circDENND4C, a novel serum marker for epithelial ovarian cancer, acts as a tumor suppressor by downregulating miR-200b/c

Figures & data

Figure 1. Expression of circDENND4C and miR-200b/c and its correlation in EOC tissue and serum specimens. (a–c) qRT-PCR was performed to determine the relative expression of circDENND4C and miR-200b/c in clinical tissue specimens. (d–f) qRT-PCR was performed to measure the relative expression of circDENND4C and miR-200b/c in clinical serum specimens. (g–j) The correlation between circDENND4C and miR-200b/c expression in EOC tissue and serum specimens. *** vs normal tissue/serum, p < 0.001; ^### vs benign tissue/serum, p < 0.001.

Figure 2. CA125 and HE4 expressions and the correlation between circDENND4C and CA125/HE4 in EOC serum specimens. (a,b) Serum CA125 and HE4 levels in clinical serum specimens. (c,d) The correlation between circDENND4C and CA125/HE4 expression in EOC serum specimens was determined using Spearman correlation coefficient analysis. *** vs normal serum, p < 0.001; ^### vs benign serum, p < 0.001.

Figure 3. Serum circDENND4C expression differs between patients with benign ovarian tumors/EOC and healthy women. (a,b) ROC analysis shows the sensitivity and specificity of serum circDENND4C in distinguishing patients with benign ovarian tumors/EOC from healthy women. ROC: receiver operating characteristic; AUC: area under the curve.

Table 1. The expression of circDENND4C in tissues/serums of EOC patients was related to the disease progression.

Parameters	Tissular circDENND4C		p Values	Serumal circDENND4C
	High expression (n = 30)	Low expression (n = 30)		High expression (n = 30)	Low expression (n = 30)	p Values
Age
<50	11	13	0.598	10	14	0.292
≥50	19	17		20	16
Menopause
Yes	18	16	0.602	19	15	0.297
No	12	14		11	15
FIGO stage
I/II	18	8	0.006	17	9	0.030
III/IV	12	22		13	21
TNM stage
I/II	19	7	0.001	18	8	0.006
III/IV	11	23		12	22
Histologic grade
G1/G2	17	8	0.013	16	9	0.059
G3	13	22		14	21
Tumor size
<5 cm	18	9	0.020	18	9	0.020
≥5 cm	12	21		12	21
Differentiation degree
Low	8	15	0.032	9	14	0.147
Middle	10	9		10	9
High	12	6		11	7
Lymph node metastasis
Yes	11	20	0.020	12	19	0.071
No	19	10		18	11

circDENND4C: circ-DENN domain containing 4C; EOC: epithelial ovarian cancer; FIGO: International Federation of Obstetrics and Gynecology; TNM: tumor node metastasis.

Table 2. Analysis of auxiliary diagnostic value of serum circDENND4C in EOC.

Binary regression analysis
Classifications	Regression coefficient	p Value
Normal vs. benign	−29.144	0.000
Benign vs. EOC	−151.231	0.994
Normal vs. EOC	−13.077	0.000
Normal + benign vs. EOC	−40.112	0.99
ROC curve analysis
Classifications	AUC	p Value	95% CI	Critical predictive value
Normal vs. benign	0.675	<0.0001	0.6029–0.7471	0.7
Benign vs. EOC	–	–	–	–
Normal vs. EOC	0.675	<0.0001	0.6029–0.7471	0.4
Normal + benign vs. EOC	–	–	–	–

EOC: epithelial ovarian cancer; ROC: receiver operating characteristic; AUC: area under the curve; CI: confident interval.

Table 3. Analysis of auxiliary diagnostic results of serum circDENND4C, CA125 and HE4 for epithelial ovarian cancer.

Parameters		Confirmed results (n)
		Positive	Negative	Total
circDENND4C	Positive	60	5	65
	Negative	0	115	115
CA125	Positive	57	59	116
	Negative	3	61	64
HE4	Positive	57	51	108
	Negative	3	69	72
Total	–	60	120	180

circDENND4C: circ-DENN domain containing 4C; CA125: carbohydrate polypeptide antigen 125; HE4: human epididymal secretory protein 4.

Table 4. The diagnostic performance comparison between serum circDENND4C and CA125/HE4 for epithelial ovarian cancer.

Diagnostic markers	Sensitivity (%)	Specificity (%)	Accuracy (%)
Single marker
CA125^+	95.00 (57/60)	50.83 (61/120)	65.56 (118/180)
circDENND4C^+	100.00 (60/60)	95.83 (115/120)	97.22 (175/180)
X^2	0.039	9.722	6.160
P value	0.844	0.002	0.013
HE4^+	95.00 (57/60)	57.50 (69/120)	70.00 (126/180)
circDENND4C^+	100.00 (60/60)	95.83 (115/120)	97.22 (175/180)
X^2	0.039	6.587	4.368
p Value	0.844	0.010	0.037
Combination of two markers
CA125^++HE4^+	91.67 (55/60)	42.50 (51/120)	58.89 (106/180)
CA125^++circDENND4C^+	95.00 (57/60)	50.00 (60/120)	65.00 (117/180)
X^2	0.018	0.499	0.335
P value	0.892	0.480	0.563
CA125^++HE4^+	91.67 (55/60)	42.50 (51/120)	58.89 (106/180)
HE4^++circDENND4C^+	95.00 (57/60)	55.83 (67/120)	68.89 (124/180)
X^2	0.018	1.457	0.860
p Value	0.892	0.227	0.354
Combination of two/three markers
CA125^++HE4^+	91.67 (55/60)	42.50 (51/120)	58.89 (106/180)
CA125^++HE4^++circDENND4C^+	91.67 (55/60)	41.67 (50/120)	58.33 (105/180)
X^2	0.000	0.007	0.003
p Value	1.000	0.933	0.956

circDENND4C: circ-DENN domain containing 4C; CA125: carbohydrate polypeptide antigen 125; HE4: human epididymal secretory protein 4.

+: positive.

Figure 4. circDENND4C expression and the role on miR-200b/c expression in EOC cells. (a) circDENND4C expression in EOC cells was assessed using qRT-PCR. * vs HOSEPiCs, p < 0.05. (b) EOC cells overexpressing circDENND4C were established via the transfection of a pcDNA-based circDENND4C overexpression vector. (c,d) Overexpression of circDENND4C significantly downregulated miR-200b/c expression in EOC cells. ** vs pcDNA, p < 0.01.

Figure 5. Overexpression of circDENND4C inhibited EOC cell proliferation by regulating the expression of miR-200b/c. (a–d) A CCK-8 proliferation assay was performed to measure the OD value of SKOV3 and OVCAR3 cells at 450 nm and 24 h, 48 h, and 72 h following the transfection of different constructs. * vs pcDNA, p < 0.05; ^# vs circDENND4C, p < 0.05; $vs NC mimic, p < 0.05; % vs miR-200b/c mimic, p < 0.05.

Figure 6. Overexpression of circDENND4C promoted cell apoptosis by regulating the expression of miR-200b/c. (a) Representative images of flow cytometry-based apoptosis analysis. (b) Histogram of results from the flow cytometry apoptosis experiment. * vs pcDNA, p < 0.05; ^# vs circDENND4C, p < 0.05; $vs NC mimic, p < 0.05; % vs miR-200b/c mimic, p < 0.05.

Data availability statement

All the data produced or analyzed in the present work are available from the corresponding author on reasonable request.