Clinical diagnostic value of circ-ARHGER28 for breast cancer and its effect on MCF-7 cell proliferation and apoptosis

doi:10.21203/rs.3.rs-4149825/v1

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Clinical diagnostic value of circ-ARHGER28 for breast cancer and its effect on MCF-7 cell proliferation and apoptosis

https://doi.org/10.21203/rs.3.rs-4149825/v1

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published 26 Jun, 2024

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Objective

To investigate the clinical diagnostic value of circ-ARHGER28 in breast cancer (BC), and the biological functiona of circ-ARHGER28 on the proliferation and apoptosis of MCF-7 cells.

Methods

Human circRNA microarray was performed to analysis the expression of circRNAs in BC patients. RT-qPCR combined with bioinformatic analysis was applied to verify the candidate circRNAs in BC tissues and peripheral blood. Circ-ARHGER28 was chosen as the candidate gene for further research. The clinical diagnostic value and biological functions of circ-ARHGER28 were analyzed. The over-expression and inhibit vector of circ-ARHGER28 were constructed and transfected to MCF-7 cells, the CCK-8 assay and clone formation experiments were applied to detect the cells proliferative and migratory abilities. Flow cytometry analyzed cell apoptosis and cell cycle distribution. RTqPCR and western blot were performed to detect the expression of apoptosis and PI3K/AKT/mTOR associated genes and proteins.

Results

Over-expression of circ-ARHGER28 inhibited the proliferation, colony formation and migration of the MCF-7 cells, as well as increased the population of the cells G₂/M phase and apoptotic rate. The apoptosis associated genes and proteins were significantly increased, whereas the gene and protein of PI3K, AKT and mTOR were decreased in the cells.

Conclusion

circ-ARHGER28 has a good correlation with BC. Circ-ARHGER28 inhibited MCF-7 cells proliferation and increased its apoptotic rate. The function of circ-ARHGER28 was associated with the PI3K/AKT/mTOR signaling pathway. Circ-ARHGER28 proved to be an ideal biomarker for BC diagnostic and a novel target for BC therapy.

circular RNA

breast cancer

biomarker

proliferation

apoptosis

Breast cancer (BC) is one of the most frequently occurring cancer in women, and is the leading cause of cancerassociate mortality among women worldwide[1–2]. Despite the improvements that have been made in diagnostic and treatment for BC, the mortality rate remains unacceptably high [3]. To identify an efficient and sensitive diagnostic marker and therapeutic target for BC is an urgent task.

Circular RNA(circRNA) is a type of noncoding RNA that has a continuous closed-loop structure[4]. circRNAs often stably expressed in different tissues and fulfill important roles in different types of cancer[5]. Previous study has shown that circRNAs involved in oncogenesis and influenced the proliferation, migration, apoptotic and metastatic of cancer cells through “sponging” to their target microRNAs (miRNAs). For example, circTFF1 was shown to act as a competing endogenous RNA (ceRNA) of TFF1 by sponging miR-326, subsequently accelerating BC progression via the miR-326/TFF1 axis [6]. CircRNF20 acts as a miRNA sponge to miR-487a and promoting its transcription [7]. In spite of the advances have been made, the potential functions of circRNAs in cancer are still need more research.

In this study, circRNA expression profiling in BC tissue and paracarcinoma tissues were analyzed by microarray. The candidate genes were subsequently selected based on the results. Therefore, Circ-ARHGER28 was identified as a candidate gene for further research. Cell Counting Kit (CCK)-8, colony formation, wound healing, flow cytometry, RTqPCR, and western blotting assays were utilized to investigate the differential expression of circ-ARHGER28 and its effect on cell proliferation and apoptosis of BC cells. Taken together, the results of the study have provided that circ-ARHGER28 could be an ideal biomarker for BC diagnostic and treatment.

