Circular RNA AGFG1 motivates breast cancer cell proliferation, invasion, migration, and glycolysis by controlling microRNA-653-5p/14-3-3 protein epsilon

A recent Pairwise meta-analysis confirmed that circular RNA AGFG1 (circAGFG1) is abnormally highly expressed in breast cancer (BC) and may be associated with death risk. The purpose of this study was to elucidate the biological role of circAGFG1 in BC and to explore its potential downstream molecular mechanisms. CircAGFG1, miR-653-5p and YWHAE expression in BC tissues and cells were analyzed by RT-qPCR or western blot. Gene expression was regulated by transfection of plasmids or oligonucleotides and the biological behaviors of BC cells were analyzed by a series of assays. The ring structure of circAGFG1 was analyzed by RNase R and actinomycin D treatment. Dual luciferase reporter assay and RNA-pull down were used to verify the targeting relationship of circAGFG1 and downstream factors. A nude mouse xenograft experiment was performed to verify the effect of circAGFG1 on cancer cells in vivo . The results showed that circAGFG1 and YWHAE were highly expressed in BC while miR-653-5p was lowly expressed. Both circAGFG1 and YWHAE had a targeting relationship with miR-653-5p. Knockdown of circAGFG1 inhibited BC cell proliferation, invasion, migration, and glycolysis. The inhibitory effect of circAGFG1 knockdown on BC was reversed by silencing miR-653-5p. The inhibitory effect of overexpression of miR-653-5p on malignant behaviors of BC cells was reversed by overexpression of YWHAE. Knock-down of circAGFG1 inhibited tumor growth in vivo . Taken together, these data suggest that circAGFG1 acts as a sponge for miR-653-5p to mediate YWHAE expression to promote the malignant behaviors of BC.


INTRODUCTION
Breast cancer (BC) is a malignant tumor in the mammary gland epithelial tissue, which seriously threatens the survival and health of women worldwide (Wang et al., 2021).Surgery, endocrine therapy, chemotherapy, radiotherapy and targeted therapy are applicable for BC treatment to reduce the mortality rate (Li et al., 2021).However, due to high recurrence and easy metastasis, patients' prognosis is still poor, and the mortality rate is still high (Xie et al., 2021).Hence, an in-depth study of the latent molecular mechanisms of BC is crucial to provide new and effective therapeutic targets.
Circular RNAs (circRNAs) are a cluster of closed circular RNAs (Li et al., 2019).A study has shown that circRNAs are available to control gene expression and the biological behavior of cancer cells (Wu et al., 2019).Meanwhile, multiple circRNAs have been revealed to take part in regulating BC progression (Zhang et al., 2021;Qi et al., 2021;Cui et al., 2021).CircAGFG1 is a novel circRNA that has been confirmed to be elevated in BC and may be implicated in the diagnosis and prognosis of BC (Ma et al., 2021).However, it is unclear whether circAGFG1 affects the biological behavior of BC cells.circRNAs can perform as competing endogenous RNAs (ceRNAs) for microRNAs (miRNAs) to control downstream targets (Chu et al., 2021).In the light of the ceRNA theory, it predicted a miRNA (miR-653-5p) associated with circAGFG1.Recently, miR-653-5p has been tested to involve diversified cancers, such as non-small cell lung cancer (Han et al., 2019) and gastric cancer (Li et al., 2021).However, it is necessary to further elucidate other downstream target genes regulated by miR-653-5p in BC.
YWHAE, also known as 14-3-3ε, is a member of the YWHA protein family (Wu et al., 2021).YWHAE which is extensively expressed in most cancers is a transcription factor [ (Li et al., 2021).Previous studies have shown that YWHAE is elevated in BC and can motivate the proliferation, metastasis and chemotherapy of BC cells, and may become a latent therapeutic target for BC (Leal et al., 2016;Cimino et al., 2008;Park et al., 2019).
This study hypothesized that circAGFG1 might indirectly regulate YWHAE expression by adsorbing miR-653-5p to participate in BC cell biological behaviors.Our aim was to explore the biological function of cir-cAGFG1 in BC and to elucidate its downstream underlying mechanisms.
pital of Kunming Medical University and written informed consent was collected from all participants.

