#These authors contribute equally to this work
To investigate the characteristics of circular RNA hsa-circ-0006969 in breast cancer and identify it as a novel biomarker for breast cancer.
Three breast cancer (BC) patient tissues were selected to perform human circRNA microarray analysis. GeneSpring 13.0 (Agilent) software was applied for analyzing the data. Another 116 BC patients were recruited for verification. Hsa-circ-0006969 was found as a potential circRNA for BC diagnostic biomarker. The structure of hsa-circ-0006969 was predicted by circPrimer1.2 software. MiRanda v3.3, RNA hybrid 2.1, and Cytoscape 3.6.0 were used for predicting the networks of circRNA-miRNA. T-test, Curve regression, and ROC analysis were applied to certify the diagnostic values of hsa-circ-0006969. Using SPSS25.0 software to perform the Statistical analysis.
546 higher expression and 1475 lower expression circRNAs were identified. Four circRNAs were filtrated for further verification. Hsa-circ-0006969 was significantly low expressed in BC tissues and peripheral blood. Hsa-circ-0006969 was confirmed as higher diagnostic correlation with BC tissues (AUC = 0.965) and peripheral blood (AUC = 0.842), with Grade 1 (AUC = 0.639), ER-positive (AUC = 0.612), TNM I (AUC = 0.693), TNM II (AUC = 0.712), TNM III (AUC = 0.757), TNM early stage(I, II) (AUC = 0.709). Hsa-circ-0006969 presented more effective diagnostic values for tumor metastasis (AUC = 0.784) compared with CA153 (AUC = 0.752).
Hsa-circ-0006969 could be a novel biomarker for the diagnosis and treatment of BC.
Breast cancer (BC) is the most common cancer which is a leading cause of mortality in women [
Circular RNA(circRNA) is one type of covalently closed noncoding RNA which have a closed loop structure [
In this study, the circRNAs was profiled in breast cancer and para-carcinoma tissue using microarray analysis. Then verified the differential expressions of circRNAs in other cohorts of BC tissues and peripheral blood specimens. On account of the association studies of the target circRNA and the BC patient’s clinical characteristics, we confirmed hsa-circ-0006969 as a potential biomarker for breast cancer. Further analysis is being performed to evaluate the diagnostic value of hsa-circ-0006969 in breast cancer.
Both cancer tissues and para-carcinoma samples were obtained from the oncology surgery department of the General Hospital of Ningxia Medical University. The study was approved by the General Hospital of Ningxia Medical University Ethics Committee (No. 2018-118). Every patient has signed the informed consent to authorize the use of their samples.19 pair of cancer tissues and para-carcinoma samples and 100 BC peripheral blood was collected from March 2020 to August 2021. The age of patients ranged between 25–75 years old (average 48.26 ± 8.32 years). Every patient has not received radiotherapy and chemotherapy treatment before sample collection.
Peripheral blood samples were obtained from the vein with an EDTA plain tube (2 mL) after overnight fasting. Cancer tissues and para-carcinoma samples were obtained from patients who underwent surgical breast resection. The para-carcinoma samples were located >5 cm from the tumor part. Healthy persons were recruited from the physical examination department in the General Hospital of Ningxia Medical University as control.
Tissue RNA was extracted from cancer and para-carcinoma tissues with trizol reagent (Invitrogen, Carlsbad, USA). Peripheral blood RNA was extracted using a total RNA rapid extraction kit (Bioteke, Beijing, China). The RNA was measured using nanodrop 2000 (Thermo Scientific, Waltham, USA). RNA integrity test was performed by 1.3% agarose gel electrophoresis (120 V, 15 min, 1 × buffer). Total RNA was incubated with 10 U of RNase R (Geneseed, Guangzhou, China) at 37°C for 30 min. Finally, the expression of β-actin, ARHGEF28, and hsa-circ-0006969 was detected by RT-qPCR.