Participants and specimens. The study was approved by the Ethics Committee of General Hospital of Ningxia Medical University (No.2018 − 118).Both tumor tissues and plasma samples were collected from the oncology surgery department, the General Hospital of Ningxia Medical University. All patients involved in the study signed the informed consent form for donation of their samples. A total of 20 BC tissues and 80 BC peripheral blood samples were collected from January 2019 to August 2022. The paracarcinoma tissues were located > 5 cm from the tumors. The patients were including (1) women; (2) without previous cancer history; (3) without HIV/AIDS; (4) > 18 years old; (5) after mastectomy. The age of patients ranged between 25–75 years old (average 49.94 ± 9.97 years). None of the patients were treated with radiotherapy or chemotherapy before specimen collection. Healthy persons (as controls) were recruited from the physical examination department of the General Hospital of Ningxia Medical University and signed informed consent form accordingly.

Antibodies and reagents. The protein antibodies ofrabbit antibodies against human Bax,Bcl-2, β-Actin were purchased from AB Clonal.Whereas cleaved caspase 8, cleaved caspase 9, cleaved caspase 3), poly ADP ribose polymerase 1 (PARP 1), mTOR, p-mTOR (Ser 2448), PI3K p85, p-PI3K p85 (Tyr 458)/p55 (Tyr 199), AKT, p-AKT (Ser 473) and the HRPconjugated goat antirabbit/mouse IgG secondary antibody were all purchased from Cell Signaling Technology, Inc.The PAGE Gel Preparation kit, TrisglycineSDS electrophoresis liquid and transfer buffer were purchased from Epizyme, Inc.

Cell culture and transfection. Human mammary epithelial cell line MCF-10A, and human breast cancer cell lines MDAMB-231, MCF-7 and HCC-1937 were purchased from the Typical Culture Preservation Committee Cell Bank of Chinese Academy of Sciences. MCF-10A cells were cultured in Complete™ Minimum Essential Medium (MEM) (Gibco, USA), whereas MDAMB-231 and MCF-7 cells were cultured in DMEM containing Gibco®10% FBS (Thermo Fisher Scientific, Inc.) at 37°C in 5% CO₂. HCC-1937 cells were cultured in RPMI-1640 medium (Thermo Fisher Scientific, Inc.) with 10% FBS at 37°C in 5% CO₂. Cell lines were stored in liquid nitrogen until required for experiments.

Circ-ARHGER28 over-expression lenti-viral vector (pEXcirc) and nonspecific negative control (pEXNC) were constructed by Guangzhou Gene seed Biotech Co Ltd.1.5x10⁶ cells/well were seeded into 6well plates for 12 h, then transfected using 20 multiplicity of infection for 48h. In order to enhance transfection efficiency, the transfected cells were screened with 2 µg/ml puromycin for 2 days.

RNA isolation and amplification. Total RNA from BC tissues were isolated with TRIzol™ reagent (Thermo Fisher Scientific, Inc.). The RNA from BC peripheral was isolated with total RNA rapid extraction kit (Bioteke Corporation, Inc.). The quality of RNA was measured at 260 nm using a Nanodrop 2000™ spectrophotometer (Thermo Fisher Scientific, Inc.). RNA integrity was determined using agarose gel electrophoresis (1.3% gels; the gels were run at 120V for 15 min). Total RNA (2.5 µg) was incubated at 37°C,30 min with 10 U RNase R (Guangzhou Geneseed Biotech Co., Ltd.). After treatment with RNase R, the total RNA were amplified with PCR (Eppendorf,German). The PCR products were determined using 1.2% agarose gel electrophoresis at 120 V for 40 min.

