Identification of circular RNAs hsa-circ-000696 as a novel biomarker for breast cancer

Background : To investigate the characteristic expression of circular RNAs between breast cancer and para-carcinoma tissue, identified hsa-circ-000696 as a novel biomarker for breast cancer. Methods: 136 patients were recruited in General Hospital of NingXia Medical Universit. Of which 3 patients tissue for microarray analysis, 20 and 113 patients for circRNA expression in tissue and peripheral blood, respectively. The candidate circRNAs was verified by qPCR in BC patients’ tissue and peripheral blood. GeneSpring 13.0(Agilent) software were used for analysis the circRNA array data. CircRNA structure were performed by circPrimer1.2 software. MiRanda v3.3,RNAhybrid 2.1 and Cytoscape 3.6.0 were utilized as tools to predict the circRNA-miRNA networks. T-test, Curve regression analysis and ROC analysis were performed to determine the diagnostic values of candidate circRNAs. Statistical analysis was performed using SPSS23.0 software. Results: 2021 differentially expressed circRNAs were identified, of which 546 were upregulated and 1475 were downregulated. Four circRNAs were selected with filter criteria for further verify in BC tissues. Hsa-circ-0006969 was significantly downregulated both in breast cancer tissues and peripheral blood. Hsa-circ-0006969 proved a highest diagnostic value in breast cancer tissues(AUC=0.889), peripheral blood(AUC=0.823) for Grade 1(AUC=0.672), Grade 3(AUC=0.675), ER positive(AUC=0.648), TNM Ⅰ(AUC=0.687) and TNM Ⅱ(AUC=0.769), TNM Ⅲ(AUC=0.757), TNM early stage(Ⅰ, Ⅱ) (AUC=0.751). Hsa-circ-0006969 show more effective diagnostic values of for tumor metastasis (AUC=0.742) compared with CA153 (AUC=0.712). Conclusion: Has-circ-0006969 characters: 5592 Sample Word Doc to HTML Online Converter Word Document to HTML Online Converter Free online Word to HTML converter with built-in code cleaning features and easy switch between the visual and source editors. It works perfectly for any document conversion, like Microsoft Word, Excel, PowerPoint, PDF, Google Docs, Sheets, and many more. You can also use this tool for composing web content from scratch or just to tidy up the dirty markup.


Background
Breast cancer is one of the most frequently occurring women cancer and the leading cause 4 of cancer-related death among worldwide [1]. Current studies have shown that age, obesity, abnormal estrogen, late childbirth, genetic and epigenetic are associated with an increased risk of breast cancer [2,3]. Despite the development of diagnosis and treatment of BC, the mortality remains unsatisfactory for BC patients [4]. This discrepancy is mainly because the current diagnostic methods cannot simultaneously achieve high sensitivity and convenience of breast cancer. We need an efficient and sensitivity diagnostic marker and therapeutic target.
Circular RNA(circRNA) is a type of non-coding RNA which have a continuous closed loop structure [5]. circRNA have a large number of miRNA binding sites, which provide its function of regulating miRNA by "sponge" with them. CircRNA are tissue-specific and play important roles in different cancer [5,6]. Recent reports have showed that cirRNA involved in the cancer cells metastasis by directly bounding(sponge) to its target miRNA. For example, Human ciRS-7 is a typical example as miRNA sponge functions with 74 miR-7 binding sites [7]. In Esophageal Squamous Cell Carcinoma, circ-ITCH Inhibit cell proliferation through binding miR-7, miR-17, and miR-214, and active Wnt/β-catenin signaling pathway [8]. However, the potential functions of a large proportion of circRNAs in cancer still not clear.
In this study, we first screening the circRNAs profile in breast cancer and para-carcinoma tissue by microarray analysis. Then confirmed the differentially expression circRNAs in larger independent cohorts including breast cancer tissue and patients' peripheral blood specimens. Based on the correlation analysis of the target circRNA and patient's clinic characteristic, we identified hsa-circ-0006969(CIRC6969) as a candidate gene for breast cancer diagnostic. Further analysis being used to evaluate the diagnostic value of CIRC6969 in breast cancer. Collect the clinical, pathological, and molecular characterization data of all participants.

Cell culture
Human breast cancer cell line MDA-MB-231 were purchased from The Typical Culture Preservation Committee Cell Bank of Chinese Academy of Sciences. The cells were 6 cultured in DMEM (Gibco, USA) with 10% fetal bovine serum (FBS, Gibco, USA) at 37 °C in 5% CO 2 .

