Circ_0002232 Acts as a Potential Biomarker for AML and Reveals a Potential ceRNA Network of Circ_0002232/miR-92a-3p/PTEN

Introduction

Acute myeloid leukemia (AML), the most common malignant myeloid disease in adults, is characterized by loss of differentiation of blasts (myeloid progenitor cell) and clonal amplification in the peripheral blood and bone marrow.^1,2 It had a poor prognosis in the past.² Cytogenetics analyses play a crucial role to identify subgroups of AML with different outcomes.³ Meanwhile, identifying molecular genetic markers also helps to divide AML patients into different groups and refine their prognosis.³

In recent years, non-coding RNAs have increasingly caught researchers’ attention. A wide variety of studies have shown that non-coding RNAs participate in the process of controlling cell differentiation through regulating expression of the gene.⁴

Circular RNAs (circRNAs) are an emerging class of non-coding RNAs and are characterized by having covalent binding between the 3ʹ and 5ʹ ends which are generated by the mechanism of reverse splicing.⁵ Due to the conserved characteristic across species and tissue, circRNAs have been found to be ideal diagnostic and prognostic biomarkers for disease, especially cancer.⁶ For example, according to Xia et al, their study indicated that high-expressed of circ_0067934 in esophageal cancer was related with poor proliferation. Up-regulated expression of circ_0067934 was an unfavorable factor for esophageal squamous cell carcinoma.⁷ Shao et al revealed that circ_0014717 expression significantly decreased in gastric carcinoma. The level of its expression was related to tumor staging and distal metastasis. Due to the stable expression of circ_0014717, it had been regarded as an ideal biomarker for clinical detection of gastric cancer.⁸

Moreover, circular RNAs, which have also been named as competing endogenous RNAs (ceRNAs), could regulate cell biological function through acting as miRNA sponges.⁵ Actually, circRNAs play an essential regulatory role in diseases through interacting with disease-related miRNAs.⁹ For example, Weng et al illustrated that over-expressed ciRs-7 acted as miRNA sponge to abolish the tumor suppressive effect of miR-7 and promoted tumorgenesis in colorectal cancer.¹⁰ Circ_FBLIM1 had been found to serve as ceRNA and regulate the process of hepatocellular cancer through binding with miR-346. This process promoted the progression of hepatocellular cancer.¹¹ But there are few studies focused on the diagnostic and prognostic value of circular RNAs or their function acting as ceRNA in malignant hematonosis.

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) serves as a classic tumor suppressor.¹² It mainly participates the homeostasis of the phosphatidylinositol 3 kinase (PI3K)/AKT pathway.¹² And losing the suppressive function of PTEN plays an essential role in the occurrence of cancer. PTEN has been found to be down-expressed in several solid cancers, like prostate cancer and breast cancer.^13,14 Furthermore, some research illustrated that the expression of PTEN transcript was remarkably lower in AML than controls and inactivation of PTEN promoted AML progression.^15,16

To our knowledge, PTEN-dependent circular RNAs have seldom been studied in cancer, let alone AML. Circ_0002232 is one of circRNAs of PTEN. The purpose of this research was to analyse circ_0002232 expression in AML and to investigate its clinical relevance. We wanted to find whether it could serve as a biomarker for diagnosis and prognosis of AML and reveal the potential ceRNA network behind it.

Materials and Methods

Patients and Samples

A total of 163 samples, including 48 controls and 115 de novo AML patients, were provided by the Affiliated People’s Hospital of Jiangsu University. This study was approved by Human Research Ethics Committee of the Affiliated People’s Hospital of Jiangsu University and was conducted in accordance with the Declaration of Helsinki. Patients involved in this study were clearly diagnosed and classified according to guidelines of World Health Organization (WHO) and French-American-British (FAB) criteria.^17,18 To refine the group, the total 115 AML samples we used included 98 non-acute promyelocytic leukemia AML (non-APL AML) samples and 51 normal cytogenetic AML (CN-AML) samples. All of the enrolled patients have to meet the following conditions: 1) age ≥ 18 years old; 2) patients were diagnosed with AML for the first time; 3) patients had not received any form of treatment; and 4) patients had complete pathological data in our hospital. 5) participatants signed informed consent and patients who failed to meet above criteria were excluded from the study. Bone marrow (BM) specimen was collected after every participant signed informed consent. Extraction of bone marrow mononuclear cells (BMNCs) was conducted by using Lymphocyte Separation Medium (TBD sciences corporation, Tianjin, China). The vital clinical and laboratory features of these patients are listed in Table 1 and demographic data of controls are shown in Supplemental File 2

Table 1 Comparison of clinical and laboratory characteristics between AML patients with low and high circ_0002232 expression

RNA Isolation and Reverse Transcription

The process of isolating total RNA from BMNCs was conducted by using Trizol reagent (Thermo Fisher Scientific, Waltham, MA, USA). Reverse transcription mixture contains 2μg of total RNA from each sample, 10mM of dNTPs, 10μM of random hexamers, 80U of RNase inhibitor, and 200U of reverse transcriptase (Thermo Fisher Scientific, Waltham, MA, USA). The reverse transcript system was incubated at 25°C for 10 min, at 42°C for 60 min and stored at −20°C.

