Identification of deregulated circular RNA circ-0008102 as novel biomarker in pediatric β-thalassemia patients

doi:10.21203/rs.3.rs-1984616/v1

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Identification of deregulated circular RNA circ-0008102 as novel biomarker in pediatric β-thalassemia patients

https://doi.org/10.21203/rs.3.rs-1984616/v1

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Circular RNA circ-0008102 has previously been found upregulated in β-thalassemia (β-thal) in circRNAs microarray. Our study aimed to identify whether circ-0008102 could be a novel biomarker in β-thal. qRT-PCR confirmed that the expression levels of circ-0008102 in pediatric β-thal patients with HbF ≥ 5.0% (n = 26) were significantly higher than that in pediatric β-thal patients with HbF < 5.0% (n = 33) and healthy controls (n = 30). ROC curves analysis showed that the AUC of circ-0008102 for differentiating patients with HbF ≥ 5.0% from patients with HbF < 5.0% and healthy controls with an AUC of 0.774 and 0.702, respectively. Furthermore, circ-0008102 expression was positively correlated with the levels of HbF, GGT, β-globin and γ-globin mRNA, but was negatively corrected with the levels of MCV, MCH, HbA and Cr. circ-0008102 was mainly located in the cytoplasm, and its five highest-ranking candidates miRNAs were miR-372-3p, miR-329-5p, miR-198, miR-152-5p and miR-627-3p. 651 mRNAs regulated by these miRNAs were found based on bioinformatics analysis, and enrichment analysis of circ-0008102/miRNAs/mRNAs network showed these mRNAs were involved in DNA binding and transcription regulatory region binding, and were associated with Th17 cell differentiation and stem cell pluripotency signaling pathways. In conclusion, we preliminary proved that peripheral blood deregulated circ-0008102 might be an effective biomarker for detection of pediatric β-thal with high HbF. circ-0008102 participates in the pathogenesis of β-thal through regulating γ-globin expression, which needs to be investigated further.

circ-0008102

β-thal

biomarker

circ-0008102/miRNAs/mRNAs

γ-globin

β-thalassemia (β-thal) is an autosomal recessive hereditary anemia characterized by reduced or absent β-globin chain synthesis, which is most widespread among Mediterranean countries such as the Central Asia, Middle East, India, South America, and countries alongside the North coast of Africa and Southern China. It has been assessed that approximately 68,000 children born with various thalassemia syndromes each year worldwide¹. According to clinical manifestations, there are three main forms of β-thal, including β-thal major (TM), β-thal intermedia (TI), and β-thal carrier (TC). The β-globin gene, located on chromosome 11p15.5, is responsible for the coding of β-globin chain, patients with TM or TI are mostly homozygotes or compound heterozygotes for β⁰ or β⁺ mutation, and patients with TC are heterozygotes for β⁺ mutation. Although no treatment is needed for individuals who diagnosed as TC, patients with TM or TI are mainly treated by long-term blood transfusion and its high cost has brought a heavy economic burden to the family and society². Currently, bone marrow or stem cell transplantation is a radical treatment for patients with TM or TI, but it is difficult to popularize clinically due to the limitation of matching and marrow source³. Thus, an increased knowledge about the pathophysiology of β-thal may contribute to identify novel treatment methods for this disease.

Circular RNAs (CircRNAs), firstly identified in 1976, are commonly regarded to be derived from precursor mRNAs, with a covalently closed loop structure and non poly-adenylated tail^4,5. Because of this unique structure, circRNAs are stable in the cells and are not easily degraded by Ribonuclease R (RNase R). In recent years, With the development of high-throughput sequencing analysis and bioinformatic methods, thousands of circRNAs have been identified⁶. circRNAs are evolutionarily conserved and exhibit cell and tissue-type specific expression across species, and have shown great potential in diagnosis, therapy and prognosis for a variety of human diseases, including hematological diseases^7,8. For example, circ-MYBL2, a circRNA from MYBL2 gene, is reported to facilitate the progression of acute myeloid leukemia by regulating fms related receptor tyrosine kinase 3 (FLT3) translation through recruiting polypyrimidine tract binding protein 1 (PTBP1)⁹. circ-BUB1B_544aa, generated from BUB1 mitotic checkpoint serine/threonine kinase B (BUB1B) gene, has the potential to aggravate multiple myeloma malignancy through evoking chromosomal instability¹⁰. Besides, in myelodysplastic syndrome, peripheral blood circ-100352, circ-104056 and circ-102817 are valuable for diagnosis and prognosis, and are linked with some important cancer-related functions and pathways¹¹. Nowadays, increasing study has revealed that some circRNAs are abnormally expressed in β-thal and play important roles in epigenetic modification and gene expression regulation¹². However, the roles and mechanism of many unknown circRNAs in β-thal still remain to be explored.

