Circular RNA RHOT1 promotes progression and inhibits ferroptosis via mir-106a-5p/STAT3 axis in breast cancer

To explore the effect of circRHOT1 on breast cancer progression and the underlying mechanism. Significantly, our data revealed that the depletion of circRHOT1 was able to repress the proliferation and induce the apoptosis of breast cancer cells. CircRHOT1 knockdown could remarkably inhibit the invasion and migration in the breast cancer cells. Meanwhile, the depletion of circRHOT1 enhanced the erastin-induced inhibition effect on cell growth of breast cancer cells. The circRHOT1 knockdown notably increased the levels of reactive oxygen species (ROS), iron, and Fe2+ in breast cancer cells. Mechanically, circRHOT1 was able to sponge microRNA-106a-5p (miR-106a-5p) and inhibited ferroptosis by down-regulating miR-106a-5p in breast cancer cells. Besides, miR-106a-5p induced ferroptosis by targeting signal transducer and activator of transcription 3 (STAT3) in the system. Moreover, the overexpression of STAT3 and miR-106a-5p inhibitor could reverse circRHOT1 knockdown-mediated breast cancer progression. Functionally, circRHOT1 promoted the tumor growth of breast cancer in vivo. In conclusion, we discovered that circRHOT1 contributed to malignant progression and attenuated ferroptosis in breast cancer by the miR-106a-5p/STAT3 axis. Our finding provides new insights into the mechanism by which circRHOT1 promotes the development of breast cancer. CircRHOT1 and miR-106a-5p may serve as potential targets for breast cancer therapy.


INTRODUCTION
Breast cancer is the most frequent lethal cancer in women globally [1]. Despite the advancement of various treatments, breast cancer is still the second principal reason for tumor-related death in women [2]. Adjuvant therapy, radical surgery, and early diagnosis improve patients' survival times and prognosis with breast cancer, but mortality incidence is still unsatisfactory [3]. To develop the practical therapeutic strategy, the comprehensive understanding of the molecular signalings in breast cancer pathogenesis is urgently required [4]. Ferroptosis serves as a distinctive type of Regulated cell death (RCD) and is identified initially when a small molecule called erastin restrains the RCD process, especially in RAS-mutated cancer cells [5]. It has been identified that the inhibition of ferroptosis can enhance cancer progression, and ferroptosis plays a crucial role in breast cancer development [6][7][8]. However, the molecular mechanism of the ferroptosis process in breast cancer progression remains elusive.
With the rapid development of next-generation sequencing technology, circular RNAs (circRNAs), as a crucial sort of regulatory RNAs that forms a loop structure without 5′-3′ polyadenylated or polarities tails, have been identified [9]. CircRNAs demonstrate essential role in the modulation of breast cancer progression in various investigations [10,11]. For example, it has been reported that circABCB10 contributes to progression and proliferation of breast cancer cells by targeting miR-1271 [12]. Circ001783 is able to modulate the malignant development of breast cancer through regulating miR-200c-3p [13]. Moreover, circRHOT1 has been reported to promote cancer AGING progression in multiple cancer models, such as liver cancer and pancreatic cancer [14,15]. It has been identified that circRHOT1 is abnormally expressed in the and promotes the proliferation, migration, and invasion, and inhibits apoptosis of cancer cells [15]. However, the role of circRHOT1 in breast cancer development remains unreported. Accordingly, we were interested in the effect of circRHOT1 on breast cancer.
MicroRNAs (miRNAs) have been well-recognized to regulate gene expression in various biological processes, such as invasion, migration, apoptosis, and proliferation [16]. Multiple investigations have demonstrated miRNAs are involved in the regulation of breast cancer. For instance, it has been reported that miR-183-5p reduces apoptosis and contributes to proliferation of human breast cancer by regulating the PDCD4 [17]. MiR-802 inhibits the proliferation of breast cancer by targeting FoxM1 [18]. Meanwhile, a recent study shows that miR-106a-5p as a tumor suppressor in breast cancer progression [19]. Moreover, STAT3 is a well-identified oncogenic regulator in breast cancer [20]. We identified the potential interaction of circRHOT1 with miR-106a-5p, miR-106a-5p with STAT3 in the bioinformatic analysis. However, the correlation of circRHOT1 with miR-106a-5p and STAT3 in the development of breast cancer is still elusive.
In this study, we focused on the exploration of the role and the underlying mechanism of circRHOT1 in the development of NSCLC. We identified a novel function of circRHOT1 promoting malignant progression and inhibiting ferroptosis in breast cancer by regulating the miR-106a-5p/STAT3 axis.

