EIF4A3-regulated circ_0087429 can reverse EMT and inhibit the progression of cervical cancer via miR-5003-3p-dependent upregulation of OGN expression

Abstract

Background: Circular RNAs (circRNAs) are noncoding RNAs with stable structures with high expression and tissue-specific expression. Studies have shown that circRNA dysregulation is closely related to the progression of tumours. However, the function and regulatory mechanism of most circRNAs in cervical cancer are still unclear. METHODS: CircRNAs related to cervical cancer were screened through the Gene Expression Omnibus (GEO) database. qRT-PCR was used to verify the expression of circ_0087429 in cervical cancer tissues and cells. Then, in vivo and in vitro experiments were conducted to evaluate the role of circ_0087429 in the progression of cervical cancer. The role of the circ_0087429/miR-5003-3p/osteoglycin (OGN) axis in the epithelial to mesenchymal transition (EMT) was confirmed by rescue experiments, fluorescence in situ hybridization, luciferase reporter assays, immunofluorescence staining and western blotting. The inhibitory effect of Eukaryotic initiation factor 4A-III (EIF4A3) on the biogenesis of circ_0087429 was verified by RNA immunoprecipitation (RIP) assays and qRT-PCR.

Results: circ_0087429 is significantly downregulated in cervical cancer tissues and cells and negatively correlated with International Federation of Gynecology and Obstetrics (FIGO) staging and lymphatic metastasis in cervical cancer patients. circ_0087429 can significantly inhibit the proliferation, migration, invasion and angiogenesis of cervical cancer in vitro and tumour growth and metastasis in vivo. OGN is significantly downregulated in cervical cancer tissues and cells. circ_0087429 can upregulate the expression of OGN by competitively binding with miR-5003-3p, thereby reversing EMT and inhibiting the progression of cervical cancer. EIF4A3 can inhibit circ_0087429 expression by binding to its flanking regions.

Conclusions: As a tumour suppressor, circ_0087429 regulated by EIF4A3 can reverse EMT and inhibit the progression of cervical cancer through the miR-5003-3p/OGN axis. It is expected to become a potential target for the treatment of cervical cancer.

Keywords: Cervical cancer; Circ_0087429; EIF4A3; Epithelial to mesenchymal transition; MiR-5003-3p; OGN.

Fig. 1

The expression and characteristics of circ_0087429 in cervical cancer. a. Differentially expressed circRNAs between cervical cancer and adjacent tissue samples in the GSE102686 dataset. b. Differentially expressed circRNAs between cervical cancer and normal cervical epithelial cell lines in the GSE103696 dataset. c. Heatmap of circRNAs with significant differential expression in two datasets (GSE102686 and GSE103696). circ_0087429 (hsa_circRNA_104814) is marked with a red box. d. Differential expression of circ_0087429 in 44 pairs of cervical cancer and adjacent tissue samples. e. Differential expression of circ_0087429 between cervical cancer and normal cervical epithelial cell lines. f. The Sanger sequencing results showed that circ_0087429 was formed by head-to-tail splicing of the second exon of the parental gene SPIN1. g. The expression of circ_0087429 and SPIN1 after RNaseR treatment was detected by qRT-PCR. h. The expression changes in circ_0087429 and SPIN1 at different time points after adding actinomycin D were determined by qRT-PCR. i. A subcellular fractionation assay was used to detect the relative expression of circ_0087429 in the cytoplasm and nucleus. U6 was used as the internal reference for the nuclear fraction, and GAPDH was used as that for the cytoplasmic fraction. j. FISH staining confirmed the expression of circ_0087429 in the cytoplasm. Scale bar, 100 μm. ***p < 0.001

Fig. 2

circ_0087429 inhibits the proliferation of cervical cancer in vitro. a. The expression of circ_0087429 in HeLa cells stably transfected with pcDNA5.1-circ_0087429 was detected by qRT-PCR. b. The expression of circ_0087429 in SiHa cells treated with siRNA was detected by qRT-PCR. c-g. CCK-8 assays and EdU assays were used to detect changes in cell proliferation after overexpression or knockdown of circ_0087429. Scale bar, 50 μm. h-i. Colony formation assays were used to detect the proliferation ability of cells after overexpression or knockdown of circ_0087429. **p < 0.01, ***p < 0.001

Fig. 3

circ_0087429 inhibits migration, invasion and angiogenesis of cervical cancer in vitro. a-b. Wound healing assays determined the migration ability of cells after overexpression or knockdown of circ_0087429. Scale bar, 100 μm. c-d. Transwell invasion assays detected the invasion ability of cells after overexpression or knockdown of circ_0087429. Scale bar, 50 μm. e–f. After HUVECs were cocultured with circ_0087429-overexpressing or circ_0087429-knockdown cells, the tube-forming ability was detected. Scale bar, 50 μm. g. The expression of circ_0087429 in HUVECs after coculture with circ_0087429 overexpression or knockdown cells was detected by qRT-PCR. **p < 0.01, ***p < 0.001

Fig. 4

circ_0087429 can competitively bind with miR-5003-3p. a-b. The expression changes in candidate miRNAs after circ_0087429 overexpression or knockdown were determined by qRT-PCR. c. The differential expression of miR-5003-3p between cervical cancer and normal cervical epithelial cell lines was detected by qRT–PCR. d. The differential expression of miR-5003-3p between cervical cancer and adjacent tissue samples was detected by qRT–PCR. e. Spearman’s correlation analysis confirmed the correlation between circ_0087429 and miR-5003-3p expression in cervical cancer tissue samples. f. The colocalization of circ_0087429 and miR-5003-3p in cells was detected by FISH staining. Scale bar, 50 μm. g. The binding sites of circ_0087429 and miR-5003-3p were predicted by circBank. h-i. A luciferase reporter assay was used to examine the relative luciferase activity of the circ_0087429WT and circ_0087429MUT. **p < 0.01, ***p < 0.001, NS means no statistical significance