Here, circ-0008102, as known as circ-100654, is deriving from ligand dependent nuclear receptor corepressor (LCOR) gene, is largely functionally unknown. A previous expression profile of circRNAs by microarray from the GSE196682 dataset has revealed that circ-0008102 expression is upregulated in the peripheral blood from β-thal carriers with high HbF (n = 3), compared to that in healthy controls (n = 3). Therefore, whether increased circ-0008102 expression is involved in the development of β-thal is worth investigating. The present study aimed to confirm the expression levels of circ-0008102 in large cohort of pediatric β-thal patients and healthy controls, and analyzed its relationship with clinical characteristics. And receiver operating characteristic (ROC) curves analysis was performed to evaluate the diagnostic value of circ-0008102 for pediatric β-thal. Moreover, based on bioinformatics analysis, the circ-0008102/miRNAs/mRNAs interaction network was established to help us better understand its biological roles and underlying mechanisms in pediatric β-thal.

Comparison of baseline characteristics in pediatric β-thal patients and healthy controls. The genotypes of pediatric β-thal patients were shown in Table 1, and a total of 19 genotypes were observed in the pediatric β-thal patients (n = 59), and the pathogenic mutations of β-globin gene were mainly concentrated in the β^{CD41–42(−TCTT)}, β^−28(A>G), β^{IVS−II−654(C>T)}, β^CD26(G>A), β^{CD71–72(+A)}, β^{CD27–28(+C)}, β^Int(ATG>AGG), β^{IVS−I−1(G>T)} and β^CD17(A>T) sites. No pathogenic mutations of β-globin gene was detected in the healthy controls (n = 30).

Table 1

The genotypes of pediatric β-thal patients
Case no.	Sex	Age (year)	Genotypes
1	male	7	β^{CD41–42(−TCTT)}/β^−28(A>G)
2	male	10	β^{IVS−II−654(C>T)}/β^−28(A>G)
3	female	7	β^{IVS−II−654(C>T)}/β^{IVS−II−654(C>T)}
4	male	8	β^{CD41–42(−TCTT)}/β^CD26(G>A)
5	female	6	β^{IVS−II−654(C>T)}/β^{CD71–72(+A)}
6	male	9	β^{CD41–42(−TCTT)}/β^{CD41–42(−TCTT)}
7	male	9	β^{CD41–42(−TCTT)}/β^{CD27–28(+C)}
8	male	4	β^{IVS−II−654(C>T)}/β^Int(ATG>AG)
9	female	9	β^{CD41–42(−TCTT)}/β^−28(A>G)
10	male	7	β^{CD41–42(−TCTT)}/β^{IVS−I−1(G>T)}
11	female	10	β^{CD41–42(−TCTT)}/β^{CD41–42(−TCTT)}
12	male	7	β^{CD41–42(−TCTT)}/β^CD17(A>T)
13	female	9	β^CD17(A>T)/β^CD17(A>T)
14	female	11	β^{CD41–42(−TCTT)}/β^CD17(A>T)
15	female	13	β^{CD41–42(−TCTT)}/β^−28(A>G)
16	female	6	β^{CD41–42(−TCTT)}/β^CD17(A>T)
17	male	10	β^{CD41–42(−TCTT)}/β^−28(A>G)
18	male	5	β^CD17(A>T)/β^{CD71–72(+A)}
19	male	7	β^{CD41–42(−TCTT)}/β^{CD71–72(+A)}
20	male	9	β^{CD41–42(−TCTT)}/β^{CD41–42(−TCTT)}
21	male	9	β^CD17(A>T)/β^CD17(A>T)
22	male	9	β^CD17(A>T)/β^CD17(A>T)
23	male	7	β^{CD41–42(−TCTT)}/β^−28(A>G)
24	male	9	β^{CD41–42(−TCTT)}/β^{CD41–42(−TCTT)}
25	female	11	β^{CD41–42(−TCTT)}/β^{CD41–42(−TCTT)}
26	male	5	β^{CD41–42(−TCTT)}/β^CD17(A>T)
27	female	8	β^{IVS−II−654(C>T)}/β^CD17(A>T)
28	male	8	β^{IVS−II−654(C>T)}/β^CD26(G>A)
29	male	11	β^{IVS−II−654(C>T)}/β^CD17(A>T)
30	female	9	β^CD17(A>T)/β^CD26(G>A)
31	female	8	β^{CD41–42(−TCTT)}/β^CD17(A>T)
32	female	6	β^{CD41–42(−TCTT)}/β^{CD41–42(−TCTT)}
33	female	7	β^{CD41–42(−TCTT)}/β^{CD41–42(−TCTT)}
34	female	10	β^{CD41–42(−TCTT)}/β^{IVS−II−654(C>T)}
35	male	10	β^{CD41–42(−TCTT)}/β^{CD41–42(−TCTT)}
36	female	10	β^{CD41–42(−TCTT)}/β^CD17(A>T)
37	female	8	β^CD17(A>T)/β^−28(A>G)
38	male	6	β^{CD41–42(−TCTT)}/β^{CD41–42(−TCTT)}
39	female	5	β^{CD41–42(−TCTT)}/β^CD17(A>T)
40	male	5	β^{CD41–42(−TCTT)}/β^CD17(A>T)
41	female	7	β^CD17(A>T)/β^CD17(A>T)
42	male	9	β^{CD41–42(−TCTT)}/β^{CD41–42(−TCTT)}
43	female	6	β^{CD41–42(−TCTT)}/β^{IVS−II−654(C>T)}
44	male	6	β^{CD41–42(−TCTT)}/β^CD17(A>T)
45	male	11	β^{CD41–42(−TCTT)}/β^CD17(A>T)
46	female	7	β^CD17(A>T)/β^−28(A>G)
47	male	6	β^{IVS−II−654(C>T)}/β^CD26(G>A)
48	female	8	β^{CD41–42(−TCTT)}/β^CD17(A>T)
49	male	7	β^{CD41–42(−TCTT)}/β^{CD41–42(−TCTT)}
50	female	7	β^{CD41–42(−TCTT)}/β^{CD41–42(−TCTT)}
51	female	11	β^−28(A>G)/β^{IVS−I−1(G>T)}
52	male	7	β^{CD41–42(−TCTT)}/β^{IVS−II−654(C>T)}
53	male	8	β^{CD41–42(−TCTT)}/β^{CD41–42(−TCTT)}
54	male	5	β^CD17(A>T)/β^CD26(G>A)
55	female	11	β^{CD41–42(−TCTT)}/β^−28(A>G)
56	male	11	β^{CD41–42(−TCTT)}/β^{CD71–72(+A)}
57	female	13	β^{CD41–42(−TCTT)}/β^{IVS−II−654(C>T)}
58	female	10	β^{CD41–42(−TCTT)}/β^CD17(A>T)
59	female	5	β^{CD41–42(−TCTT)}/β^{CD41–42(−TCTT)}
β-thal: β-thalassemia