Real-Time Quantitative PCR

The expression of circ_0002232, miR-92a-3p and PTEN was detected by real-time quantitative PCR (RQ-PCR) with specific primers listed in supplemental File 1. The PCR reaction systems of detecting circ_0002232 and PTEN were SYBR Premix Ex Taq II (TaKaRa, Japan) and the reaction system of detecting the expression of miR-92a-3p was miScript SYBR green PCR kit (Qiagen, Duesseldorf, Germany). 7500 Thermocycler (Thermo Fisher Scientific, Waltham, MA, USA) was used to perform reaction system. A housekeeping gene (ABL) was used to calculate the quantity of circ_0002232 and PTEN. And the quantity of miR-92a-3p was valued by U6. Relative expression level of circ_0002232, miR-92a −3p and PTEN were calculated by using 2^−ΔΔCT formula.

Gene Mutation Detection

Mutations of gene NPM1, N/K-RAS, DNMT3A, c-KIT, U2AF1, IDH1/2 and SRSF2 were detected by High Resolution Melting analysis.^19–22 Direct DNA sequencing was used to detect mutations of gene CEBPA and FLT3-ITD.

Bioinformatics and Statistical Analysis

Micro RNAs which might bind with circ_0002232 were predicted by circRNA-miRNA interaction prediction websites, miRanda (http://miranda.org.uk) and RNAhybrid (https://bibiserv.cebitec.uni-bielefeld.de/rnahybrid/submission.html). Target genes of miR-92a-3p were predicted by miRTarBase (http://mirtarbase.mbc.nctu.edu.tw/php/index.php), miRDB (http://www.mirdb.org) and TargetScan (http://www.targetscan.org/vert_72/).

Statistical analysis was conducted by using spss software version 22.0. The diagnostic value of circ_0002232 expression was evaluated by receiver operating characteristic (ROC) curve and area under the ROC curve (AUC). The differences of categorical variables between the two groups were analysed by using Pearson Chi-square analysis or Fisher exact test and the differences of continuous variables were evaluated by using Mann–Whitney U-test. To explore prognostic potential of circ_0002232, Kaplan-Meier curves were used to analyse the impact of circ_0002232 for overall survival (OS) and Cox regression analysis was used to assess its independent prognostic value. Spearman correlation analysis was used respectively to examine the correlationship among the expression of circ_0002232, miR-92a-3p and PTEN. P value less than or equal to 0.05 (two-sided) was considered statistically significant in all analyses.

Results

Circ_0002232 Expression in AML and Controls

In our experiment, the expression level of circ_0002232 in de novo AML (median 0.0486, range 0.000215–0.953) was notably decreased compared with that in controls (median 0.468, range 0.00693–40.518; P<0.001, Figure 1). In addition, compared with controls, circ_0002232 expression level was remarkably down-regulated in non-APL AML patients (P=0.0022, Figure 1) and was low-expressed in CN-AML patients (P=0.027, Figure 1).

Figure 1 Relative expression level of circ_0002232 in controls and AML. The expression of circ_0002232 in controls, whole AML, non-APL AML and CN-AML patients was measured by using RQ-PCR. Each dot represents a single sample and horizontal line represents the median level of expression.

Differentiating Ability of Circ_0002232 Expression

The capacity of circ_0002232 expression to distinguish AML patients from controls was analysed by ROC curve (AUC:0.851, 95% CI:0.788–0.915, P<0.001, Figure 2A). It indicated that cric_00022232 could act as a significant marker in differentiating between AML patients and controls. In addition, the remarkable significance was found in distinguishing non-APL AML patients from controls (AUC:0.848, 95% CI:0.781–0.914, P<0.001, Figure 2B).

Figure 2 ROC curve analysis of circ_0002232 for distinguishing AML patients from controls: (A) Whole AML; and (B) non-APL AML.