RNA Isolation
Tissue RNA were isolated from BC tissue and adjacent normal tissue with Trizol reagent (Invitrogen, Inc., USA). Peripheral blood RNA were isolated with Total RNA Rapid Extraction Kit (Bioteke, Inc., China). The quality of RNA were measured using Nanodrop 2000 (Thermo Scientific, USA). RNA integrity was determined by 1.3% agarose gel electrophoresis (120V, 15 min, 1×FA buffer ).

circRNA Microarray
Human CircRNA Array v2 microarray(Beijing Capital Bio Biotechnology Corporation, China) has been used for circRNA microarray expression profiling. GeneSpring 13.0 software(Agilent) were used for circRNA array data analysis. Three pairs of BC tissues and para-carcinoma tissues were collected for circRNA microarray analysis. The labelled RNAs were hybridized on the microarray containing 162351 human circRNA probes. In order to improve the screening efficiency, differentially expressed of circRNAs were screened by the filter criteria FC ≥4, P-value<0.05, Original Fluorescence value ≥100.
qPCR The cDNA were synthesized by the RevertAid First strand cDNA Synthesis kit (Thermo Fisher Scientific, Inc., USA). qPCR were performed using LightCycler480II quantitative system(Roche, USA). The primers were synthesized Sangon biotech(Shanghai) Co.,Ltd, all circRNA primer was list in Table S1. The reaction in a total volume of 20 μL system including 1 µl total RNA (500 ng), 1 µl random primer, 10 µl ddH2O, 2 µl dNTP, 4µl Reaction buffer, 1µl Ribolock RNase Inhibitor, 1µl RevertAid M-MuLV RT. The cycling program is 25℃ 5min, 42℃ 60min, 70℃ 5min. GAPDH Expression were used as an internal control. Each reaction was repeated three times and the mean relative expression of genes were calculated using ΔΔCT method [9]. The PCR production was stored at -80°C before use.

Microarray data analysis
The circRNA microarray data were extracted using Feature Extraction software(CapitalBio). Normalization, Fold change and P value were performed using GeneSpring software V13.0 (Agilent). Heat map were analysed using Cluster 3.0 software.

Statistical analysis
Data statistical analysis were performed by SPSS23.0 software (IBM,USA), GraphPad Prism version 8.0 (GraphPad Software, USA), data were presented as mean ±SD. The categorical variables were tested by the Chi-square test, and the continuous variables were tested by the t-test. ROC were performed to verify the clinical diagnostic value of circRNA. The correlation between circRNA expression and the breast cancer pathological characteristics 8 were evaluated by Pearson`s correlation test and Curve regression analysis. Differences were considered significant if p<0.05 (* p < 0.05; **p < 0.01).

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circRNAs Expression profiles in the BC patients
To identify the specific circRNA expressed in breast cancer, 3 paired BC tissues samples (CA1-CA3) and para-carcinoma tissues samples (AP1-AP3) were collected for circRNA microarray assay. Based on the circRNA microarray results, 2021 differential expressed circRNAs being identified. With the filter criteria as FC ≥2 and P-value <0.05, 546 were upregulated and 1475 were downregulated ( Figure S1). The cluster analysis divided the differential expressed circRNA in two groups ( Figure S1A). According to the different fluorescence signal values to screening differential expressed circRNAs in two group ( Figure S1B).
We use the filter criteria as FC ≥4, P-value < 0.05, Original fluorescence value ≥100 to verify the candidate circRNA. Symbol gene functional annotation was associated with tumor occurrence and development. The four candidate circRNAs selected included two upregulated circRNAs, Hsa-circ_0044513 and Hsa-circ_0101692, two downregulated circRNAs, Hsa-circ-0006969 and Hsa-circ_0054020 as candidate circRNAs in a subsequent validation utilizing a larger cohort (Table S1).

Verify the candidate circRNAs by qPCR
To verifiy the four selected circRNA, 20 pairs of breast carcinoma and para-carcinoma tissues samples were used in an independent cohort. qPCR proved that hsa-circ-0006969, hsa-circ-0054020 were significantly decreased while another hsa-circ-0044513 and hsacirc-0101692 were increased in breast carcinoma. The expression FC of hsa-circ-0006969 was 5.4, hsa-circ-0054020 was 3.1, hsa-circ-0044513 was 5.7 and hsa-circ-0101692 was 9.6, respectively. ROC curve analysis performed that hsa-circ-0006969(CIRC6969) have higher AUC and lower P values than other three circRNAs, indicated that expression of CIRC6969 was significantly correlated with BC ( Figure 1).
Further validation of the expression of CIRC6969 in peripheral blood samples from 113 patients with BC and 50 healthy person. The results proved the expression of CIRC6969 was down-regulated in BC patients compared with healthy person (Figure 2A, Table S2).
Based on the ROC curve analysis, AUC of CIRC6969 was 0.823 with sensitivity of 82.3% and specificity of 68.0% ( Figure 2B). The results suggested that CIRC6969 was a bloodspecific circRNA in breast cancer.

