Up-Regulation of circRNA hsa_circ_0003074 Expression is a Reliable Diagnostic and Prognostic Biomarker in Patients with Osteosarcoma

Figures & data

Figure 1 Flow chart of patient selection in the study and main study procedures.

Table 1 Twenty up-regulated and 20 down-regulated circRNA in patients with osteosarcoma

circRNA	Regulation	Fold Change	Chrom	circRNA_Type	P-value
hsa_circ_0007418	up	1.02	chr1	exonic	0.01
hsa_circ_0000654	up	1.63	chr15	exonic	0.02
hsa_circ_0007390	up	1.05	chr15	exonic	0.02
hsa_circ_0031460	up	1.11	chr14	exonic	0.01
hsa_circ_0055451	up	1.53	chr2	exonic	0.01
hsa_circ_0007822	up	1.29	chr1	exonic	0.02
hsa_circ_0005753	up	2.71	chr6	exonic	0.03
hsa_circ_0001445	up	1.23	chr4	exonic	0.04
hsa_circ_0002076	up	1.54	chr2	exonic	0.02
hsa_circ_0004103	up	2.33	chr11	exonic	0.01
hsa_circ_0000566	up	1.25	chr14	exonic	0.03
hsa_circ_0003074	up	3.83	chr1	exonic	0.01
hsa_circ_0090868	up	2.64	chr16	exonic	0.03
hsa_circ_0001616	up	1.66	chr6	exonic	0.04
hsa_circ_0002863	up	1.75	chr2	exonic	0.03
hsa_circ_0007458	up	2.55	chr11	exonic	0.02
hsa_circ_0035226	up	2.35	chr10	exonic	0.04
hsa_circ_0001551	up	2.77	chr5	exonic	0.01
hsa_circ_0004976	up	2.22	chr2	exonic	0.03
hsa_circ_0044949	up	2.03	chr17	exonic	0.02
hsa_circ_0047552	down	1.24	chr8	exonic	0.03
hsa_circ_0011933	down	1.10	chr1	exonic	0.04
hsa_circ_0000546	down	1.28	chr14	exonic	0.02
hsa_circ_0047135	down	1.01	chr18	exonic	0.01
hsa_circ_0006063	down	1.30	chr10	exonic	0.03
hsa_circ_0026134	down	1.53	chr12	exonic	0.04
hsa_circ_0055377	down	1.67	chr2	exonic	0.02
hsa_circ_0075826	down	2.71	chr6	exonic	0.01
hsa_circ_0047151	down	1.56	chr18	exonic	0.03
hsa_circ_0067524	down	1.44	chr3	exonic	0.04
hsa_circ_0000246	down	2.19	chr10	exonic	0.02
hsa_circ_0046753	down	1.35	chr18	exonic	0.04
hsa_circ_0045801	down	1.61	chr17	exonic	0.03
hsa_circ_0002554	down	1.31	chr3	exonic	0.01
hsa_circ_0019799	down	1.68	chr10	exonic	0.0
hsa_circ_0072797	down	2.14	chr5	exonic	0.05
hsa_circ_0049637	down	2.66	chr19	exonic	0.04
hsa_circ_0002971	down	1.30	chr6	exonic	0.03
hsa_circ_0018758	down	2.25	chr10	exonic	0.04
hsa_circ_0075829	down	1.45	chr6	exonic	0.01

Figure 2 hsa_circ_0003074 expression in osteosarcoma cancer tissues and osteosarcoma cell lines. (A) is a heat map of circRNA chip detection in osteosarcoma cancer tissues and adjacent tissues. (B) shows the results of qRT-PCR detection of hsa_circ_0003074 in 60 pairs of osteosarcoma cancer tissues and adjacent tissues. (C) shows the results of qRT-PCR detection of hsa_circ_0003074 in different osteosarcoma cell lines. *The difference is statistically significant compared with adjacent tissues and the hFOB1.19 cell line (p <0.05), respectively. All experiments are repeated three times.