There was no statistical difference of age and sex distribution between pediatric β-thal patients and healthy controls. As expected, compared with healthy controls, pediatric β-thal patients displayed significantly decreased levels of hematological parameters, including the levels of RBC (P < 0.001), Hb (P < 0.001), MCH (P < 0.001) and HbA (P < 0.001) (Table 2). And the levels of HbA2 (P = 0.008) and HbF (P < 0.001) in pediatric β-thal patients were significantly higher than that in healthy controls. Pediatric β-thal patients showed abnormal biochemical indicators, including TBIL, DBIL, ALT, AST and SF, and the levels of these parameters were markedly enhanced compared with that in healthy controls (all P < 0.001); however, the level of Cr in pediatric β-thal patients was decreased compared with healthy controls (P < 0.001). These data confirmed that pediatric β-thal patients have phenotypes of anemia and abnormal liver function and iron metabolism.

Table 2

Baseline characteristics of pediatric β-thal patients and healthy controls
Characteristics	β-thal patients (n = 59)	Healthy controls (n = 30)	P values
Age (years)	8.12 ± 2.12	7.22 ± 1.87	0.078
Sex (male/female)	31/28	18/12	0.504
RBC (×10¹²/L)	3.46 ± 0.63	4.64 ± 0.21	< 0.001*
Hb (g/L)	91.39 ± 18.16	132.97 ± 5.68	< 0.001*
MCV (fL)	80.16 ± 10.97	83.31 ± 2.20	0.124
MCH (pg)	26.33 ± 1.78	28.65 ± 0.85	< 0.001*
HbA (%)	87.16 ± 11.62	96.74 ± 0.96	< 0.001*
HbA₂ (%)	3.79 ± 2.76	2.80 ± 0.17	0.008*
HbF (%)	9.05 ± 11.67	0.17 ± 0.34	< 0.001*
BUN (mmol/L)	5.27 ± 2.00	4.79 ± 0.97	0.310
Cr (µmol/L)	30.77 ± 8.54	39.27 ± 6.07	< 0.001*
UA (µmol/L)	288.98 ± 106.32	307.79 ± 72.06	0.478
TBIL (µmol/L)	29.69 ± 6.18	8.07 ± 2.42	< 0.001*
DBIL (µmol/L)	9.59 ± 4.32	1.84 ± 0.56	< 0.001*
ALT (U/L)	45.73 ± 40.00	13.87 ± 3.44	< 0.001*
AST (U/L)	44.51 ± 28.78	25.49 ± 4.26	< 0.001*
GGT (U/L)	15.97 ± 8.35	13.78 ± 2.43	0.284
SF (µg/L)	2873.47 ± 1657.40	48.30 ± 21.90	< 0.001*
RBC: red blood cells, Hb: hemoglobin, MCV: mean corpuscular volume, MCH: mean corpuscular hemoglobin, HbA: hemoglobin A, HbA2: hemoglobin A2, HbF: fetal hemoglobin, BUN: blood urea nitrogen, Cr: creatinine, UA: uric acid, TBIL: total bilirubin, DBIL: direct bilirubin, ALT: alanine transaminase, AST: aspartate transaminase, GGT: gamma glutamyl-transferase, SF: serum ferritin. *P < 0.05 compared to healthy controls

Moreover, based on HbF level, the pediatric β-thal patients were further categorized into two groups: HbF < 5.0% group (n = 33) and HbF ≥ 5.0% (n = 26) group. The hematological parameters and biochemical indicatorsof the two groups were compared and shown in Table S1. Compared with the HbF < 5.0% group, the levels of MCH (P < 0.001) and HbA (P < 0.001) in the HbF ≥ 5.0% group were decreased, whereas the levels of TBIL (P = 0.026) and GGT (P = 0.014) were increased. However, no significant differences were observed in age, sex distribution, RBC, Hb, MCV, HbA2, BUN, Cr, UA, DBIL, ALT, AST, and SF levels in the two groups (all P > 0.05). These data demonstrated that HbF is associated with MCH, HbA, TBIL and GGT in pediatric β-thal patients.