RTqPCR analysis. The cDNAs were synthesized using a revert aid first strand cDNA synthesis kit (Thermo Fisher Scientific, Inc.). RTqPCR was performed using Light Cycler 480 II Quantitative system (Roche Diagnostics). The primers were synthesized by Sangon Biotech (Shanghai) Co.,Ltd. The thermo cycling conditions were as follows: 25°C for 5min, 42°C for 60min, and 70°C for 5min. The expression of β-actin was used as an internal control. Each reaction was repeated three times, and the mean relative expression of genes were calculated using the 2^ΔΔCq method[8]. The PCR products were stored at -80°C before use. The NCBIPrimer database was used to design specific primers for target genes, and the sequences of the specific primers are listed in Table S1.

circRNA microarray data analysis. circRNA microarray expression profiling was performed via Human CircRNA Array v2 microarray analysis (CapitalBio Corporation). Three paired BC and paracarcinoma tissues were used for the analysis. The labeled RNAs were hybridized to a microarray system containing 162,351 human circRNA probes. The primary screening filter was set as fold change (FC) ≥ 2, Pvalue < 0.05, original fluorescence value ≥ 100. The secondary filter criteria were FC ≥ 4, Pvalue < 0.01, original fluorescence value ≥ 500. The circRNA microarray data were extracted using feature extraction software (CapitalBio Corporation). Cluster 3.0 and GraphPad Prism v. 8.0 software were used to perform heat map analysis. Scatter plots and volcano plots were constructed by ggPlot 2.0 (R) software. circRNA structures were predicted using circ Primer1.2 software. miRanda v3.3a (http://miranda.org.uk) and RNAhybrid 2.1 (https://directory.fsf.org/wiki/RNAhybrid) were utilized to predict the target miRNAs. Graphs showing the circRNAmiRNA interaction networks were drawn using Cytoscape v. 3.6 (https://cytoscape.org/). Gene Ontology (GO) analysis and the Gene Cards database(https://www.genecards.org/) were used to annotate the gene symbols of all differentially expressed circRNAs.

miRNA/mRNA sequence and analysis. miRNA sequencing libraries were generated using an Illumina®NEB Next®Multiplex Small RNA Library Prep Set (New England BioLabs, Inc.). mRNA sequencing libraries were constructed following removal of the ribosomal RNA using a RiboZero Gold rRNA Removal kit (Illumina, Inc.). Subsequently, the libraries were sequenced using a HiSeq 2500 System (Illumina, Inc.) according to the manufacturer's instructions. The differentially expressed genes were subsequently separated with the filter criteria, FC ≥ 2 and Pvalue < 0.05. The functional profiles of the nonredundant genes were obtained by annotating with the GO and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Gene abundance in each sample was estimated by SOAP[9].

CCK-8 assay. 5x10³cells/well were plated in 96-well plates and cultured at 37℃,5% CO₂ for 1.5h. According to the manufacturer’s instructions, the cell viability was measured using the CCK-8 kit(Beijing Solarbio Science & Technology Co., Ltd.). The time was 0h, 12h, 24h, 36h and the results was calculated with spectrophotometry at the emission wavelength of 450 nm.

Colony formation assay. 2x10³cells/well were plated into 6-well plates and cultured at 37℃, 5% CO₂ for 5 days. Following 24h of incubation, the cells at the lower side of the chamber were fixed in 4% paraformaldehyde solution (Beijing Solarbio Science & Technology Co., Ltd.) for 20 min and dyed with 0.1% crystal violet staining solution (Beijing Solarbio Science & Technology Co., Ltd.) for 10 min. Then counting and photographing the cells in five representative fields. All experiments were repeated three times independently.

Apoptosis and cell cycle analysis.1x10⁵/ml Cells were cultured with Complete™ medium. The numbers of apoptotic cells were measured using an AnnexinV/PI kit (Jiangsu KeyGen Bio Tech Co., Ltd.). After transfected pEX-circ, the cells were centrifuged at 1000 rpm for 5 min and washed with PBS, then resuspended in 500µl binding buffer. Subsequently, 5 µl fluorochromeconjugated annexin V and 5 µl PI were added, and the mixture was incubated at room temperature for 30 min. Muse™ Cell analyzer software (Millipore, USA) was used to quantify the results.

The cell cycle distribution was also performed. The cells were harvested and stained with PI (BD Biosciences) for 30 min. Then analyzed its cell cycle with Muse™ Cell analyzer.