CircRHOT1 promotes proliferation and inhibits apoptosis of breast cancer cells
To assess the potential function of circRHOT1 in the modulation of breast cancer progression, the human breast cancer MDA-MB-231 and T47D cells were treated with control shRNA or circRHOT1 shRNA, and the efficiency of circRHOT1 depletion was validated in the cells ( Figure 1A). MTT assays revealed that the depletion of circRHOT1 inhibited the cell viability of www.aging-us.com 8117 AGING cells were treated with control shRNA or circRHOT1 shRNA. (B, C) The cell viability was measured by the MTT assays in the cells. (D, E) The cell proliferation was analyzed by the colony formation assays in the cells. (F, G) The cell apoptosis was assessed by flow cytometry analysis in the cells. Data are presented as mean ± SD. Statistic significant differences were indicated: ** P < 0.01. the MDA-MB-231 and T47D cells ( Figure 1B, 1C). Similarly, the colony formation was reduced by the circRHOT1 knockdown in the MDA-MB-231 and T47D cells ( Figure 1D, 1E). Furthermore, cell apoptosis was enhanced by the circRHOT1 depletion in the cells ( Figure 1F, 1G). Taken, together, these data suggest that circRHOT1 promotes proliferation and inhibits apoptosis of breast cancer cells.

CircRHOT1 enhances invasion and migration of breast cancer cells
Next, we investigated the effect of circRHOT1 on the migration and invasion of breast cancer cells. For this purpose, the MDA-MB-231 and T47D cells were treated with control shRNA or circRHOT1 shRNA, and the efficiency of circRHOT1 depletion was confirmed in the cells (Figure 2A). Transwell assays revealed that the migration and invasion of MDA-MB-231 and T47D cells were significantly repressed by the depletion of circRHOT1 ( Figure 2B, 2C). Similarly, the depletion of circRHOT1 remarkably enhanced the wound healing proportion in the cells ( Figure 2D, 2E), suggesting that circRHOT1 is able to enhance the migration and invasion of breast cancer cells.

CircRHOT1 reduces ferroptosis in breast cancer cells
To assess the role of circRHOT1 in ferroptosis, we analyzed the effect of circRHOT1 on the erastininduced inhibition of cell growth and the intracellular levels of reactive oxygen species, iron, and Fe 2+ , and the expression of GPX4 and SLC7A11, which was the markers for ferroptosis. To this end, the MDA-MB-231 and T47D cells were treated with control shRNA or circRHOT1 shRNA, and the efficiency of circRHOT1 knockdown was validated in the cells ( Figure 3A  The migration and invasion were examined by wound healing assays in the cells. The wound healing proportion was shown. Data are presented as mean ± SD. Statistic significant differences were indicated: ** P < 0.01.

AGING
Meanwhile, the iron levels were increased by circRHOT1 depletion in the cells ( Figure 3D). The circRHOT1 knockdown remarkably enhanced the levels of ROS in the MDA-MB-231 and T47D cells ( Figure  3E). Besides, the depletion of circRHOT1 promoted the accumulation of Fe 2+ in the cells ( Figure 3F). Moreover, the expression of GPX4 and SLC7A11 was attenuated by circRHOT1 knockdown in the cells ( Figure 3G, 3H). Together these indicate that circRHOT1 can reduce ferroptosis in breast cancer cells.