Identification and characterization of circ-0008102. Based on the annotation of circBase, circ-0008102 with a length of 515 nt is produced by back-splicing from exons 5, 6, and 7 of the LCOR gene locus (NM_032440), which is located at human chromosome 10: 98703869–98711953 position (Fig. 1A). The junction sequences of circ-0008102 were validated by Sanger sequencing (Fig. 1B). Subsequently, to verify that circ-0008102 is generated from the back-splicing instead of trans-splicing or genomic rearrangements, the divergent and convergent primers were designed to amplify circ-0008102 and LCOR mRNA, respectively. RT-PCR revealed that both cDNA and gDNA could amplify LCOR mRNA using the convergent primers, whereas only cDNA could amplify circ-0008102 using the divergent primers (Fig. 1C). Additionally, qRT-PCR showed that circ-0008102 was more stable than LCOR mRNA in 293T cells under treatment with RNAse R (Fig. 1D, P < 0.001). To summarize, circ-0008102 is confirmed to be a circular RNA.

Validation of deregulated circ-0008102 expression in pediatric β-thal patients. We explored the expression levels of circ-0008102 in peripheral blood of large cohort of subjects, and results showed that there was no statistical difference of circ-0008102 expression between pediatric β-thal patients (n = 59) and healthy controls (n = 30) (Fig. 2A, P > 0.05). And the expression levels of circ-0008102 had a dynamic change with age, with the highest expression at age 11 (Fig. 2B). Also, the expression levels of circ-0008102 in the peripheral blood from different genotypes of pediatric β-thal patients were detected. Interestingly, pediatric β-thal patients with β^−28(A>G)/β^{IVS−I−1(G>T)} showed highest expression levels of circ-0008102, followed by β^CD17(A>T)/β^CD26(G>A) and β^CD17(A>T)/β^−28(A→G) (Fig. 2C), indicating that circ-0008102 expression might be linked to the genotypes of pediatric β-thal. Surprisingly, the expression levels of circ-0008102 in peripheral blood of pediatric β-thal patients with HbF ≥ 5.0% were significantly higher than that in pediatric β-thal patients with HbF < 5.0% and healthy controls (Fig. 2D, P < 0.05). These data demonstrated that circ-0008102 expression is upegulated in pediatric β-thal patients with high HbF.

Evaluation of circ-0008102 in peripheral blood as a novel diagnostic biomarker for pediatric β-thal patients. We then evaluated the diagnostic ability of circ-0008102 to distinguish pediatric β-thal patients from healthy controls through ROC curves analysis. Figure 3A showed that the AUC value was 0.558, and 95% confidence interval (CI) was 0.433–0.682, and the cut-off value was 1.08, with sensitivity was 0.508 and specificity was 0.667 (Fig. 3A, P = 0.373), hinting that circ-0008102 has low clinical diagnostic value for the diagnosis of pediatric β-thal. As shown in Fig. 3B, peripheral blood circ-0008102 expression discriminated pediatric β-thal patients with HbF ≥ 5.0% from pediatric β-thal patients with HbF < 5.0% with an AUC of 0.774 (95% CI: 0.603–0.885, sensitivity: 0.692, specificity: 0.757) (P = 0.001). Similarly, the AUC of circ-0008102 for differentiating pediatric β-thal patients with HbF ≥ 5.0% from healthy controls was 0.702 (95% CI: 0.558–0.845), with 0.731 sensitivity and 0.70 specificity (Fig. 3C, P = 0.009). These data demonstrated peripheral blood circ-0008102 might be an effective diagnostic biomarker in pediatric β-thal with high HbF.

Analysis of correlation between circ-0008102 expression and clinical characteristics in pediatric β-thal patients. As shown in Table 3, in the association between circ-0008102 expression and hematological parameters in pediatric β-thal patients showed that the expression level of circ-0008102 was negatively correlated with the levels of MCV (r=-0.436, P = 0.001), MCH (r=-0.693, P < 0.001) and HbA (r=-0.499, P < 0.001), and was positively correlated with HbF level (r = 0.462, P = 0.001). However, no association was found in RBC, Hb, and HbA₂ (all P > 0.05). Similarly, spearman correlation analysis revealed that the expression level of circ-0008102 was negatively correlated with the level of Cr (r=-0.477, P = 0.014) in pediatric β-thal patients, and was positively correlated with the level of GGT (r = 0.434, P = 0.013). However, no association was found in BUN, UA, TBIL, DBIL, ALT, AST, and SF (all P > 0.05). Together, these data suggested that deregulated circ-0008102 expression is associated with the pathogenesis of pediatric β-thal.