Western blot analysis.Total proteins were extracted using a total protein extraction kit (Jiangsu Key Gen BioTech Co., Ltd.). The lysates were boiled in SDS sample buffer and then subjected to 10% SDS-PAGE. The protein concentration was determined using a BCA kit (Thermo Fisher Scientific, Inc.). The membranes were blocked with Super Block™ buffer (Thermo Fisher Scientific, Inc.) for 1 h at room temperature. Subsequently, the membranes were probed with Bax (dilution, 1:2,000), Bcl-2 (1:2,000), caspase-3 (1:2,000), caspase-8 (1:2,000), caspase-9 (1:2,000), PARP-1 (1:2,000), mTOR (1:2,000), pmTOR (1:2,000), PI3K (1:2,000), pPI3K (1:2,000), AKT (1:2,000), pAKT (1:2,000) and βactin (1:2,000) overnight at 4°C. The bound antibodies were then detected with HRPconjugated secondary antibodies (1:5,000 dilutions in SuperBlock™ buffer) with an incubation of the membranes at room temperature for 1 h. The densities of the bands were semi-quantified using ImageJ software v1.8.0 (National Institutes of Health). The expression of the proteins were normalized to βactin as the control.

Statistical analysis. Statistical analysis of the data was performed using SPSS version 23.0 software (IBM Corp.) and GraphPad Prism version 9.0 (GraphPad Software, Inc). Data are presented as the mean ± SD. The categorical variables were tested by the Chisquare test, whereas the continuous variables were tested by oneway ANOVA. Receiver operating characteristic (ROC) analysis was performed to verify the clinical diagnostic value of circRNAs.

Diagnostic values were considered significant if the area under the curve (AUC) was ≥ 0.6. P < 0.05 was considered to indicate a statistically significant difference.

Expression of circRNAs in BC tissues and cell lines. To identify the specific circRNA expressed in BC, three paired BC tissue samples (BC1‑BC3) and paracarcinoma tissue amples (PC1‑PC3) were collected for circRNA microarray assay. Base on the circRNA microarray results, 2,021 differentially expressed circRNAs was identified. With the primary screening filter, 546 up-regulation and 1,475 down-regulation circRNAs were found (Fig. 1 AC). With the secondary filter criteria, 27 up-regulated and 19 down-regulated circRNAs were obtained (Fig. 1D). After gene function annotation and prediction, 4 candidate circRNAs being selected, including 2 up-regulated circRNAs and 2 down-regulated circRNAs. The 4 circRNAs were subsequently validated via RTqPCR in an independent cohort that included 20 cases of BC tissues and 113 peripheral bloods. With the ROC curve analysis, circ-ARHGER28 was shown to have the high diagnostic value for BC tissues (AUC = 0.889, 95% CI = 0.780–0.998, P = 0.0001) (Fig. 1E-F).The result proved that circ-ARHGER28 is a specific differentially expressed circRNA in BC.

Based on the structural and functional annotation analysis, circ-ARHGER28 being identified that it was derived from the exon 10 and 11 regions within the human ARHGEF28 locus (Fig. 2A). The expression of circ-ARHGER28 was lower in BC cell lines, especially in MCF-7cells (Fig. 2B). The cytoplasmic and nuclear components analysis suggested that circ-ARHGER28 is a cytoplasmic highly stable expression circRNA (Fig. 2C). RNase R digestion and agarose gel electrophoresis combined RTqPCR identified the structure of circ-ARHGER28 (Fig. 2D,E).

Diagnostic value of circ-ARHGER28 in BC. A total 80 BC patients and 40 healthy person peripheral blood samples were collected. RTqPCR was used to demonstrate the expression of circ-ARHGER28 in the blood. The results suggested that circ-ARHGER28 was significantly decreased in the BC patients’ peripheral blood (Fig. 3A). Based on the ROC curve analysis, circ-ARHGER28 had higher AUC and lower Pvalue in patients with BC (AUC = 0.821, 95% CI = 0.757–0.887, P = 0.0001) (Fig. 3B). These results indicated that circ-ARHGER28 was a specific circRNA in BC. To determine the diagnostic values of circ-ARHGER28 for BC, ROC curve analysis was performed. The AUC value of circ-ARHGER28 for diagnosis of ERpositive BC was 0.648(P = 0.031), early TNM stages I and II were 0.687(P = 0.017), 0.649(P = 0.007), respectively. Compared with CA153, the AUC value of circ-ARHGER28 for the diagnosis of tumor metastasis was 0.742(P = 0.001) (Fig. 3 CH). These results indicated that circ-ARHGER28 could be a potential biomarker for BC diagnosis and treatment.