Three pair tumor and para-carcinoma tissues in BC patients were obtained for circRNA expression analysis using Human CircRNA Array v2 microarray (Beijing Capital Bio Biotechnology Corporation, Beijing, China). The circRNA microarray data were analyzed by GeneSpring 13.0 software (Agilent). To improve the screening efficiency, candidate circRNAs were screened with the filter criteria Fold change(FC) ≥4,
Total RNA was synthesized to cDNA with the revert aid first-strand cDNA synthesis kit (Thermo Scientific, Waltham, USA). Primers were synthesized by Sangon Biotech (Shanghai) Co, Ltd., primers are listed in Table S1. Light Cycler480 II quantitative system (Roche, Rotkreuz, Switzerland) was used for RT-qPCR. The reaction volume includes 2 µl cDNA, 0.8 µl sense primer, 0.8 µl reverse primer, 10 µl TB-Green Premix Ex Taq (TAKARA, Kyoto, Japanese), replenish ddH2O to 20 µl. The program is 94˚C for 10 s, 40 cycles of 94˚C for 15 s, 55˚C for 15 s, and an annealing temperature for 30 s. β-actin was used as an internal control. Each reaction was repeated 3 times and the results of relative expression were calculated using 2−ΔΔCq method [
CircRNA microarray data was obtained using Feature Extraction software (CapitalBio, Beijing, China). Normalization, fold change and
SPSS25.0 software (IBM, Almunk, USA) and GraphPad Prism version 9.0 (GraphPad Software, San Diego, USA) were applied for statistical analysis. The continuous data were presented as mean ± SD and tested using a
With circRNA microarray, the differentially expressed circRNAs in 3 paired BC tissue specimens (BC1–BC3) and para-carcinoma tissue specimens (PC1–PC3) were analyzed. A total of 2021 differentially expressed circRNAs were identified in BC tissue compared with para-carcinoma tissue. With the screening criteria as FC ≥2 and
(A) circRNA microarray assay analysis of the specific expressions of circRNAs in 3 paired breast cancer (BC) and para-carcinoma tissues (PC). Upregulated circRNA was indicated by “red”, downregulated circRNA was indicated by “blue”, and no significant difference was shown by “white”. (B) Scatter plot revealed the results of microarray assay; (C) Volcano plot showed the results of microarray assay; (D) Cluster diagram of differentially expressed circRNAs with the filter criteria as FC ≥4,
Based on the gene function annotation and prediction, four circRNA candidates were selected for further identification. The 4 circRNAs include two higher expressed circRNAs: hsa-circ-0044513, hsa-circ-0101692, and two lower expressed circRNAs: hsa-circ-0006969, hsa-circ-0054020. RT-qPCR was then performed for validation of the circRNAs in an independent cohort of 16 cases of BC samples. The results confirmed that hsa-circ-0006969 has a good consistency with BC samples. ROC curve analysis results illustrated that hsa-circ-0006969 has a high diagnostic value for BC tissues (AUC = 0.965,
After bio-information analysis, 4 circRNA candidates were selected for further identification, including 2 upregulated and 2 downregulated circRNAs. RT-qPCR validated the four circRNAs in an independent cohort including 16 cases of para-carcinoma and carcinoma tissues; (A) RT-qPCR and ROC of hsa-circ-0006969; (B) RT-qPCR and ROC of hsa-circ-0054520; (C) RT-qPCR and ROC of hsa-circ-0044513; (D) RT-qPCR and ROC of hsa-circ-0101692; ns, no significant, *
Specific primers were designed according to the reverse cleaved sites and linear sites of hsa-circ-0006969 (
(A) Schematic illustration showing junction site and linear site primers in hsa-circ-0006969. The presence of hsa-circ-0006969 was validated by RT-qPCR using junction site and linear site primers. Junction was head-to-tail hsa-circ-0006969 splicing sites; (B) Verification of the hsa-circ-0006969 expression in peripheral blood by RT-qPCR in gDNA, RNA, and cDNA. Junction site, and linear site primers were used; (C) RT-qPCR analysis of hsa-circ-0006969 expression in peripheral blood after treated or not with RNase R digestion, *