Table 3

Spearman correlation analysis the correlations between circ-0008102 expression and hematological parameters and biochemical indicators in pediatric β-thal patients.
Parameters	Circ-0008102 expression		Indicators	Circ-0008102 expression
Parameters	Correlation coefficient	P values	Indicators	Correlation coefficient	P values
RBC	0.107	0.454	BUN	-0.190	0.384
Hb	-0.187	0.188	Cr	-0.477	0.014*
MCV	-0.436	0.001*	UA	-0.202	0.322
MCH	-0.693	< 0.001*	TBIL	0.257	0.135
HbA	-0.499	< 0.001*	DBIL	0.143	0.413
HbA₂	0.101	0.471	ALT	0.072	0.675
HbF	0.462	0.001*	AST	0.062	0.718
-	-	-	GGT	0.434	0.013*
-	-	-	SF	-0.203	0.274
*P < 0.05

Analysis of β-globin and γ-globin expression in pediatric β-thal patients and its association with circ-0008102 expression. Compared with healthy controls, the mRNA expression levels of β-globin in the peripheral blood from pediatric β-thal patients were significantly downregulated with 0.05 fold changes (Fig. 4A, P < 0.001); however, the mRNA expression levels of γ-globin were significantly upregulated with 209.04 fold changes (Fig. 4B, P < 0.001). A positive correlation between the mRNA expression levels of β-globin and γ-globin was shown in Fig. 4C (r = 0.772, P < 0.001). Furthermore, in pediatric β-thal patients, circ-0008102 expression was positively correlated with the mRNA expression levels of β-globin (r = 0.608) and γ-globin (r = 0.697) (Fig. 4D and 4E, P < 0.001). All data stated that circ-0008102 might be involved in the regulation of γ-globin expression in pediatric β-thal.

Construction of circ-0008102/miRNAs/mRNAs interaction network. The results of FISH showed that circ-0008102 was mainly located in the cytoplasm of 293T cells (Fig. 5), suggesting circ-0008102 could function as a miRNA sponge. The molecular interactions between circ-0008102 and its five highest-ranking candidates miRNAs were displayed in Figure S1A, including miR-372-3p, miR-329-5p, miR-198, miR-152-5p, and miR-627-3p. The target genes of those five miRNAs were predicted by Targetscan 7.1 and mirdb V6 databases, and 651 mRNAs were found, and seventeen of them were targeted by more than one miRNAs, such as ryanodine receptor 2 (RYR2), VHL binding protein 1 (VBP1), leukocyte immunoglobulin like receptor B5 (LILRB5), FYVE and coiled-coil domain autophagy adaptor 1 (FYCO1), ferritin mitochondrial (FTMT) and sterile alpha motif domain containing 1 (SAMD1). We further constructed the circ-0008102/miRNAs/mRNAs network focused on the five miRNAs and 651 mRNAs (Figure S1B).

GO and KEGG enrichment analysis of circ-0008102/miRNAs/mRNAs network. The top 10 significantly enriched GO terms in the biological process (BP), cellular components (CC), and molecular function (MF) were shown in Figure S2A-2C. In terms of BP, the top three enriched items were “regulation of DNA-templated transcription”, “regulation of nucleic acid-templated transcription” and “regulation of RNA biosynthetic process”. In terms of CC, the top three enriched items were “transcription regulator complex”, “chromation” and “chromosome”. In terms of MF, “sequence-specific DNA binding”, “transcriptional cis-regulatory region binding” and “transcription regulatory region nucleic acid binding” were enriched for the first three places. Besides, the top 10 significantly enriched pathways of KEGG enrichment analysis were shown in Figure S2D. Among these pathways, “stem cells pluripotency”, “Th17 cell differentiation”, “osteoclast differentiation” and “cellular senescence” were closely correlated with the pathogenesis of β-thal.

With the deepening of the research on the underlying molecular basis of β-thal, its clinical treatment methods have been updated and gradually improved, and more and more biomarkers related to this disease have been reported, including erythrocyte transferrin receptor 1, alpha-hemoglobin stabilizer protein, heat shock protein 70, and CD35 and CD55^13–15. In recent years, non-coding RNAs, including miRNAs and long non-coding RNAs (lncRNAs), are regarded as a class of promising blood-based biomarkers for detection of human diseases including β-thal¹⁶. For example, Leecharoenkiat et al reported that plasma upregulated miR-451 expression is a novel hemolytic marker in β⁰-thal/HbE patients, and is associated with the degree of hemolysis and promotes erythropoiesis¹⁷. Kuno et al demonstrated that elevated miR-125b expression from activated phagocytic monocytes is a genetic modifier related to anemia severity in β-thal patients¹⁸. El-Khazragy et al suggested that plasma dysregulated miR-let-7d, miR-122, and miR-200 serve as novel and non-invasive predictor biomarkers for cellular damage under condition of tissue iron excess in transfusion-dependent β-thal patients¹⁹. Recently, a cross-sectional study concluded that circulating MALAT1 (metastasis-associated lung adenocarcinoma transcript 1) and MIAT (myocardial infarction associated transcript) are promising predictors for blood transfusion status in β-thal patients²⁰. In this study, we found deregulated circ-0008102 expression was associated with the pathogenesis of pediatric β-thal, and firstly demonstrated that circ-0008102 might be an effective biomarker for detection of pediatric β-thal with high HbF.