CircRHOT1 inhibits ferroptosis by sponging miR-106a-5p in breast cancer cells
Then, we further explored the mechanism of circRHOT1-mediated breast cancer progression. We identified the potential interaction between circRHOT1 and miR-106a-5p in the bioinformatic analysis by using ENCORI (http://starbase.sysu.edu.cn/index.php) ( Figure  4A). Then, we treated the MDA-MB-231 and T47D cells with miR-106a-5p mimic or the control mimic, and the efficiency was verified in the cells ( Figure 4B). The miR-106a-5p mimic remarkably reduced the luciferase activities of circRHOT1 but failed to affect circRHOT1 with the miR-106a-5p-binding site mutant ( Figure 4C). Meanwhile, the depletion of circRHOT1 significantly enhanced the expression of miR-106a-5p in the MDA-MB-231 and T47D cells ( Figure 4D). Moreover, the miR-106a-5p inhibitor could rescue circRHOT1 depletion-inhibited cell growth in the erastin-treated MDA-MB-231 and T47D cells ( Figure  4E, 4F). Similarly, miR-106a-5p inhibitor reversed circRHOT1 depletion-enhanced levels of iron and ROS in the system ( Figure 4G-4I). In addition, the circRHOT1 knockdown-reduced expression of GPX4 and SLC7A11 was enhanced by miR-106a-5p inhibitor ( Figure 4J). Taken together, these data results suggest that circRHOT1 inhibits ferroptosis by sponging miR-106a-5p in breast cancer cells.

MiR-106a-5p induces ferroptosis by targeting STAT3 in breast cancer cells
Then, we further explored the role of miR-106a-5p in the modulation of ferroptosis. We identified the miR-106a-5p-targeted site in STAT3 3' UTR in a bioinformatic analysis by using Targetscan (http://www.targetscan.org/vert_72/) ( Figure  5A). Significantly, the miR-106a-5p mimic treatment attenuated luciferase activities of wild type STAT3 but AGING failed to affect the STAT3 with the miR-106a-5pbinding site mutant in the MDA-MB-231 and T47D cells ( Figure 5B). Besides, the mRNA and protein expression of STAT3 were significantly repressed by miR-106a-5p mimic in the cells ( Figure 5C, 5D), suggesting that miR-106a-5p is able to target STAT3 in the breast cancer cells. Meanwhile, the depletion of circRHOT1 significantly reduced the expression of STAT3, in which miR-106a-5p inhibitor could reverse this effect ( Figure 5E). Furthermore, the overexpression of STAT3 could rescue miR-106a-5p mimic-inhibited cell growth in the erastin-treated MDA-MB-231 and T47D cells ( Figure 5F). Similarly, STAT3 overexpression reversed miR-106a-5p mimic-enhanced levels of iron and ROS in the system ( Figure 5G, 5H). In addition, the miR-106a-5p mimic-reduced expression of GPX4 and SLC7A11 was enhanced by the overexpression of STAT3 ( Figure 5I). Together these data suggest that miR-106a-5p induces ferroptosis by targeting STAT3 in breast cancer cells.

CircRHOT1
contributes to breast cancer progression by miR-106a-5p/STAT3 axis Next, we further investigated the role of circRHOT1/miR-106a-5p/STAT3 axis in the modulation of breast cancer progression. As expected, the overexpression of STAT3 or miR-106a-5p inhibitor could enhance the circRHOT1 knockdown-reduced cell viability in the MDA-MB-231 and T47D cells ( Figure  6A, 6B). Meanwhile, the cell apoptosis was enhanced by the depletion of circRHOT1, in which the overexpression of STAT3 or miR-106a-5p inhibitor could reverse this effect in the MDA-MB-231 and T47D cells ( Figure 6C, 6D). Taken together, these data suggest that circRHOT1 contributes to breast cancer progression by miR-106a-5p/STAT3 axis.

CircRHOT1 promotes the tumor growth of breast cancer in vivo
We further investigated the impact of circRHOT1 on the breast cancer development in vivo.  Figure 7C). Besides, the expression of miR-106a-5p was increased but the expression of STAT3 was decreased by the circRHOT1 depletion in the tumor tissues of the mice ( Figure 7D, 7E). Together these indicate that circRHOT1 promotes the tumor growth of breast cancer in vivo.