The association between hsa-circ-0006969 and BC clinical characteristics was verified in 100 patients. The results show that the expression of hsa-circ-0006969 was significantly related to triple-negative breast cancer (
Parameter | N | Hsa-circ-0006969 level (Mean ± SD) | ||
---|---|---|---|---|
Age status | ||||
≥50 | 43 | 14.53 ± 0.89 | 1.130 | 0.261 |
<50 | 57 | 14.34 ± 0.80 | ||
Tumor size status (cm) | ||||
≥2c | 53 | 14.46 ± 0.86 | 0.491 | 0.625 |
<2c | 47 | 14.38 ± 0.82 | ||
Triple Negative Breast Cancer (TNBC, n) | ||||
Yes | 18 | 15.27 ± 0.74 | 2.387 | 0.039* |
No | 82 | 14.39 ± 0.81 | ||
Her-2 Positive | ||||
Yes | 43 | 14.34 ± 0.79 | -0.823 | 0.412 |
No | 57 | 14.48 ± 0.87 | ||
Estrogen Receptor (ER, n) | ||||
Positive | 81 | 14.50 ± 0.85 | 2.003 | 0.048* |
Negative | 19 | 14.06 ± 0.67 | ||
Progesterone Receptor (PR, n) | ||||
Positive | 70 | 14.45 ± 0.82 | 0.450 | 0.654 |
Negative | 30 | 14.36 ± 0.88 | ||
Androgen Receptor (AR, n) | ||||
Postive | 83 | 14.45 ± 0.87 | 0.794 | 0.429 |
Negative | 17 | 14.27 ± 0.70 | ||
Tumor grading (G, n) | ||||
G1 | 25 | 14.91 ± 0.89 | G1 |
0.021* |
G2 | 43 | 14.39 ± 0.74 | ||
G3 | 32 | 14.72 ± 0.87 | ||
TNM stage | ||||
I | 16 | 14.02 ± 0.68 | I |
0.422 |
II | 49 | 14.21 ± 0.82 | ||
III | 28 | 14.93 ± 0.62 | ||
IV | 7 | 14.81 ± 1.06 | ||
Edmondson grading | ||||
Early stages (I–II) | 65 | 14.16 ± 0.79 | −4.675 | 0.000** |
Advanced stages (III–IV) | 35 | 14.91 ± 0.71 | ||
Tumor metastasis status | ||||
Yes | 71 | 14.61 ± 0.83 | 3.612 | 0.000** |
No | 29 | 13.97 ± 0.67 | ||
CA153 status | 1.156 | 0.250 | ||
CA153 positive | 18 | 14.63 ± 0.80 | ||
CA153 negative | 82 | 14.38 ± 0.84 | ||
CEA status | ||||
CEA positive | 14 | 14.95 ± 0.78 | 1.020 | 0.310 |
CEA negative | 86 | 14.52 ± 0.82 |
Notes: The diagnostic cutoff value for metastasis of CA153 was 100 U/mL. The diagnostic cutoff value of CEA was 20 ng/mL. *
(A–E) The association analysis of hsa-circ-0006969 with clinical pathological characteristics of 100 BC patients. (A) hsa-circ-0006969
To determine the diagnostic values of hsa-circ-0006969 in BC patient peripheral blood, ROC curve analysis was applied. The results proved that the AUC of hsa-circ-0006969 for diagnosis of High differentiated tumor (Grade 1) was 0.639 (
Clinicopathological characteristics | Expressiona ( |
AUC | AUC (95%CI) | Sensitivity | Specificity | ||
---|---|---|---|---|---|---|---|
Positive | Negative | ||||||
Grade 1 | 13.92 ± 0.81 | 14.55 ± 0.73 | 0.639 | 0.525−0.761 | 0.013* | 0.687 | 0.661 |
Grade 2 | 14.31 ± 0.97 | 14.48 ± 0.85 | 0.476 | 0.369−0.584 | 0.667 | 0.764 | 0.328 |
Grade 3 | 14.59 ± 0.95 | 14.39 ± 0.90 | 0.540 | 0.417−0.63 | 0.527 | 0.667 | 0.527 |
ER positive | 14.81 ± 0.82 | 14.42 ± 0.73 | 0.612 | 0.511−0.741 | 0.029* | 0.346 | 0.947 |
Metastasis (circRNA) | 14.50 ± 0.85 | 13.92 ± 0.87 | 0.784 | 0.652−0.865 | 0.0001** | 0.792 | 0.803 |
Metastasis (CA153) | 47.65 ± 36.20 | 13.53 ± 13.94 | 0.752 | 0.634−0.859 | 0.0001** | 0.619 | 0.761 |
TNM I | 14.51 ± 0.86 | 13.91 ± 0.91 | 0.693 | 0.590−0.873 | 0.017* | 0.650 | 0.692 |
TNM II | 14.29 ± 0.75 | 14.55 ± 0.91 | 0.712 | 0.568−0.782 | 0.006** | 0.529 | 0.781 |
TNM III | 14.52 ± 0.73 | 14.39 ± 0.87 | 0.757 | 0.660−0.853 | 0.0001** | 0.750 | 0.741 |
TNM IV | 14.84 ± 0.96 | 14.36 ± 0.80 | 0.599 | 0.444−0.753 | 0.208 | 0.813 | 0.423 |
TNM stage |
14.30 ± 0.85 | 14.70 ± 0.97 | 0.709 | 0.665-0.858 | 0.0001** | 0.653 | 0.844 |
Note: a: The results of relative expression were calculated using ΔCt and presented as mean ± SD; *
The binding miRNA of hsa-circ-0006969 was predicted by miRanda and RNAhybrid software. The results proved that 56 microRNAs have potential binding sites with hsa-circ-0006969 (
(A) All candidate binding miRNAs of hsa-circ-0006969; (B) The top 6 candidate binding miRNAs and binding sites of hsa-circ-0006969 associated with cancer cell cycle, proliferation and apoptosis; (C–D) GO and KEGG database predicts the main signal biological process and pathway of top 6 candidate target miRNAs; miRNA target mRNA positive correlation was indicated by “orange”, miRNA target miRNA negative correlation was indicated by “blue”.