LCOR, as a master transcription factor, is involved in converting haemogenic endothelium into haematopoietic stem and progenitor cells²¹. It is also reported that LCOR negatively regulates early adipogenesis by inhibiting C/EBPβ transcriptional activity²². circ-0008102 is derived from LCOR gene locus, which is a exonic circRNA with 515 nt in length. Recently, dysregulated circRNAs has received more attention as diagnostic or predictive biomarkers for malignant tumors, nervous system diseases, cardiovascular disease, and endocrine diseases^23,24. However, the role of circRNAs in β-thal is poorly known. Herein, 59 pediatric β-thal patients were collected and theirs hematological parameters and biochemical indicators were consistent with the phenotypes of TM or TI. Encouragingly, we found that the expression levels of circ-0008102 in peripheral blood from pediatric β-thal patients with HbF ≥ 5.0% were significantly higher than that in pediatric β-thal patients with HbF < 5.0% and healthy controls. The data were consistent with uprgulated circ-0008102 expression in a circRNA microarray profile of GSE196682 dataset (adult β-thal patients vs. healthy controls), indicating high credibility of our data. Furthermore, we found circ-0008102 expression was positively correlated with the levels of HbF and GGT, and was negatively corrected with the levels of MCV, MCH, HbA and Cr. All these strong unraveled deregulated circ-0008102 expression is associated with the pathogenesis of pediatric β-thal. However, the detailed biological role of circ-0008102 in pediatric β-thal deserved further studies.

circRNAs display good diagnostic performance, not only because of their high specificity and sensitivity but also because of their great stability in peripheral blood²⁵. Currently, the predictive role of circRNAs in β-thal remains to be blank. To our knowledge, it is the first time to identify the diagnostic power of circ-0008102 in pediatric β-thal patients in our study. The peripheral blood circ-0008102 revealed a significant diagnostic value on pediatric β-thal patients with HbF ≥ 5.0% when discriminated with pediatric β-thal patients with HbF < 5.0% and healthy controls (AUC : 0.774 and 0.702, respectively). These results suggested that circ-0008102 has potential as a novel biomarker in the detection of pediatric β-thal with high HbF. Without doubt, future multi-center, large-scale study is needed to verify the diagnostic value of circ-0008102 in pediatric β-thal.

Epigenetic factors can influence the severity of β-thal, leading to differing presentations despite similar underlying mutations. Increasing study suggested that reactivating of HbF is a effective therapeutic target for β-thal^26,27. In β-thal patients, the γ-globin gene is activated to elevate the expression of HbF, resulting in reduced hemolysis and relieved the symptoms of anemia. The regulatory roles of non-coding RNAs on γ-globin expression have gradually been identified, such as miRNAs: miR-29b/miR-96/miR-326/miR-2355-5p^28–31 and lncRNAs: hemoglobin subunit beta pseudogene 1 (HBBP1)/HMI-LNCRNA/BGLT3-lncRNA^32–34. In our study, we found an increased expression of γ-globin mRNA in pediatric β-thal patients, and γ-globin mRNA expression level had a positive correlation with β-globin mRNA expression, confirming the activated γ-globin gene in β-thal. Moreover, we found circ-0008102 expression was positively correlated with the mRNA levels of γ-globin and β-globin, indicating circ-0008102 might be involved in the regulation of γ-globin expression. While the more details need further study in future.

Increasing evidence showed that exonic circRNAs serve as sponges of miRNAs to regulate the expression of target mRNAs³⁵. We also found circ-0008102 was located in cytoplasm of 293T cells by FISH assay, confirming circ-0008102 functioned as miRNA sponges. To better understand the regulatory mechanism of circ-0008102 in this study, we constructed the circ-0008102/miRNAs/mRNAs network based on bioinformatics analysis, and found circ-0008102 might act as ceRNAs to capture miR-372-3p, miR-329-5p, miR-198, miR-152-5p and miR-627-3p, and subsequently regulate the 651 mRNAs expression. The GO and pathway enrichment analysis indicated that these mRNAs involved in DNA binding and transcription regulatory region binding, and were associated with Th17 cell differentiation and stem cell pluripotency signaling pathways. However, as the present study was an in silico research, a further in vitro and in vivo experiments are required for validation of these pathways.

In conclusion, our data provide preliminary evidence that the expression level of circ-0008102 is deregulated in peripheral blood of pediatric β-thal, and circ-0008102 might be an effective biomarker for detection of pediatric β-thal with high HbF. circ-0008102 participates in the pathogenesis of β-thal through regulating γ-globin expression, which needs to be investigated further.

Study participants. 59 pediatric β-thal patients (age: 4–13 years, average age: 8.12 ± 2.12 years) (33 cases with HbF < 5.0% and 26 cases with HbF ≥ 5.0%) were prospective collected in Fujian Province, China between January 2017 and December 2020. Inclusion criteria: pediatric patients with anemia symptoms and diagnosed with β-thal by genetic diagnosis. 30 age- and gender-matched healthy controls (age: 3–15 years, average age: 7.22 ± 1.87 years) was obtained from Fujian Maternity and Child Health Hospital College of Clinical Medicine for Obstetrics & Gynecology and Pediatrics. Inclusion criteria: pediatrics with no symptoms of anemia, and no pathogenic mutation in the β-globin gene by genetic diagnosis. Exclusion criteria for all subjects: pediatrics with various chronic and congenital diseases, acute lung infection, abnormal blood coagulation. All subjects had no genetic relationship, and the three common deletional mutations (--^SEA, -α^3.7, and -α^4.2) and three non-deletional mutations (α^QSα, α^CSα, and α^WSα) of α-thal were excluded in subjects enrolled in this study. This study was approved by the Ethics Review Committee of Fujian Maternity and Child Health Hospital College of Clinical Medicine for Obstetrics & Gynecology and Pediatrics, Fujian Medical University (no: 201,2018) and was conducted in conformity to the Declaration of Helsinki. Written informed consent was obtained from participants’ parents following a detailed description of the purpose of the study.