Ribonuclease R (RNase R) and actinomycin D
To test RNase R resistance, total RNA (2 μg) from MDA-MB-231 cells was collected and incubated with 3 U/μg RNase-R (07250, Epicentre Technologies, USA) at 37°C for 30 min.Actinomycin D experiment: MDA-MB-231 cells were treated with 2 mg/mL actinomycin D (129,935, Millipore, USA) to block transcription.Then, cellular RNA was obtained to measure circAGFG1 and AGFG1 expression by RT-qPCR.

Subcellular localization analysis
Cytoplasmic and nuclear RNAs of MDA-MB-231 cells were isolated using the PARIS Kit (Invitrogen).Then, circAGFG1 in cytoplasmic RNA and nuclear RNA was checked by RT-qPCR.U6 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were applied as nuclear and cytoplasmic controls, respectively.

RT-qPCR
Total RNA was extracted from BC tissues and cells using Trizol reagent (Invitrogen) PrimeScript RT reagent kit (Takara, Tokyo, Japan) and miRNA First Strand Synthesis kit (Takara, Japan) were employed for reverse transcription of circRNA/mRNA and miRNA, respectively.RT-qPCR was performed with SYBR Green kit (Thermo Fisher Scientific, MA, USA) and Mx3005P QPCR system (Agilent Technologies, Santa Clara, CA, USA).U6 and GAPDH were employed as internal controls for miRNA and mRNA/circRNA, respectively (Ma et al., 2021).The primer sequences were manifested in Table 1.The relative expression of genes was analyzed by 2 -ΔΔCt .

Flow cytometry
Apoptosis rates of transfected MDA-MB-231 and MDA-MB-157 cells were analyzed by Annexin V-Fluorescein isothiocyanate (FITC) propidium iodide (PI) apoptosis kit (BD Biosciences).Briefly, cells were resuspended in 1 × binding buffer (500 μl) and mixed with 5 μl Annexin V-FITC and 5 μl PI in the dark for 15 min.Finally, the percentage of apoptotic cells was evaluated by a flow cytometer (FACS Calibur).

Transwell migration and invasion analysis
MDA-MB-231 and MDA-MB-157 cells were placed in a serum-free medium at a density of 5×10 4 cells/well, and the medium containing 10% fetal bovine serum (Invitrogen) was added to the bottom chamber.Then after 24 h, cells were fixed with methanol for 30 min, stained with 0.1% crystal violet, and viewed in an inverted mi-

RNA-pull down assay
Biotin-labeled miR-653-5p (Bio-miR-653-5p) probe and control probe (Bio-NC) were gained from Ribo-Bio.After transfection with Bio-NC or Bio-miR-653-5p, MDA-MB-231 cells were lysed after 48 h and incubated with Dynabeads M-280 Streptavidin (Invitrogen) at 4°C for 2 h.The RNA complexes on the beads were washed and the enrichment of circAGFG1 and YWHAE was tested by RT-qPCR.

In vivo tumor growth assay
Twelve BALB/c male nude mice (5 weeks old, body weight of 15-20 g) were purchased from Vital River (Beijing, China).MDA-MB-231 cells (5×10 6 cells/mouse) with or without stable knockdown of circAGFG1 were injected subcutaneously into each mouse (6 per group).Tumor volume was calculated in the light of the formula 0.5×length×width 2 and measured every 3 d.After 23 d, the mice were euthanized.Tumor tissues were excised for further analysis.This study was permitted by the Animal Research Committee of The First Affiliated Hospital of Kunming Medical University (Ding et al., 2021).

Statistical analysis
All experiments in this study were biologically replicated at least three times.SPSS 21.0 (SPSS, Inc, Chicago, IL, USA) statistical software was applied for analysis of the data.After the Kolmogorov-Smirnov test, the data were normally distributed and expressed as mean ± standard deviation (S.D.).The two-group comparison was done by t-test, while the multiplegroup comparison was performed by one-way analysis of variance (ANOVA) and Fisher's least significant difference t-test (LSD-t).The chi-square test was used to analyze the correlation between circAGFG1 and the clinicopathological data of patients and Kaplan-Meier analysis to examine the relationship between circAGFG1 and patients' survival P<0.05 was considered statistically significant.