Clinical and Laboratory Characteristics of AML Patients

For the purpose of exploring the relationship between clinical parameters and circ_0002232 expression, we obtained the cut-off value, which had the maximum sum of sensitivity and specificity according to ROC curve analysis, and divided 115 AML patients into low-expressed group (circ_0002232^low) and high-expressed group (circ_0002232^high). Hence, we used 0.165 as the cut-off value, whose sensitivity was 0.813 and specificity was 0.757. There were no significant discrepancies between the two groups in sex, hemoglobin, BM blasts, complete remission (CR), cytogenetic abnormalities and nine gene mutations (P>0.05, Table 1).

However, remarkable differences were observed in FAB classifications (P=0.036), white blood cell (WBC) count (P=0.041) and platelet count (P=0.021) between circ_0002232^low and circ_0002232^high groups. Age of the patients in circ_0002232^low group was notably younger than those in circ_0002232^high group (P=0.004). Moreover, we found that there was a positive correlation between circ_0002232 expression and patients’ age (Pearson r=0.256, P=0.0057, Figure 3A). And a positive correlation between the expression of circ_0002232 and platelet count was also found (Pearson r=0.202, P=0.031, Figure 3B).

Figure 3 Pearson correlation analysis: (A) correlationship between patients’ age and circ_0002232 expression in AML; and (B) correlationship between platelet count and circ_0002232 expression in AML.

Correlation Between Circ_0002232 Expression and Patients’ Clinical Outcome

Survival analysis included 88 AML patients and excluded 27 patients who failed to follow up. Median follow-up time of included patients was 8 months, which ranged from 1 month to 90 months. According to Kaplan-Meier analysis, circ_0002232^low group had significantly longer OS compared with circ_0002232^high group in whole AML (P=0.030, Figure 4A). In non-APL AML, patients in low-expressed circ_0002232 group tended to have better prognosis (P=0.014, Figure 4B). However, in low age group (age<40y), we found that patients with high circ_0002232 expression tended to have better OS, but it was not statistically significant (P=0.214, Figure 4C).

Figure 4 Kaplan-Meier analysis showed the differences in overall survival between circ_0002232low and circ_0002232high group: (A) overall survival among whole AML; (B) overall survival among non-APL AML; and (C) overall survival among AML (age<40y).

Univariate analysis, including age (≤60y or >60y), WBC count (≥30×10⁹/L or <30×10⁹/L), cytogenetic abnormalities’ classification, circ_0002232 expression, RAS mutation, U2AF1 mutation with P<0.05, showed that expression of circ_0002232 could be used as a valuable factor for AML patients’ prognosis. However, according to multivariate analysis, expression of circ_0002232 could not act as an independent factor for OS (P=0.727) among AML patients (Table 2).

Table 2 Univariate and multivariate analyses of prognostic variables for overall survival in whole AML patients

Correlation Between Expression of Circ_0002232 and PTEN in AML

The expression of PTEN in AML (median 1.984, range 0.00701–88.0896) was remarkably down-regulated compared with controls (median 3.330, range 0.842–103.788) (P=0.0057, Figure 5A). Furthermore, the expression of circ_0002232 was positively correlated with its parental gene PTEN (Spearman r=0.678, P<0.001, Figure 5B).

Figure 5 (A) Relative expression level of PTEN in controls and whole AML. (B) Spearman correlation analysis between the expression of PTEN and circ_0002232 in AML.

Potential Interaction Network of Circ_0002232/miR-92a-3p/PTEN

CircRNA-miRNA interaction prediction websites were used to predict miRNAs which might bind with circ_0002232. Through searching literature, we finally choose miR-92a-3p (Figure 6A and B). The expression of miR-92a-3p was detected in controls and AML patients. MiR-92a-3p was notably up-expressed in AML (median 6.215, range 0.0610–218.199) compared with controls (median 0.472, range 0.00815–2.964) (P=0.0087, Figure 6E). Spearman correlation analysis revealed that circ_0002232 expression was negatively correlated with miR-92a-3p expression in AML (Spearman r=−0.301, P=0.016, Figure 6F). Moreover, prediction websites showed the potential binding sites between miR-92a-3p and PTEN (Figure 6C and D). According to the result of Spearman analysis, miR-92a-3p had negative correlation with PTEN (Spearman r=−0.324, P=0.034, Figure 6G).

Figure 6 (A) Venn results of microRNAs which could bind with circ_0002232 predicted by miRanda and RNAhybrid. (B) Binding sites between circ_0002232 and miR-92a-3p predicted by miRanda. (C) Venn results of genes which could bind with miR-92a-3p predicted by TargetScan, miRTarBase and miRDB. (D) Binding sites between miR-92a-3p and PTEN predicted by miRTarBase. (E) Relative expression level of miR-92a-3p in controls and AML. (F) Spearman correlation analysis between the expression of circ_0002232 and miR-92a-3p in AML. (G) Spearman correlation analysis between the expression of miR-92a-3p and PTEN in AML.