Peripheral blood samples. The peripheral blood (5 ml each) was collected into PAXgene Blood RNA Tubes (Qiagen, Hilden, Germany) and was stored immediately at -80°C for further RNA extraction. The hematological parameters, including red blood cells (RBC), hemoglobin (Hb), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), hemoglobin A (HbA), hemoglobin A2 (HbA2) and fetal hemoglobin (HbF), were analyzed using a Sysmex XN-3000 automatic hematology analyzer (Sysmex Corporation; Shanghai, China) and an automated capillary S2 electrophoresis system version 6.2 (Sebia, Paris, France).

Blood biochemical analysis. The biochemical parameters, including renal function [(blood urea nitrogen (BUN), creatinine (Cr) and uric acid (UA)], hepatic function [(alanine transaminase (ALT), aspartate transaminase (AST), gamma glutamyl-transferase (GGT), total bilirubin (TBIL) and direct bilirubin (DBIL)] and serum ferritin (SF), were determined using chemiluminescent microparticle immunoassay under an automatic biochemical analyzer (Abbott Diagnostics; Abbott Park, IL, USA).

Molecular analysis for β-thal genotypes. Human genomic DNA (gDNA) was extracted from the peripheral blood samples using a genomic DNA isolation kit (Qiagen) following the manufacturer's instruction. The DNA samples were quantified by a Bio Photometer MULTISKAN GO (Thermo Scientific, Waltham, MA, USA), and the concentrations greater than 100 ng/µL and the purity with 1.8-2.0 optical densities at 260/280 nm indicated good quality. The 17 common mutations of β-thal, including {codons (CD)41/42(-TCTT), IVS-II-654(C > T), -28(A > G), CD71/72(+ A), CD17(A > T), HbE[β26(B8)Glu-Lys, GAG > AAG or CD26(G > A)], CD31(-C), CD27/28(+ C), CD43(G > T), -32(C > A), -29(A > G), -30(T > C), CD14/15(+ G), Capt40 to t43(-AAAC), initiation CD(T > G), IVS-I-1(G > T) and IVS-I-5(G > T)}, were detected using reverse dot blot hybridization (RDBH) with a β-thal gene detection kit (Yishengtang Biological Products Co., Ltd, Shenzhen, China), following the manufacturer's instruction.

RNA extraction and reverse transcription. Total RNA from the peripheral blood samples was isolated using a PAXgene Blood RNA Kit (Qiagen) according to the manufacturer’s protocol. The purity of the RNA samples were assessed using a Bio Photometer MULTISKAN GO, and the OD260/280 ratio at 1.9 to 2.0 indicated good quality. The RNA integrity was determined by 1% formaldehyde denatured gel electrophoresis. After calculating the concentration, RNA was frozen in -80°C refrigerator. RNA was reverse transcribed into cDNA using a SuperScript™ III Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA) with random primers, according to the manufacturer’s protocol. The cDNA was preserved in -20°C for further use.

Reverse transcription PCR (RT-PCR). RT-PCR was carried out with cDNA and gDNA as templates to amplify circ-0008102 and LCOR mRNA using a PrimeScript™ RT-PCR kit, according to the manufacturer’s instruction (Takara Bio, Inc., Shiga, Japan). The procedures for RT-PCR were shown as following: 1) Denaturation at 98°C for 10 min; 2) 35 step-cycles of incubation at 98°C for 10 sec, 60°C for 30 sec, and 72°C for 30 sec; 3) a final extension at 72°C for 5 min. The divergent primers were used to detect backsplice junction of circ-0008102 and convergent primers were applied to detect LCOR mRNA. Divergent primers: sense, 5’-GACGGTGTACTTGATCTGTCCA-3’ and antisense, 5’-GTTGGATCATTCGCTGCATGAT-3’; convergent primers: sense, 5’-GACGGACTTCGGAGTGGTGATG-3’ and antisense, 5’-GAGCCAGTGGAACTTTGAGTGATG-3’. After the reaction, 6 µL portion of each PCR product was analyzed by electrophoresis through a 2% agarose gel in 1×Tris-borate-EDTA buffer at 15 V/cm for 50 min.

Quantitative real-time PCR (qRT-PCR). qRT-PCR was carried out with cDNA as template to detect the expression levels of circ-0008102, LCOR, β-globin, and γ-globin. The StepOnePlus™ Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) was adopted to perform qRT-PCR with a PrimeScript™ RT-PCR kit (Takara Bio, Inc.,), according to the manufacturer’s instruction. The circ-0008102 and LCOR primers were the same as the divergent and convergent primers, respectively. The primers for other genes were shown as following: β-globin: sense, 5’-TGTCCACTCCTGATGCTGTTATG-3’ and antisense, 5’-GGCACCGAGCACTTTCTTG-3’; γ-globin: sense, 5’-CTGGGAAGGCTCCTGGTTG-3’ and antisense, 5’-CAGAGGCAGAGGACAGGTTG-3’; β-actin: sense, 5’-GCACAGAGCCTCGCCTT-3’ and antisense, 5’-GTTGTCGACGACGAGCG-3’. The β-actin gene was used as a internal control for quantitative analysis. qRT-PCR were performed in triplicate for both target and internal control genes, and the negative control (non-cDNA) were run with every experimental plate to assess specificity and to rule out contamination. The relative fold-change of each target gene with respect to β-actin was calculated by the 2^−ΔΔCt method.