Bioinformatic analysis of circ-ARHGER28. With MiRanda and RNAhybrid software analysis, 56 microRNAs being identified as potential target ceRNA of circ-ARHGER28(Fig. 4A). Based on the overlapping information of the two databases, 8 miRNAs were identified (Fig. 4B). The gene function and related signaling pathway analysis showed that these miRNAs were associated with ‘cell cycle’, ‘cell proliferation’ and ‘apoptosis’ (Fig. 4C and D). These results suggested that circ-ARHGER28 was associated with cell proliferation and apoptosis and may intervene with tumor occurrence and development through “sponging” to its specific miRNAs.

Circ-ARHGER28 influence MCF-7 cells proliferation and migration. To explore the biological function of circ-ARHGER28 in the regulation of BC cells, an circ-ARHGER28 over-expression lentiviral vector (pEX-circ) was constructed and transfected into MCF-7 cells. RTqPCR results showed that the expression of circ-ARHGER28 was significantly up-regulated in the pEX-circ group compared with the control and NC groups (P < 0.05) (Fig.S1A and B). Cell viability and colony formation assays revealed that over-expression of circ-ARHGER28 inhibited the proliferative and colony formation abilities of MCF-7 cells (Fig. 5A and B). Wound healing assay demonstrated that over-expression of circ-ARHGER28 inhibited the migration of MCF-7 cells (Fig. 5C). Flow cytometry proved that over-expression of circ-ARHGER28 increased the number of G₂/M phase (P < 0.05) and the apoptotic rate of MCF-7 cells(P < 0.05) (Fig. 5D and E). Collectively, these results suggested that over-expression of circ-ARHGER28 could inhibit proliferation and migration of MCF-7 cells via inducing cell apoptosis and cell cycle arrest.

circ-ARHGER28 promoted apoptosis of MCF-7 cells via PI3K/AKT signaling pathway. To investigate the underlying mechanism of how circ-ARHGER28 promoted MCF-7 cell apoptosis, miRNA/mRNAseq analysis was performed. Based on the results, 22 genes were up-regulated and 10 genes were down-regulated in response to over-expression circ-ARHGER28. KEGG analysis showed that the specific genes were mainly involved in PI3K/AKT and mTOR signaling pathways (Fig. 6A-C). Additionally, the miRNAseq results showed that there were 28 up-regulated miRNAs and 5 down-regulated miRNAs in response to circ-ARHGER28 over-expression (Fig. S1 C). Bioinformation analysis demonstrated that the miRNAs were also associated with ‘apoptosis’, ‘PI3K/AKT and mTOR’signaling pathway(Fig. S1 D and E).

RTqPCR and western blot were performed to detect the expression of apoptosis and PI3K/AKT/mTOR signaling pathway associated genes and proteins. The results showed that the expression of PI3K, AKT, mTOR and Bcl-2 were down-regulated, whereas PARP-1, caspase-3, caspase-8 and caspase-9 were up-regulated in pEX-circ group(Fig. 6D). The protein expression of cleavedcaspase 3, cleavedcaspase 8, cleavedcaspase 9, PARP-1 and BAX/Bcl2 ratio were significantly increased, whereas PI3K, AKT and mTOR were significantly down-regulated in pEX-circ group (Fig. 6E and F).

