Expression Profiles of Circular RNA in Granulosa Cells from Women with Biochemical Premature Ovarian Insufficiency

Figures & data

Table 1. Clinical characteristics of patients with biochemical premature ovarian insufficiency and controls.

Variable	bPOI (n = 23)	Control (n = 23)	p-value
Age (year)	32.22 ± 4.86	32.13 ± 4.20	0.949
BMI (kg/m^2)	20.29 ± 2.58	21.02 ± 2.06	0.307
AMH (ng/ml)	0.56 ± 0.31	2.99 ± 1.56	<0.001
AFC	4.29 ± 1.94	12.64 ± 2.53	<0.001
Basal FSH (mIU/ml)	15.93 ± 6.04	6.79 ± 0.96	<0.001
Basal LH (mIU/ml)	6.43 ± 3.47	5.49 ± 1.59	0.246
Basal FSH/LH	2.87 ± 1.34	1.33 ± 0.39	<0.001
Basal estradiol (pg/ml)	36.12 ± 30.31	34.90 ± 12.15	0.860
Basal progesterone (ng/ml)	0.55 ± 0.41	0.49 ± 0.22	0.495
Number of oocytes retrieved	3.04 ± 1.82	14.05 ± 6.72	<0.001

Data are presented as the mean ± SD; p < 0.05 is considered to be statistically significant.

AFC: Antral follicle count; bPOI: Biochemical premature ovarian insufficiency; AMH: Anti-Müllerian hormone; FSH: Follicle-stimulating hormone; LH: Luteinizing hormone; SD: Standard deviation.

Figure 1. Differential expression of circular RNAs in granulosa cells from eight patients with biochemical premature ovarian insufficiency and eight control patients.

(A) Hierarchical clustering analysis of differently expressed circular RNAs (circRNAs; fold change ≥ 2, p < 0.05). Red = upregulated, green = downregulated. (B) Volcano plot visualizing the differentially expressed circRNAs. The red point in the plot represents the differentially expressed circRNAs (fold change ≥ 2, p < 0.05). (C) Scatter plot visualizing the differentially expressed circRNAs. The circRNAs above the top green line and below the bottom green line indicate more than twofold change of circRNAs.

circRNA: Circular RNA.

Table 2. Characterization of the top 20 upregulated/downregulated circular RNAs in patients with biochemical premature ovarian insufficiency.