CircAGFG1 is elevated in BC
Firstly, the circAGFG1 expression pattern in BC was detected by RT-qPCR.CircAGFG1 expression was higher in BC tissues compared with normal tissues (Fig. 1A).The link between circAGFG1 and clinicopathological characteristics of BC patients was then analyzed.The elevation of circAGFG1 was associated with tumor-node-metastasis stage, tumor size, lymph  node metastasis and distant metastasis, but not with age (Table 2).Kaplan-Meier survival analysis clarified that the elevation of circAGFG1 was associated with worse overall survival (Fig. 1B).
CircAGFG1 expression was higher in all BC cell lines (MDA-MB-231, MCF-7, SK-BR-3, and MDA-MB-157) than in normal cells (MCF-10A) (Fig. 1C).Among them, MDA-MB-231 cells and MDA-MB-157 cells with the highest CircAGFG1 expression were selected for follow-up experiments.The circular structure of circAGFG1 was confirmed by RNase R and actinomycin D experiments, and it came out that circAGFG1 was resistant to RNase R digestion and was more stable than linear AGFG1 mRNA, and actinomycin D did not affect the stability of circAGFG1 (Fig. 1D, E).Furthermore, subcellular localization analysis clarified that circAGFG1 was majorly distributed in the cytoplasm of MDA-MB-231 cells (Fig. 1F).
The above data suggest circAGFG1 is stably elevated in BC and may take part in BC progression.

Repressing circAGFG1 restrains MDA-MB-231 cell biological behaviors and glycolysis
To figure out circAGFG1's biological function in BC, circAGFG1 was silenced in MDA-MB-231 and MDA-MB-157 cells.CircAGFG1 knockdown efficiency was shown in Fig. 2A.CCK-8 and colony formation experiments showed that the knockdown of circAGFG1 inhibited the proliferation ability of BC cells (Fig. 2B,  C).Flow cytometry showed that the knockdown of cir-cAGFG1 promoted apoptosis in cells (Fig. 2D).Transwell experiments demonstrated that the knockdown of circAGFG1 increased the migratory and invasive abili-ties of cells (Fig. 2E).Most fast-growing malignant cells have active glycolysis and gain more energy through glycolysis (Zhang et al., 2022).Next, the effect of cir-cAGFG1 on glycolysis was explored.It was examined that knockdown of circAGFG1 inhibited cellular glucose consumption, lactate production and ATP/ADP ratio (Fig. 2F-H).Western blot detected that knockdown of circAGFG1 suppressed the protein expression of MMP9 and GLUT1 and promoted the protein expression of Bax (Fig. 2I).The above data indicated that knockdown of circAGFG1 refrains BC cell biological functions and glycolysis.
The above data suggest that circAGFG1 affects BC cell progression via targeting miR-653-5p.
The above data indicate that YWHAE is the downstream gene of miR-653-5p.

Elevation of miR-653-5p depresses BC cell effects via targeting YWHAE
For further investigating the link between miR-653-5p and YWHAE, a functional rescue experiment was performed.YWHAE expression was significantly increased after transfection of the YWHAE overexpression vector, and the inhibitory effect of miR-653-5p mimic on YWHAE was reversed by the YWHAE overexpression vector (Fig. 6A, B).CCK-8 and colony formation experiments showed that the inhibitory effect of overexpression of miR-653-5p on cell proliferation was reversed by overexpression of YWHAE (Fig. 6C, D).In addition, the promoting effect of overexpression of miR-653-5p on apoptosis was reversed by overexpression of YWHAE (Fig.

6E
). Transwell experiments showed that overexpression of miR-653-5p inhibited cell invasion and migration, but overexpression of YWHAE prevented this phenomenon (Fig. 6F).Commercial kit results showed that the inhibitory effects of overexpression of miR-653-5p on cellular glucose consumption, lactate production, and ATP/ADP ratio were reversed by overexpression of YWHAE (Fig. 6G-I).Western blot showed that overexpression of miR-653-5p inhibited the expression of MMP9, GLUT1 and promoted the expression of Bax, and overexpression of YWHAE reversed the changes of these proteins (Fig. 6J).
The above data suggest that miR-653-5p affects BC cell progression by targeting YWHAE.