circRNA is a type of noncoding RNA which has a closed continuous loop and lack of 3’ and 5’ ends. circRNA often acted as ceRNA or miRNA “sponge” to regulate mRNA expression and involved in mulitiple biological process [10–11]. Previous study indicated that circRNA expression has tissuespecific and occurs stably in most types of cancer tissues and plasma [12]. Due to the development of circRNA research, the abnormal expression of circRNAs in tumorigenesis and tumor development has become one of the most important scientific discoveries [13]. Yuan et al.[14] showed that hsa_circ_0068033 was down-regulated and acted as a tumor suppressor gene in BC. Li et al.[15]identified that hsa_circ_0104824 may serve as a promising predictive biomarker and therapeutic target for patients with BC. Lei et al.[16] reported that circIFI30 could promote triplenegative breast cancer (TNBC) progression and may become a novel diagnostic marker and therapeutic target for patients with TNBC.

BC was one of the most commonly occurring types of cancer that was associated with high mortality and morbidity rates in women. For patients with BC, early diagnosis and determining the state of differentiation are crucial for effective treatment of the disease [17]. Due to the specific molecular structure and its high expression in BC cells and tissues, circRNAs is considered to be an ideal biomarker and target for BC diagnosis and treatment [18]. Zhang et al.[19] reported that circUBR1 was up-regulated in BC. Decrease of circUBR1 led to an inhibition of BC cells proliferation, invasion while promoted the cells apoptosis in vitro. Misir et al.[20] found that miRNA associated circRNAs may have the ability to discriminate patients with BC from healthy subjects, the results makes it possible to become a potentially tumor predictive biomarkers.

In this study, circRNA microarray analysis was used to screen the profiling of differentially expressed circRNAs in BC. circ-ARHGER28 being verified Among them. The stablity expression of circ-ARHGER28 in BC patients’ tissues and peripheral samples made it became a candidate circRNA for research [21]. The bio-informatics analysis revealed that circ-ARHGER28 was encoded by the ARHGEF28 gene and located in sequence positions73128162-73136585 of the sense strand on human chromosome 5. Previous study have indicated that ARHGEF28 gene was involved in cell adhesion formation, cell motility, aggregation and apoptosis through regulating integrin and growth factor receptor[22]. Another study reported that circ-ARHGER28 was found in the cerebellum and in Hela and HepG2 cells [23]. However, there are no research identified that circ-ARHGER28 has any role in BC diagnostic and proliferation.

To illustrate the putative role of circ-ARHGER28 in BC, the distribution and association of circ-ARHGER28 with clinical features of BC were analyzed. The results revealed that the expression of circ-ARHGER28 in peripheral blood was negatively correlated with CA153 (P < 0.05). CA153 is one of the most important specific biomarkers of breast cancer. It is commonly used for the diagnosis, metastasis, and monitoring of postoperative recurrence of breast cancer [24]. About 30–60% of BC patients show an increase of CA153, and this increase may be up to 80% in cases of metastatic BC[25].Therefore, circ-ARHGER28 may be used as a collaborative monitoring index for CA153, and improve the diagnostic accuracy of CA153. In addition, the expression of circ-ARHGER28 differed significantly according to tumor staging, ERpositivity, and tumor metastasis (P < 0.05). Circ-ARHGER28 had a high diagnostic accuracy in BC patients’ peripheral blood as far as monitoring ERpositivity, tumor metastasis and tumor staging.

circRNA often exert their role in tumor occurrence and development through binding their target miRNAs in a “sponging” process. A recent study has shown that CircBCAR3 could promote the proliferation, migration, invasion, and ferroptosis of esophageal cancer cells through targeting miR-27a-3p [26]. Furthermore, it has been reported that circ-ABCB10 plays a crucial role in promoting the progression of gastric cancer by targeting the miR-1915-3p/Rac1 axis [27]. In this study, 8 possible circ-ARHGER28 target miRNAs were identified followed by target miRNAs predictive analysis. The biological processes of these 8 miRNAs were all associated with cell cycle, proliferation and apoptosis. The results confirmed that circ-ARHGER28 may affect BC cells proliferation and metastasis by targeting its miRNA.

