/MMP16 pathway