CircAGFG1 knockdown restrains tumor growth in vivo
To determine whether circAGFG1 silencing restrained tumor growth in vivo, sh-NC or sh-CircAGFG1-transfected MDA-MB-231 cells were implanted into nude mice.sh-CircAGFG1 reduced the tumor volume and weight of mice (Fig. 7A, B).Meanwhile, circAGFG1 knockdown suppressed circAGFG1 and YWHAE expression in tumor tissues, while elevated miR-653-5p expression (Fig. 7C, D).IHC analysis illustrated that repression of circAGFG1 repressed Ki67 and MMP9 expression in tumor tissues (Fig. 7E).All in all, it is suggested that cir-cAGFG1 knockdown restrains BC tumor growth in vivo.

DISCUSSION
BC is an extremely familiar cancer worldwide and a momentous cause of death in women.Its high morbidity, high recurrence rate and unpleasing prognosis are the main reasons of patient death.Hence, in-depth studies of the molecular mechanisms of BC are urgently needed to confirm effective targets for BC therapy.With the in-depth research on regulatory mechanism in cancer, the study direction has gradually shifted from coding RNA to ncRNAs.BC development is complexly controlled by diversified ncRNAs, including circRNAs, and miRNAs (Liu et al., 2021).In the research, it was discovered for the first time that circAG-FG1 combined with miR-653-5p, which targeted YWHAE, thereby facilitating MDA-MB-231 cell biological behaviors and glycolysis in vitro and accelerating tumor growth in vivo.
CircAGFG1 has been confirmed to be elevated in BC and may be implicated in BC patients' diagnosis and prognosis.A study has shown that circRNAs are more stable in tissues and exosomes compared with traditional linear RNAs (He et al., 2021).Likewise, in the research, circAGFG1 expression was elevated in BC, was not easily degraded by RNase R and was abundantly exhibited in the cytoplasm.In addition, this study also found that circAGFG1 elevation was associated with poor pathological features and prognosis of BC patients.CircAGFG1 expression is enhanced in various cancers like esophageal cancer (Fu et al., 2021), cervical cancer, and triple-negative BC (Zhang et al., 2021), and repressing its expression can restrain the malignant progression of cancer.In the research, the knockdown of circAGFG1 restrained BC cell biological behaviors and glycolysis, and depressed tumor growth in vivo.This suggests that circAGFG1 may offer a therapeutic target for BC.It has been reported that cells can secrete circRNAs through exosomes to regulate tumor growth and metastasis.As exampled, in BC, has_circ_0000615 is packaged by exosomes and transported into the circulating blood (Mashouri et al., 2019).Therefore, subsequent studies need to detect BC cell exosomes to detect the role of circAGFG1.
CircRNAs perform as ceRNAs to interact miRNAs.Through bioinformatics analysis, it was found that Cir-cAGFG1 can sponge various miRNAs.Among these miRNAs, miR-653-5p was selected as circAGFG1's target in the research.Elevation of miR-653-5p refrains the proliferation and migration of melanoma (Liu et al., 2020), together with the proliferation of cervical cancer cells (Wu et al., 2020).MiR-653-5p's functions in BC have been initially clarified, and this miRNA is lowly expressed in BC and has anti-tumor effects.In the research, miR-653-5p expression was inhibited in BC.Furthermore, miR-653-5p inhibitor could partially suppress the effects of circAGFG1 silencing on BC cells.
YWHAE acts as a transcription factor to activate or depress the transcription of target genes.For example, targeting YWHAE to activate apoptotic signaling can suppress cutaneous squamous cell carcinoma (Holmes et al., 2021).Previous studies have shown that YWHAE is elevated in BC and motivates the metastasis and chemotherapy of BC cells, and may become a latent therapeutic target for BC.However, the mechanism by which the circAGFG1/miR-653-5p/YWHAE axis affects BC has not been investigated.In the research, it was also found that YWHAE expression was elevated in BC.In addition, it was originally discovered that YWHAE was a downstream gene of miR-653-5p.Meanwhile, YWHAE can integrate multiple signaling pathways by interacting with different proteins in cancer progression (Ou et al., 2021).In addition, YWHAE-NUTM2 has been reported to deregulate both v-raf-leukemia viral oncogene 1/v-Raf murine sarcoma viral oncogene homolog B and Hippo pathways, resulting in elevation of cyclin D1, which accelerates the progression of high-grade endometrial stromal sarcoma.Therefore, it was speculated that YWHAE might interact with NUTM2 protein to integrate signaling pathways, and then participate in the progression of BC, which needs to be further explored in follow-up studies.
Notably, circAGFG1 can affect MMP9, GLUT1, and Bax expression in BC cells by targeting and regulating the miR-653-5p/YWHAE axis.MMP9 can degrade matrix proteins, destroy extracellular matrix, and promote tumor cell invasion and metastasis (Augoff et al., 2022).In addition, high GLUT1 expression is associated with metabolic reprogramming of tumor cells, including increased glucose uptake, lactic acid production, and acidified microenvironment (Pezzuto et al., 2020).Bax activation can increase the permeability of the mitochondrial outer membrane and release apoptosis signaling molecules such as cytochrome C in mitochondria, thus inducing apoptosis (Edlich et al., 2018).In this study, we speculate that circAGFG1's function in promoting BC cell invasion and migration is significantly related to the increased expression of MMP9.In addition, circAGFG1 can increase the glycolytic capacity of BC cells by promoting GLUT1 expression, which will provide powerful energy for the proliferation and metastasis of BC cells.The inhibitory effect of circAGFG1 on Bax may reduce the permeability of the mitochondrial membrane of BC cells, thereby preventing the release of apoptosis signaling molecules, which will help to reduce the apoptosis rate of cancer cells.
However, this study is limited in that the effect of circAGFG1 in multiple models of each BC subtype requires further investigation.In addition, the mechanism of YWHAE regulating BC remains to be further explored.Future multicenter trials are required to illustrate circAGFG1's function in BC.