DISCUSSION
Breast cancer is the prevailing women cancer affecting modern people and resulting in high mortality [1]. CircRNAs have been identified to exert crucial functions in the modulation of breast cancer [12]. Nevertheless, the role of circRHOT1in the development of breast cancer is still unreported. In this study, we firstly identified that circRHOT1 could inhibit ferroptosis and promote the progression of breast cancer by modulating the miR-106a-5p/STAT3 axis.
Previous studies have identified several circRNAs in the regulation of breast cancer pathogenesis. It has been reported that circ100219 enhances the progression of breast cancer by sponging miR-485-3p [21]. CircMTO1 represses cell viability of and attenuates monastrol resistance by modulating the TRAF4/Eg5 signaling in breast cancer [22]. Circ0052112 increases invasion and migration of breast cancer cells by targeting miR-125a-5p [23]. Circ0001982 enhances the carcinogenesis of breast cancer by inhibiting miR-143 [24]. Circ0072309 suppresses invasion and proliferation by regulating miR-492 in breast cancer [25]. Moreover, it has been found that circRHOT1 contributes to the malignant AGING progression of liver cancer by inhibiting NR2F6 expression [14]. The elevated circRHOT1 enhances invasion and proliferation of pancreatic cancer cells [15]. In this study, we demonstrated that circRHOT1 promoted proliferation and invasion, and migration, and inhibited apoptosis and ferroptosis in the breast cancer cells. CircRHOT1 enhanced the tumor growth of breast cancer in vivo. Our data elucidate a novel role of circRHOT1 in the breast cancer progression, providing crucial evidence of the function of circRNAs in modulating breast cancer. Our data are consistent with the previous reports that circRHOT1 is able to promotes proliferation and invasion, and migration, and inhibit apoptosis of cancer cells. Besides, we firstly identified that circRHOT1 can attenuate ferroptosis of breast cancer, presenting a novel function of circRHOT1 in cancer progression.
MiRNAs play crucial function in breast cancer progression. It has been reported that miR-135a enhances invasion and migration of breast cancer cells by regulating HOXA10 [26]. MiR-421 is able to target PDCD4 and regulate cell proliferation of breast cancer [27].
The inhibition of miR-141 regulates cell invasion and migration in breast cancer cells by targeting ANP32E [28]. MiR-218 represses invasion and migration of breast cancer cells by Slit2/Robo1 signaling [29]. Moreover, it has been reported that miR-106a-5p/SCN3A axis is involved in long noncoding RNA HOXA-AS2-regulated breast cancer [19]. Our mechanical investigation revealed that circRHOT1 repressed ferroptosis by sponging miR-106a-5p and miR-106a-5p induced ferroptosis by targeting STAT3 in the breast cancer cells. The overexpression of STAT3 and miR-106a-5p inhibitor reversed circRHOT1 depletion-inhibited proliferation and circRHOT1 depletion-enhanced apoptosis of breast cancer cells. These data uncover an unreported correlation of circRHOT1 with miR-106a-5p and STAT3 and provide new evidence that circRHOT1/miR-106a-5p/STAT3 signaling is critical for the modulation of breast cancer.

CONCLUSIONS
In conclusion, we discovered that circRHOT1 contributed to malignant progression and attenuated AGING ferroptosis in breast cancer by the miR-106a-5p/STAT3 axis. Our finding provides new insights into the mechanism by which circRHOT1 promotes the development of breast cancer. CircRHOT1 and miR-106a-5p may serve as potential targets for breast cancer therapy.

MTT assays
The cell proliferation was tested by MTT assays in the MDA-MB-231 and T47D cells. Sgotly, about 2×10 5 cells were plated in 96-well plates and incubated for 24 hours. To assess the cell viability, the cells were cultured with the MTT solution (5 mg/mL) and incubated for 4 hours. And then 150 μL DMSO was applied to treat the cells. The cell viability was measured at 570nm absorbance by applying ELISA browser (Bio-Tek EL 800, USA).