To verify the biological functions of circ-ARHGER28 in the regulation of MCF-7 cells, circ-ARHGER28 over-expression lenti-viral vector (pEX-circ) was constructed and transfected into MCF-7 cells. CCK-8, colony formation and flow cytometry assay revealed that over-expression of circ-ARHGER28 inhibited BC cell proliferation and migration. Meanwhile, increasing of circ-ARHGER28 also arrested cell cycle in G₂/M phase and induced BC cells apoptosis. The miRNA/mRNAseq analysis results showed that circ-ARHGER28 was closely associated with apoptosis and PI3K/AKT/mTOR signaling pathway. PI3K/AKT/mTOR signaling pathway is a wellknown pathway that is involved in the regulation of cell survival and apoptosis [28]. PI3K is an intracellular phosphatidylinositol kinase and AKT is a serine/threonine kinase. Phosphating of PI3K activates AKT, which regulates several downstream molecules, including mTOR [29, 30]. Somatic mutations of PI3K/Akt/ mTOR related genes can induce the activation of constituent pathways in various types of cancer, affecting the proliferation, differentiation, apoptosis and other functional disorders of tumor cells[31]. In this study, RTqPCR and western blot results suggested that circ-ARHGER28 may activate of BC cells apoptosis through regulated by PI3K/AKT/mTOR signaling pathway.

In conclusion, the present study proved that circ-ARHGER28 was lowly expressed in BC patients’ tissues and peripheral blood. circ-ARHGER28 may serve as a promising predictive biomarker for BC diagnosis and treatment. Meanwhile, over-expression of circ-ARHGER28 inhibited proliferation, migration and induced apoptosis of MCF-7 cells. The function of circ-ARHGER28 was associated with the PI3K/AKT/mTOR signaling pathway. The specific mechanisms of circ-ARHGER28 involved in BC proliferation needing further study.

Ethics approval and consent to participate

The study protocol was reviewed and approved by the General Hospital of Ningxia Medical University Ethics Committee (No.2018-118). Informed consent was obtained from all individual participants included in the study.

Availability of data and materials

We guarantee the authenticity and validity of all data and results. Open up some of the raw data uploaded as supplementary files.

Conflict of Interest

The authors declare that there are no conflicts of interest exist in the paper.

Acknowledgments

All authors contributed to the study. Li-bin Wang designed the project, revised the manuscript and coordinated all aspects of work. Xiang Tao participated in all experiment and wrote manuscript. Li Na & Xu Zhang screened circRNA expression profiling in peripheral blood specimens, En-xi Hu do ROC curve analysis. Li-gang Wu collected tissue and peripheral blood samples and extracted total RNA and cDNA. Jia Wang prepared reagent and instrument. All authors read and approved the manuscript for publication.

This work was supported by grants of the National Natural Science Foundations of China (No.81860470, 82260610); The Ningxia Natural Science Foundation (No. 2023AAC02059, 2021AAC0339); The fifth group of Ningxia Young Scientific and Technological Talents lifting Project (NXKJTJGC2020080).

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No competing interests reported.

TableS1PrimersequencesofquantitativerealtimePCR.docx
FigS1.tiff
Figure S1. MicroRNAs sequencing profiles between circ-ARHGER28 over-expression and NC in MCF-7 cell line. (A) Validation of lentiviral vector transfection efficiency in MCF-7 cells. (B) The efficacy of circ-ARHGER28 over-expression was analyzed by qRT-PCR. (C) Volcano plot of the targets miRNA, different miRNAs means fold-change ≥2 and a P-value <0.05, higher expression levels are indicated by “red”, lower expression levels are indicated by “green”, no significant difference is indicated by “black”. (D) The enriched GO and terms of miRNA targets. (E) The enriched KEGG pathway of targets miRNA.

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Clinical diagnostic value of circ-ARHGER28 for breast cancer and its effect on MCF-7 cell proliferation and apoptosis

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Objective

Methods

Results

Conclusion

Figures

Introduction

Materials and methods

Results

Discussion

Declarations

References

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1