Fig. 5

The tumour suppressor effect of circ_0087429 in cervical cancer can be reversed by miR-5003-3p. HeLa cells were transfected with pcDNA5.1-NC, pcDNA5.1-circ, pcDNA5.1-circ + mimics NC or pcDNA5.1-circ + miR mimics for subsequent detection. a-b. The expression of circ_0087429 and miR-5003-3p in each group was tested by qRT-PCR. c-g. CCK-8, EdU and colony formation assays were used to detect the proliferation ability of each group of cells. Scale bar, 50 μm. h-i. Wound healing assays determined the migration ability of each group of cells. Scale bar, 100 μm. j-k. Transwell invasion assays detected the invasion ability of each group of cells. Scale bar, 50 μm. **p < 0.01, ***p < 0.001

Fig. 6

circ_0087429 downregulates the expression of OGN through competitive binding with miR-5003-3p in cervical cancer. a. The databases miRDB, miRWalk, mirDIP and TargetScan predicted the target genes of miR-5003-3p b. The expression changes in OGN after circ_0087429 overexpression and miR-5003-3p knockdown were determined by qRT-PCR. c. The expression changes in OGN after circ_0087429 knockdown and miR-5003-3p overexpression were determined by qRT-PCR. d. The expression of OGN was detected by western blotting after the expression of circ_0087429 and miR-5003-3p was changed. e. The fluorescence intensity of OGN after circ_0087429 overexpression or knockdown was confirmed by immunofluorescence staining. Scale bar, 25 μm. f. The binding sites of circ_0087429 and miR-5003-3p were predicted by miRWalk. g. A luciferase reporter assay was used to examine the relative luciferase activity of OGN-WT and OGN-MUT. h-i. The differential expression of OGN between cervical cancer and normal cervical epithelial cell lines was detected by western blotting and qRT-PCR. j-k. The differential expression of OGN between cervical cancer (T) and adjacent tissue (P) samples was detected by western blotting and qRT-PCR. l. Spearman’s correlation analysis confirmed the correlation between circ_0087429 and OGN expression in cervical cancer tissue samples. **p < 0.01, ***p < 0.001, NS means no statistical significance

Fig. 7

The cancer-promoting effect of miR-5003-3p in cervical cancer can be reversed by OGN. HeLa cells were transfected with mimics NC, miR mimics, miR mimics + oe-NC or miR mimics + oe-OGN for subsequent detection. a-b. The expression of miR-5003-3p and OGN in each group was tested by qRT-PCR. c-g. CCK-8, EdU and colony formation assays were used to detect the proliferation ability of each group of cells. Scale bar, 50 μm. h-i. Wound healing assays determined the migration ability of each group of cells. Scale bar, 100 μm. j-k. Transwell invasion assays detected the invasion ability of each group of cells. Scale bar, 50 μm. **p < 0.01, ***p < 0.001

Fig. 8

EIF4A3-regulated circ_0087429 can inhibit EMT in cervical cancer cells through the circ_0087429/miR-5003-3p/OGN axis. a-b. Immunofluorescence staining detected the fluorescence intensities of N-cadherin or E-cadherin upon circ_0087429 overexpression or knockdown, respectively. Scale bar, 25 μm. c-d. EMT related proteins in each group of cells were detected by western blotting. e. The binding sites for EIF4A3 in the flanking sequences of the SPIN1 mRNA transcript were predicted using CircInteractome. f. RIP assay demonstrated the binding between SPIN1 and EIF4A3. g. The differential expression of EIF4A3 between cervical cancer and normal cervical epithelial cell lines was detected by western blotting. h-i. The expression of EIF4A3 in the two groups of SiHa cells of oe-NC and oe-EIF4A3 was detected by qRT-PCR and western blotting. j-k. The expression of EIF4A3 in Caski cells treated with siRNA was detected by qRT-PCR and western blotting. l. The expression of circ_0087429 and EIF4A3 in SiHa cells stably overexpressing EIF4A3 were detected by qRT-PCR. m. The expression of circ_0087429 and EIF4A3 in Caski cells treated with si-EIF4A3 were detected by qRT-PCR. *p < 0.05, **p < 0.01, ****p < 0.0001

Fig. 9

Overexpression of circ_0087429 in vivo can inhibit the progression of cervical cancer a. Image of subcutaneous tumour tissues in the circ_0087429-overexpressing group and control group. b-c. Volume and weight of subcutaneous tumour tissues in each group. d. The expression levels of circ_0087429, miR-5003-3p and OGN in subcutaneous tumour tissues of each group were detected by qRT-PCR. e–f. The expression levels of OGN, N-cadherin and E-cadherin in subcutaneous tumour tissues of each group were detected by western blotting. g-h. The expression levels of OGN, N-cadherin, E-cadherin, Ki67 and CD31 in subcutaneous tumour tissues of each group were detected by immunohistochemical staining. Scale bar, 30 μm. i-l. Representative images and bar graphs of liver (i-j) and lung (k-l) metastases with circ_0087429-overexpressing group and control group in a nude mouse metastatic tumour model. Metastatic nodules are indicated by arrows. Scale bar, 60 μm. m. A schematic diagram showing how circ_0087429 regulates the expression of OGN by sponging miR-5003-3p and thereby reverses EMT. MET: mesenchymal to epithelial transition. *p < 0.05, **p < 0.01, ***p < 0.001