Bioinformatics analysis of CIRC6969
It was all known that circRNA derived from the reverse splicing of exons of different mRNA. In this study, circPrimer 1.2 software being used to identified the origin mRNA of hsa-circ-0006969. The results demonstrated that hsa-circ-0006969 was made by reverse splicing of exon 10 and 11 of ARHGEF28 gene ( Figure 3A). The circ-0006969 RT-qPCR product can not be digested by RNase R, proved its continuous closed loop structure( Figure 3B). We further used miRanda and RNAhybrid softwares to predicted the binding miRNA of CIRC6969. The results showed that 56 microRNAs were found as a potential binding target of hsa-circ-0006969 ( Figure 3C). Based on overlapping of two databases, 8 miRNAs with the significant scores and P-value were identified( Figure 3D ).
The information of the 8 miRNAs with Hsa-circ-0006969 are shown in Table 1, the biological processes of these miRNAs were all related to cell cycle and cell proliferation.
The results suggested that Hsa-circ-0006969 related to cell proliferation and differentiation, and may intervene in tumor occurrence and development through binding these miRNAs as a "sponge".

The diagnostic values of CIRC6969 as a biomarker in breasrt cancer
The clinical characteristics of 113 breast cancer patients were obtained to verify the correlation between CIRC6969 expresssion and breast cancer classification and prognosis.
Association analysis demonstrated that CIRC6969 expression were significantly related with tumor grading(P<0.05), ER positive(P<0.05), tumor metastasis(P<0.01)and TNM stage(P<0.01)( Table 2). Pearson`s correlation test and Curve regression analysis indicated that CIRC6969 expression was significantly correlated to CA153 level in peripheral blood (P<0.05) and no obvious correlation with CEA level ( Figure S2). It is suggested that CIRC6969 was specific expression in blood, and have high correlation with BC tumor stage, grade and metastasis.  Table S3). This suggested that CIRC6969 have diagnostic values in BC.

Discussion
CircRNA was a kind of non-coding RNA with a closed continuous loop and lack of 3' and 5' ends. CircRNA worked as a ceRNA or miRNA"sponge" to regulate the mRNA expression. It involved in many biological process, like gene transcription, protein translation and miRNA expression regulation through multiple mechanism [12]. Present studies indicated that circRNA expression are tissue-specific and stability in most cancer tissue and plasma [13,14]. These properties providing circRNA have the potential to become biomarkers for diagnosis and prognosis of cancers.
The most important functions of circRNAs is working as miRNA 'sponge' through engaging in competitive combination with miRNA. Based on the sponge function, circRNA could regulate cancer cell proliferation, differentiation, migration, angiogenesis, protein cracking and apoptosis [15,16]. Recent research demonstrated that circRNA was related to the malignant characteristics of the cancer through regulating miRNA. The first characterized circRNA to support this model was ciRS-7, which could inhibited normal miR-7's function through acting as an miRNA sponge [17]. CircIRAK3 promoted cell migration, 13 invasion and metastasis in vitro and in vivo by targeting miR-3607 [18].
The high morbidity of breast cancer and its recurrence severely threaten human health.
Early diagnosis and type differentiation are helpful to the treatment of breast cancer patients. Gao et al. found hsa-circ-0006528 was upregulated and related to chemotherapeutic resistance in BC [19]. Yin WB, et al. reported that hsa-circ-0001785 can be a diagnostic biomarker for breast cancer [20]. Previous research reported that circ-ABCB10 promotes BC cells proliferation through sponging miR-1271 [21].
Overexpression of hsa-circ-0136666 will promote BC cells progression by sponging miR-1299 and targeting CDK6 [22]. Zhang HD et al. found hsa-circ-0072995 promotes breast cancer cell migration and invasion through sponge for miR-30c-2-3p [23]. These suggest that circRNA plays an important role in the occurrence and development of breast cancer, which is worthy of us to further screening and functional verification.
In this study, circRNA microarray was using to profile the differentially expressed circRNAs apoptosis by regulating integrins and growth factor receptors [24]. Present research indicated that CIRC6969 was found in cerebellum, Hela cell, HepG2 cell [25,26]. But there is no evidence that CIRC6969 could play any functions in metabolism and tumor. To our knowledge, this is the first positive demonstration the relationship of CIRC6969 and BC.
With the bioinformatics analysis, 8 possible CIRC6969 targets miRNA being selected.
Functional analysis revealed that the biological processes of these 8 miRNAs were all related to cell cycle and cell proliferation, proved that CIRC6969 related to cell proliferation and differentiation, and may intervene in tumor occurrence and development through binding these miRNAs as a "sponge". ROC curve analysis revealed CIRC6969 have higher diagnostic accuracy in BC tissues and patients's peripheral blood samples. The results proveded that CIRC6969 have high correlation with BC metastasis and classification, and CIRC6969 can be used as a potential biomarker for BC diagnosis and treatment.

Conclusions
In summary, the study proveded that circRNA hsa-circ-0006969 was downregulated both in the BC tissues and peripheral samples.   Figure 1 The  ROC curve analyses of hsa-circ-0006969 in peripheral blood with breast cancer.
The AUC values are given on the graphs. sensitivity and specificity were showed in  Table S2.docx   Table S3.docx   Table S1.docx Figure S1.tif microarray data.xlsx Figure S2.tif