CircRNA	Regulation	Fold change	p-value	Gene symbol	Chromosome	Strand	Start	End
hsa_circRNA_104503	Up	4.3348856	0.031039861	TRIM24	chr7	+	138203933	138255748
hsa_circRNA_003785	Up	4.3191191	0.016443185	XLOC_012658	chr18	+	32902351	32907027
hsa_circRNA_104616	Up	4.1071068	0.0218736	SPIDR	chr8	+	48320422	48353104
hsa_circRNA_103903	Up	3.9442081	0.023707735	PAPD4	chr5	+	78952780	78964851
hsa_circRNA_101258	Up	3.9246349	0.020856298	VWA8	chr13	-	42385360	42393522
hsa_circRNA_036088	Up	3.8659703	0.029294692	RPLP1	chr15	+	69745158	69747884
hsa_circRNA_100236	Up	3.6513049	0.039709019	TMEM59	chr1	-	54506428	54509198
hsa_circRNA_402819	Up	3.4858104	0.030949297	GLB1	chr3	-	33114035	33118729
hsa_circRNA_001153	Up	3.4033414	0.028277148	MARCH1	chr4	+	164514761	164515093
hsa_circRNA_104299	Up	3.3711272	0.009393462	USP42	chr7	+	6150755	6155154
hsa_circRNA_103689	Up	3.339481	0.024133026	PTPN13	chr4	+	87693930	87696805
hsa_circRNA_405558	Up	3.3032197	0.040345806	SSH2	chr17	-	28120137	28120955
hsa_circRNA_403691	Up	3.296719	0.035614529	LOC101927768	chr6	+	114313056	114317224
hsa_circRNA_002131	Up	3.21306	0.035508723	BNIP3L	chr8	+	26248758	26265892
hsa_circRNA_001729	Up	3.2130321	0.012576279	ZNF646	chr16	-	31093273	31093358
hsa_circRNA_059571	Up	3.1824205	0.020124964	RALGAPA2	chr20	-	20553518	20620544
hsa_circRNA_407331	Up	3.1416441	0.043787336	ENOX2	chrX	-	129917520	129943235
hsa_circRNA_101115	Up	3.1134423	0.048630939	METAP2	chr12	+	95879654	95897937
hsa_circRNA_001065	Up	3.0765417	0.000857261	GYPC	chr2	-	127453811	127454077
hsa_circRNA_083948	Up	3.0694227	0.027726197	EIF4EBP1	chr8	+	37888019	37917883
hsa_circRNA_405965	Down	50.0742673	0.000388304	CCNT2-AS1	chr2	-	135625089	135626611
hsa_circRNA_000102	Down	34.546511	0.000170047	AKNAD1	chr1	-	109479800	109479932
hsa_circRNA_008389	Down	34.065194	0.000321687	DNAJC11	chr1	-	6727768	6741097
hsa_circRNA_402094	Down	18.2204676	0.00030518	HKR1	chr19	+	37838091	37838774
hsa_circRNA_030448	Down	16.1072968	0.000363936	LMO7	chr13	+	76301164	76415337
hsa_circRNA_004077	Down	13.7486624	0.000233947	VAT1L	chr16	+	77850817	77859358
hsa_circRNA_400027	Down	9.1762641	0.001987192	SNURF-SNRPN	chr15	+	25325262	25326442
hsa_circRNA_403482	Down	9.0520716	0.000680145	RBM22	chr5	-	150076352	150078193
hsa_circRNA_005198	Down	8.4638383	0.000280205	PARP4	chr13	-	25072253	25077915
hsa_circRNA_000542	Down	8.1853461	0.000344817	ARID4A	chr14	+	58785259	58796887
hsa_circRNA_000407	Down	8.1180835	0.000295619	SMARCC2	chr12	+	56558216	56558309
hsa_circRNA_000499	Down	7.964529	0.001099349	RBM26	chr13	+	79940776	79940857
hsa_circRNA_092523	Down	7.9593586	0.000980014	PANX2	chr22	+	50618381	50618469
hsa_circRNA_001405	Down	7.734526	0.000819786	PREX1	chr20	-	47316514	47316617
hsa_circRNA_001937	Down	7.4899568	0.002365583	CHD9	chr16	+	53155459	53155541
hsa_circRNA_001587	Down	7.0488002	0.000427757	XLOC_001374	chr2	+	19042277	19042456
hsa_circRNA_001481	Down	6.9676678	0.000368557	EMB	chr5	-	49694940	49707217
hsa_circRNA_103670	Down	6.9533279	0.037835328	CNOT6L	chr4	-	78694234	78697546
hsa_circRNA_100719	Down	6.8982817	0.000930526	DOCK1	chr10	+	128768965	128836080
hsa_circRNA_043602	Down	6.8486592	0.000239464	JUP	chr17	-	39679868	39925459

Figure 2. Comparison of the expression of circular RNAs between patients with biochemical premature ovarian insufficiency and controls using quantitative real-time PCR.

(A) has_circ_003785 (B) has_circ_103903 (C) has_circ_008389 (D) has_circ_103670. *p < 0.05.

bPOI: Biochemical premature ovarian insufficiency.

Figure 3. Receiver operating characteristic curve.

Receiver operating characteristic curve analysis of the expression levels of has_circ_003785 (red line), has_circ_103903 (green line) and has_circ_008389 (blue line) in biochemical premature ovarian insufficiency.

Figure 4. The circular RNA–miRNA networks.

(A) The network comprising the top 20 upregulated (red nodes) and top 20 downregulated (deep blue nodes) circular RNAs and their target miRNAs (blue nodes) (miRNAs matched with more one circular RNAs are marked in yellow). (B) Binding sites of hsa-let-7c-3p, hsa-miR-18a-5p, hsa-miR-18b-3p, hsa-miR-323a-3p and hsa-miR-1301-3p in 3′UTR of has_circ_103903. (C) Hsa-let-7c-3p, hsa-miR-18a-5p, hsa-miR-18b-3p, hsa-miR-323a-3p and hsa-miR-1301-3p mimic or negative control oligonucleotide was co-transfected with pmiR-RB-Report vector with or without the 3′UTR sequence of has_circ_103903 to evaluate the effect of those miRNAs on the luciferase activities by luciferase reporter assay. *p < 0.05; **p < 0.01; ***p < 0.001.

circRNA: Circular RNA.

Figure 5. Prediction of miRNA-mediated signaling pathways.

(A) KEGG pathway analysis for the top ten upregulated circRNA-targeted genes. The vertical axis shows the annotated functions of the target genes. The horizontal axis shows the enrichment score (-log10 transformed p-value). (B) Mapping of the FoxO signaling pathway. (C) Mapping of cellular senescence pathway. Yellow marked nodes are associated with differentially expressed circRNAs.

circRNA: Circular RNA; KEGG: Kyoto encyclopedia of gene and genome.