Discussion

CircRNAs, known as a novel category of non-coding RNAs, exist widely in mammalian cells.²³ They have been considered as ideal biomarkers for disease because of their conservative feature across species. There are a few studies concentrated on the role of circRNAs in hematological malignancies. For instance, circ_0004277 expression had been reported to be down-regulated in AML. And the expression of circ_0004277 tended to be up-regulated when the patients had complete remission and down-regulated again when they relapsed. Circ_0004277 expression changed dynamically with process of AML, which proved that it could be used as AML biological marker.²⁴

As far as we know, this is the first report focused on the expression of the circular RNA transcribed from PTEN in AML. In this study, circ_0002232 expression in AML was notably down-regulated compared with that in controls. The same results were found in groups of non-APL AML and CN-AML. According to ROC curve analysis, circ_0002232 could act as a valuable marker to identify AML patients and control groups.

Identifying the relationship between the expression of circ_0002232 and clinical characteristics, we found that the expression level of circ_0002232 was positively correlated with platelet count. Circ_0002232^low group tended to have lower platelet count. There have already been several reports focused on the abnormal platelet count and dysfunction in AML.²⁵ Low platelet count was associated with poor prognosis and recovery of platelet was concerned with relapse-free survival rate after chemotherapy in AML.^26,27 Moreover, circ_0002232^low group also tended to have lower hemoglobin and higher percentage of blast compared with circ_0002232 high expression group. This means circ_0002232^low group have more severe myelosuppression and more serious infiltration in BM. Hence, low expression of circ_0002232 is an adverse factor of AML.

Because PTEN, parental gene of circ_0002232, plays a role of tumor suppressor in many diseases, at the beginning of the experiment we proposed that the circular RNA transcribed from PTEN might also have tumor suppressive effect and down-regulation of circ_0002232 might lead to shorter overall survival time. Unexpectedly, results of Kaplan-Meier analysis revealed that OS of patients with low-expressed circ_00002232 were longer than that of patients with high-expressed circ_00002232 in whole AML. This result was obviously in contrast with our initial prospect.

However, our study indicated that patients in circ_0002232^high group were significantly older than those in circ_0002232^low group. Pearson analysis was used to confirm this result, which revealed that the circ_0002232 expression was positively correlated with patients’ age. Age is an important risk factor for AML. Survival time of AML patients tends to decrease with increased age.^28,29 We suppose that it may help us to understand this conflicting result. High-expressed circ_0002232 group tends to have poor prognosis because of the increase of age.

Then according to the expression level of circ_0002232, we divided the patients (age<40y) into two groups and compared the differences in survival time. The result in the subgroup under 40 showed that circ_0002232^high group tended to have better OS compared with circ_0002232^low group. This result confirmed our conjecture. But due to the limitation of our experiment size, this result was not statistically significant. In the future, additional experiments are needed to enlarge sample size and identify the relationship between circ_0002232 expression and OS in different age subgroups.

Moreover, the phenomenon of circRNAs acting as miRNA sponges in regulating proliferation, metastasis and relapse of gastrointestinal cancer have been reported in some studies.⁵ In this research, prediction websites revealed that there were potential binding sites among circ_0002232, miR-92a-3p and PTEN. MiR-92a-3p expression has been revealed to be up-regulated in several solid cancers including breast cancer and brain glioma.^30,31 According to our experiment, miR-92a-3p expression of AML patients was obviously up-regulated compared with controls and was negatively correlated with the expression of circ_0002232.

Furthermore, high-expressed miR-92a have been found to regulate colorectal cell migration and invasion by suppressing the expression of PTEN.³² Alteration of miR-92a also promoted its effect on metastatic behavior of nasopharyngeal carcinoma cell by targeting PTEN.³³ Notably, we found that the expression level of miR-92a-3p was also negatively correlated with PTEN in AML. Hence, we proposed that circ_0002232 might affect the process of AML and the expression of PTEN through sponging miR-92a-3p. In the future, we plan to design more experiments to explore the mechanism of this pathway in AML.

Conclusion

Our experiment revealed circ_0002232, one of circRNAs of PTEN, was remarkably down-regulated in AML and could act as a promising biomarker for the diagnosis of AML. In addition, there might be a potential ceRNA interaction network of circ_0002232/miR-92a-3p/PTEN in AML.