Peripheral Blood circRNA Microarray Profiling Identities hsa_circ_0001831 and hsa_circ_0000867 as Two Novel circRNA Biomarkers for Early Type 2 Diabetic Nephropathy

Figures & data

Table 1 Characteristics and Clinical Features of Study Population

	ET2DN (n = 20)	T2DM (n = 20)	Control (n=20)	P-values
				ET2DN vs	ET2DN vs
				T2DM	Control
Age	56.5±12.48	54.9±11.49	54.8±6.45	0.989	0.532
Male	12	13	12
Female	8	7	8
Duration of diabetes (years)	9.9±6.37	6.8±5.89	0	0.389	0.004**
SBP (mmHg)	130.6±16.11	127.6±11.23	110.2±9.20	0.577	0.033*
DBP (mmHg)	89.2±13.16	80.2±10.61	73.2±6.69	0.067	0.013*
Height (cm)	164.01±9.89	163.6±4.5	164.1±7.2	0.805	0.998
Weight (kg)	65.9±13.78	66.2±17.5	58.8±13.91	0.419	0.219
WC (cm)	91.3±10.69	89.9±14.2	89.2±11.17	0.903	0.437
HC (cm)	97.1±12.04	96.9±11.3	94.1±9.07	0.634	0.458
BMI (kg/m2)	24.4±4.41	23.8±6.32	23.0±3.19	0.786	0.112
FBG (mmol/L)	8.7±1.32	9.3±2.36	4.1±0.21	0.109	0.002*
HbA1c (%)	9.75±4.54	9.8±1.22	5.1±2.79	0.915	0.014*
Cr (μmoI/L)	80.5±23.1	70.9±13.14	69.9±7.23	0.053	0.051
UAER (mg/24h)	98.9±52.78	<5	<5	0.014*	0.011*
TC (mmol/L)	4.14±0.24	4.19±1.44	4.42±0.76	0.732	0.059
LDL-C(mmol/L)	2.3±0.78	2.52±1.19	1.74±0.79	0.409	0.157
HDL-C(mmol/L)	1.06±0.23	1.07±0.22	1.27±0.21	0.964	0.045*
TG (mmol/L)	1.76±0.83	1.26±0.49	1.84±0.34	0.098	0.808
Hb (g/L)	136.5±21.59	142.1±17.42	136.9±19.98	0.507	0.908

Notes: *, **Denotes significance at a P value of <0.05 and 0.01, respectively.

Abbreviations: T2DM, Type 2 diabetes mellitus; ET2DN, early type 2 diabetic nephropathy; SBP, systolic blood pressure; DBP, diastolic blood pressure; WC, waist circumference; HC, hips circumference; BMI, body mass index; FBG, fasting blood glucose; HbA1c, glycosylated hemoglobin, type A1c; Cr, creatinine; UAER, urinary albumin excretion rate; TC, total cholesterol; LDL-C, low-density lipoprotein cholesterol; HDL-C, high-density lipoprotein cholesterol; TG, triglyceride; Hb, hemoglobin.

Figure 1 DECs in ET2DN, T2DM and control groups. (A) The Box Plot displayed the normalized intensity distribution of all data. (B) Hierarchical clustering for DECs between ET2DN, T2DM, and control. High relative expression in red, low relative expression in green. Scatter plots were performed to analyze DECs in ET2DN vs T2DM group (C), T2DM vs control group (D) and ET2DN vs control group (E). CircRNAs above the top green line and below the bottom green line indicated more than two-fold change in circRNAs between the two groups.

Figure 2 Volcano Plots in ET2DN, T2DM and control groups indicated the DECs between ET2DN vs T2DM group (A), T2DM vs control group (B) and ET2DN vs control group(C). Fold change values are marked by the vertical lines. A green vertical line corresponds to a 1-fold change, while a horizontal green line marks a p-value <0.05, red point represents DECs.