CONCLUSION
Taken together, this study demonstrates that circAG-FG1 targeted regulation of YWHAE expression via acting as a sponge of miR-653-5p, thereby promoting BC progression.CircAGFG1/miR-653-5p/YWHAE axis may offer as a latent therapeutic target for BC.

Figure 1 .
Figure 1.CircAGFG1 is elevated in BC (A) RT-qPCR detection of circAGFG1 in normal and tumor tissues.(B) Kaplan-Meier analysis of survival prognosis of BC patients.(C) RT-qPCR detection of circAGFG1 expression in four BC cell lines (MDA-MB-231, MCF-7, SK-BR-3, and MDA-MB-157) and normal breast cell line (MCF-10A) (D/E) The ring structure of circAGFG1 was determined by RNase R and Act D methods.(F) The subcellular localization of circAGFG1 in MDA-MB-231 cells.Measurement data were clarified as mean ± S.D. (N=3); *P<0.05.

Figure 2 .
Figure 2. circAGAP1 regulates BC cell proliferation, invasion and migration, glycolysis and apoptosis sh-circAGFG1 was transfected into MDA-MB-231 and MDA-MB-157 cells.(A) RT-qPCR detection of circAGFG1 knockdown or overexpression transfection efficiency.(B/C) Cell proliferation activity was tested by the CCK-8 method and colony formation assay.(D) Cell apoptosis detected by flow cytometry.(G) Transwell assay to measure cell migration and invasion.(F-H) glucose consumption, lactate production, and cellular ATP/ADP ratio.I.Western blot detection of GLUT1, Bax, and MMP9.Measurement data were clarified as mean ± S.D. (N=3); *P<0.05.

Figure 7 .
Figure 7. Deletion of circAGFG1 restrains tumorigenesis in vivo (A) Changes in tumor volume in mice.(B) The tumor weight of mice was detected after 23 days.(C) RT-qPCR to test circAGFG1 and miR-653-5p in tumor tissues.(D) Western blot to test YWHAE in tumor tissue.(E) Ki67 and MMP9 in tumor tissue detected by IHC.Measurement data were clarified as mean ± S.D. (n=6); *P<0.05.