CircRNA is a non-coding RNA with a closed loop and the structure lack of 3’ and 5’ ends. The expression of circRNA has tissue-specific stability in most cancer tissue and blood [
The higher morbidity and recurrence of BC are a serious threat to human health. Early diagnoses are helpful in the treatment of BC patients. Due to the unique molecular structure and expression specificity, circRNAs have the potential to be diagnostic markers or therapeutic targets for BC. Jahani et al found that circRNAs were expressed in BC cells in a cell-type and stage-specific manner [
In this study, the circRNA expression profiling in Breast cancer was conducted by circRNA microarray. After the cluster analysis and GO analysis, 546 higher expressed and 1475 lower expressed circRNAs were identified in BC tumor tissues. With the filter criteria, 4 candidate circRNAs were screened for further study. RT-qPCR validation results suggest hsa-circ-0006969 has good consistency in BC tissue and peripheral blood. ROC curve analysis identified the diagnostic value of hsa-circ-0006969 in BC. The bioinformatics analysis revealed that hsa-circ-0006969 has 6 target miRNAs and the molecular biological function of these 6 miRNAs was associated with cell cycle and cell proliferation. The result indicated that hsa-circ-0006969 intervenes in BC cell differentiation and proliferation by binding the 6 target miRNAs as a “sponge”.
Furthermore, the structural and functional annotation analysis revealed that hsa-circ-0006969 was transcribed from the exon 10 and 11 regions of the human ARHGEF28 gene. ARHGEF28 was a RhoA-specific guanine nucleotide exchange factor that was involved in cell aggregation, apoptosis, and motility by influencing growth factor receptors [
In conclusion, this study provided evidence that hsa-circ-0006969 is a lower expression both in BC tissues and peripheral blood samples. hsa-circ-0006969 may play a role as a promising potential biomarker for BC diagnosis and treatment. However, further study is needed to elucidate the underlying mechanisms.
Tumor Node Metastasis
Human epidermal growth factor receptor-2
Carcino-embryonic antigen
Carbohydrate antigen 15-3
Area under the curve
Confidence interval
NOT was applicable
Wang LB planned the project, revised and polished the article, and adjusted all aspects of the work. Zhang X planned the project, executedall experiments, and wrote and revised the article. Li JP planned the project, revised the article, participated in circRNA Microarray analysis and screening candidate circRNAs. Li XH circRNA expression in specimens, and did ROC curve analysis. Tian JH did the circRNA Microarray data analysis and screening specific circRNAs. Yu JJ did the circRNA genechip analysis and statistical analysis. Huang Q prepared a table and supplementary file. Ma R collected tissue and peripheral blood samples and total RNA and cDNA extraction. Wang J prepared reagent and instrument, designed and screened primers. Cao J collected clinicopathological characteristics of patients with breast cancer.
The study protocol was reviewed and approved by the Ethics Committees of the General Hospital of Ningxia Medical University Ethics Committee (No: 2018-118). Informed consent was obtained from all individual participants included in the study.
We guarantee the authenticity and validity of all data and results. Open up some of the raw data uploads as supplementary files.
I would like to declare on behalf of my co-authors that the work described was original research that has not been published previously, and is not under consideration for publication elsewhere, in whole or in part. The manuscript is approved by all authors for publication.
This study was supported by the
The authors declare that they have no conflicts of interest to report regarding the present study.