RNAse R treatment. The RNAse R reagent (Epicentre Biotechnologies, Madison, WI, USA) was used to digest linear RNAs. Owing to circular structure, circRNAs could avoid been degraded by RNases. The total RNA was divided into 2 parts (2 µg each): one was for the digestion of 3 U/µg RNase R at 37°C for 20 min; the another was treated with equal amount of RNAse-free water under the same conditions. Subsequently, the RNA was reverse-transcribed into cDNA and qRT-PCR helped to analyze the detected LCOR mRNA and circ-0008102 follow the above experimental methods.

Sanger sequencing. Sanger sequencing was used to validate the back-splice junction sequences of circ-0008102. Brief, the partial sequence fragments of circ-0008102 contained the back-splice junction were amplify from cDNA using a PrimeSTAR® GXL Premix (Takara Bio, Inc.,), according to the manufacturer recommendation. The amplification primers were as following: sense, 5’-CCTGCCGAAAGCATCTCCAGT-3’ and antisense, 5’-TCGCTGCATGATCTCACGG-3’. The amplification primers were also used as primers for sequencing. Sanger sequencing was performed on an ABI 3730XL DNA analyzer (Applied Biosystems), and was analyzed by using a PC ChromasPro software version 2.1.5 (Applied Biosystems).

Cell culture. Human 293T cells was purchased from the Cell Center of Shanghai Institutes for Biological Sciences (Shanghai, China). The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; GibcoBRL, Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum (GibcoBRL). All cells were maintained at 37°C in a humidified atmosphere containing 5% CO₂.

Fluorescence in situ hybridization (FISH). The FISH kits of circ-0008102 and negative control were designed and purchased from Genepharma (Shanghai, China). Approximately 4×10⁴ 293T cells were seeded on cell climbing in 24-well plates overnight. Then, the cells were washed with PBS and fixed in 4% paraformaldehyde for 15 min at 37°C, and followed by permeabilized them for 15 min in 0.1% Buffer A at 37°C. After washed with PBS, 2×Buffer C was added and maintained for 30 min at 37°C, and then Buffer E was added and rehydrated for 30 min at 73°C. Subsequently, 1 µL of biotin labelled circ-0008102-probe (5’-AGTGTCCCCGTTCCCTTGAGTACTG-3’) or negative control-probe (5’-TGCTTTGCACGGTAACGCCTGTTTT-3’), 1 µL of SA-Cy3, and 8 µL of PBS were added into the cells and incubated at 37°C overnight by avoiding light. The next day, the cells were sequentially washed with 0.1% Buffer F for 10 min at 37°C, 2×Buffer C for 10 min (repeat 3 times) at 60°C, and 2×Buffer C for 10 min (repeat 3 times) at 37°C. Finally, the nucleus of cells was dyed in 4’,6’-diamidino-2-phenylindole (DAPI), and the subcellular localization of circ-0008102 was detected using a Leica TCS SP8 CARS Confocal Microscope (Leica, Wetzlar, Germany).

Bioinformatics tools. The sequences of circ-0008102 was annotated and provided from circBase (http://www.circbase.org/). The circ-0008102 sponged miRNAs was predicted with starbase (http://starbase.sysu.edu.cn/), circBANK (http://www.circbank.cn/), and circinteractome (https://circinteractome.nia.nih.gov/). The target prediction of miRNAs was based on Targetscan 7.1 (http://www.targetscan.org/vert_71/) and mirdb V6 (http://mirdb.org/miRDB/). Cytoscape v3.8.2 (http://www.cytoscape.org/) was applied to build a the circ-0008102/miRNAs/mRNAs interaction network, and the mRNAs in the network were annotated using Gene Oncology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis.

Statistical analysis. The GraphPad Prism 9 software (GraphPad Software Inc., San Diego, CA, USA) and SPSS 25.0 software (SPSS, Chicago, IL, US, USA) were used for statistical analyses. Measurement data were expressed as the mean ± standard deviation (SD), and count data were expressed as case (%). The normality of the sample distributions was calculated by the Kolmogorov-Smirnoff test. The Mann-Whitney U test, independent t-test and chi-square test were used to assess the differences between the pediatric β-thal patients and healthy controls and the differences between pediatric β-thal patients with HbF < 5.0% and pediatric β-thal patients with HbF ≥ 5.0%. The correlations between the expression level of circ-0008102 and hematological parameters, biochemical indicators, and β-globin and γ-globin mRNA expression were evaluated by Spearman correlation analysis. The receiver operating characteristic (ROC) curve analysis and the area under curve (AUC) were carried on to detect circ-0008102 as biomarker for differentiating pediatric β-thal with different HbF. P values less than 0.05 were considered statistically significant.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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No competing interests reported.

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Identification of deregulated circular RNA circ-0008102 as novel biomarker in pediatric β-thalassemia patients

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