Table 2 Upregulated of circRNAs in ET2DN Vs T2DM Group

circRNA	P-value	FDR	FC	Best Transcript	Gene Symbol
hsa_circ_0001831	0.003957062	0.234851021	62.5502519	NM_015356	SCRIB
hsa_circ_0008389	0.024164715	0.259478194	30.8906026	NM_018198	DNAJC11
hsa_circ_0000511	0.039707543	0.27804059	19.8638207	NR_002312	RPPH1
hsa_circ_0000512	0.044554324	0.280338142	17.3036901	NR_002312	RPPH1
hsa_circ_0000102	0.044549729	0.280338142	16.8341757	NM_001048210	CLCC1
hsa_circ_0007334	0.031030612	0.266424689	12.5442782	NM_138799	MBOAT2
hsa_circ_0000973	0.043018088	0.279702058	10.4860159	NM_138799	MBOAT2
hsa_circ_0000972	0.041128735	0.278202197	10.2402304	NM_138799	MBOAT2
hsa_circ_0058792	0.042438378	0.279485853	8.7270157	NM_001037131	AGAP1
hsa_circ_0026074	0.037428217	0.273841649	7.9159885	NM_001659	ARF3

Abbreviations: FDR, false discovery rate; FC, fold change.

Table 3 Downregulated of circRNAs in ET2DN Vs T2DM Group

circRNA	P-value	FDR	FC	Best Transcript	Gene Symbol
hsa_circ_0044235	0.013454974	0.243613793	6.3517711	NM_001114091	CDC27
hsa_circ_0061260	0.039743465	0.27804059	5.7977871	NM_182482	BAGE2
hsa_circ_0044234	0.0128253	0.242819714	5.7460958	NM_001114091	CDC27
hsa_circ_0070739	0.00890631	0.240416209	5.4739701	TCONS_000 08208	TCONS_000 08208
hsa_circ_0061251	0.00951574	0.240416209	5.0314561	NM_182482	BAGE2
hsa_circ_0048148	0.014944257	0.243613793	4.8032919	NM_004368	CNN2
hsa_circ_0016545	0.049044541	0.284901936	4.7501894	NM_001748	CAPN2
hsa_circ_0026309	0.041930281	0.278513226	4.3535076	NM_005653	TFCP2
hsa_circ_0004837	0.019132135	0.248178106	4.3280515	None	None
hsa_circ_0053000	0.026598072	0.259954772	4.3040147	NM_001199803	CENPO

Abbreviations: FDR, false discovery rate; FC, fold change.

Figure 3 GO and pathway analysis of DECs for their host genes in ET2DN vs T2DM group. GO analysis of upregulated (A) and downregulated (B) DECs in ET2DN vs T2DM group. Pathway analysis of upregulated (C) and downregulated (D) DECs in ET2DN vs T2DM group. (E) The common circRNAs were analyzed in ET2DN vs T2DM group and ET2DN vs control group.

Figure 4 Verification of DECs by qRT-PCR. The expression of hsa_circ_0001831(A) and hsa_circ_0000867(B) in three groups; The expression of hsa_circ_0061260(C) and hsa_circ_0044235(D) in ET2DN vs T2DM group; The expression of hsa_circ_0063503(E) and hsa_circ_0000658 (F) in ET2DN vs control group; The expression of hsa_circ_0044235 (G) in T2DM vs control group. n=20, *p<0.05, **p<0.01, ***p<0.001.

Figure 5 Prediction of hsa_circ_0001831 and hsa_circ_0000867–microRNA–mRNA interaction network. The possible microRNAs of hsa_circ_0001831 (A) and hsa_circ_0000867 (B) were predicted by Arraystar’s home-made software based on TargetScan and miRanda, next, DIANA TOOLS software was used to predict target mRNAs of microRNA, Cytoscape 2.8.2 was used to display circRNA–microRNA–mRNA interaction network.

Figure 6 ROC curve analysis of diagnostic value in hsa_circ_0001831 (A) and hsa_circ_0000867 (B) in ET2DN patients.