Figure 3 hsa_circ_0003074 expression in the serum of osteosarcoma patients. (A) is the expression of hsa_circ_0003074 in the serum of 40 patients with osteosarcoma and 30 healthy volunteers matched for age and sex. (B) is the expression of hsa_circ_0003074 in serum of 40 patients with osteosarcoma before and after surgery. (C) is the expression of hsa_circ_0003074 in serum of 40 patients with osteosarcoma before and after chemotherapy. (D) is the expression of hsa_circ_0003074 in the serum of osteosarcoma patients who are chemo-sensitive (n=25) and chemo-resistant (n=15). *Indicates that the difference is statistically significant (p <0.05).

Figure 4 Analysis of hsa_circ_0003074 in the diagnosis of osteosarcoma patients. (A) is a ROC analysis of hsa_circ_0003074 in osteosarcoma patients (n=40) and healthy volunteers (n=30). (B) is a ROC analysis of LDH in osteosarcoma patients (n=40) and healthy volunteers (n=30). (C) is a ROC analysis of ALP in osteosarcoma patients (n=40) and healthy volunteers (n=30).

Figure 5 Analysis of hsa_circ_0003074 in the prognosis of osteosarcoma patients. (A and B) show linear regression analysis of hsa_circ_0003074 with LDH and ALP. (C and D) show effects of hsa_circ_0003074 expression levels on the overall survival and progression-free survival of osteosarcoma patients (n=60), respectively.

Table 2 Correlation between clinicopathological characteristics of patients with osteosarcoma and expression of hsa_circ_0003074 in cancer tissue

	Low Expression of hsa_circ_0003074	High Expression of hsa_circ_0003074	P value
Gender			0.293
male	17	18
female	11	14
Age (years)			0.314
<25	15	18
≥25	16	11
Enneking stage			<0.01
stage I-IIA	18	7
stage IIB-III	10	25
Tumor size(cm)			<0.01
<8	17	5
≥8	11	27
Surgical site			0.308
Femur	11	15
Tibia	14	12
other	5	3
Differentiation			0.073
High	5	8
Moderate	10	13
Low	14	10
Lung metastasis			<0.01
Yes	8	25
No	18	9
Chemoresistant			<0.01
Yes	9	22
No	19	10
LDH			0.043
Normal	19	12
Abnormal	11	18
ALP			0.041
Normal	18	9
Abnormal	13	20

Abbreviations: LDH, lactate dehydrogenase; ALP, alkaline phosphatase.

Figure 6 Correlation analysis of hsa_circ_0003074 and clinicopathological characteristics in patients with osteosarcoma. (A) is the expression of hsa_circ_0003074 in the serum of osteosarcoma patients (n=40) with different tumor sizes. (B) is the expression of hsa_circ_0003074 in the serum of osteosarcoma patients (n=40) with lung metastasis and non-pulmonary metastasis. (C) is the expression of hsa_circ_0003074 in the serum of osteosarcoma patients (n=40) with different Enneking stages. (D) is the expression of hsa_circ_0003074 in the serum of osteosarcoma patients (n=40) with and without relapse. *Indicates that the difference is statistically significant (p <0.05).

Table 3 Univariate and Multivariate analysis of prognostic factors of patients with osteosarcoma

Variables	Univariate Analysis			Multivariate Analysis
	Hazard Ratio	95% CI	P value	Hazard Ratio	95% CI	P value
Age	0.943	0.345–1.244	0.532
Sex	0.895	0.724–0.938	0.634
Tumor size	2.483	1.114–2.945	0.034	3.633	2.414–4.386	0.001
Differentiation	1.473	1.023–2.473	0.336
Enneking Stage	2.394	1.734–3.398	0.025	3.877	1.335–5.394	0.003
Lung metastasis	3.545	2.235–4.325	0.013	4.862	3.548–5.521	0.003
Surgical site	1.053	1.011–1.565	0.436
Chemoresistant	2.532	1.624–3.334	0.033	3.957	2.454–4.601	0.003
LDH	1.837	1.165–2.657	0.043
ALP	1.953	1.212–2.943	0.042
hsa_circ_0003074	3.725	1.906–5.565	0.011	4.927	3.954–5.625	0.001

Abbreviations: LDH, lactate dehydrogenase; ALP, alkaline phosphatase.