Colony formation assays
About 1×10 4 MDA-MB-231 and T47D cells were put into 6-well plates and cultured in DMEM at 37° C. The cells were cleaned with PBS Buffer after 2 weeks, and made in methanol about thirty minutes, and dyed with crystal violet dye at the dose of 1%, after which the number of colonies was calculated.

Transwell assays
Transwell assays was used to analyze the cell migration and invasion of MDA-MB-231 and T47D cells based on a Transwell plate (Corning, USA) according to the manufacturer's guidance. Shortly, the upper chambers were plated with around 1 × 10 5 cells. Then solidified using paraformaldehyde (4%) and dyed using crystal violet. Invaded and migrated cells were recorded and calculated.

Wound healing assay
About 3 × 10 5 MDA-MB-231 and T47D cells were plated into the 24-well plates and incubated overnight to reach a full confluent as a monolayer. A 20μl pipette tip was applied to slowly cut a straight line across the well. Then the well was washed by PBS 3 times and changed with the serum-free medium and continued to culture. The wound healing percentage was calculated.

Analysis of cell apoptosis
About 2×10 5 MDA-MB-231 and T47D cells were plated in the 6-well dishes. Cell apoptosis was assessed by employing the Annexin V-FITC Apoptosis Detection Kit (CST, USA) using the manufacture's instruction. Shortly, about 2×10 5 collected and washed cells collected by binding buffer and were dyed at 25° C, followed by the flow cytometry analysis.

Ferroptosis analysis
The cells were co-treated with 5 mmol/L erastin or ferrostatin (1 mmol/L). The cell viability was analyzed by MTT assays in the MDA-MB-231 and T47D cells. The reactive oxygen species production was measured by flow cytometry analysis in the cells. The levels of iron and Fe 2+ were analyzed by Iron Assay Kit in the cells.

Luciferase reporter gene assay
The luciferase reporter gene assays were performed by using the Dual-luciferase Reporter Assay System (Promega, USA). Briefly, the MDA-MB-231 and T47D cells were treated with the miR-106a-5p mimic, miR-106a-5p mimic or control mimic, pmirGLO-circRHOT1, pmirGLO-circRHOT1 mutant, pmirGLO-STAT3, and pmirGLO-STAT3 mutant were transfected in the cells by using Lipofectamine 3000 (Invitrogen, USA), followed by the analysis of luciferase activities, in which Renilla was applied as a normalized control.

Western blot analysis
Total proteins were obtained from the mice tissues or cells with RIPA buffer (CST, USA). Protein concentrations were analyzed by applying the BCA Protein Quantification Kit (Abbkine, USA). Same concentration of protein was divided by SDS-PAGE (12% polyacrylamide gels), transferred to PVDF membranes (Millipore, USA) in the subsequent step. The membranes were hindered with 5% milk and hatched overnight at 4° C with the primary antibodies for STAT3 (Abcam, USA), GPX4 (Abcam, USA), SLC7A11 (Abcam, USA), and β-actin (Abcam, USA), in which β-actin served as the control. Then, the corresponding second antibodies (Abcam, USA) were used for hatching the membranes 1 hour at room temperature, followed by the visualization by using an Odyssey CLx Infrared Imaging System.

Analysis of tumorigenicity in nude mice
The effect of levobupivacaine on tumor growth of breast cancer in vivo was assessed in the Balb/c nude mice (male, 4-week-old) (n=5). About 1×10 7 cells MDA-MB-231 cells transfected with control shRNA or circRHOT1 shRNA were subcutaneously injected into the mice. After 5 days of injection, we measured tumor growth every 5 days. We sacrificed the mice after 30 days of injection, and tumors were scaled. Tumor volume was observed by estimating the length and width with calipers and measured with the method × 0.5. Animal care and method procedure were authorized by the Animal Ethics Committee of Beijing Friendship Hospital.

Statistical analysis
Data were expressed as mean ± SD, and the statistical analysis was conducted using GraphPad prism 7. The unpaired Student's t-test was used to compare two groups, and the one-way ANOVA was used to compare among multiple groups. P < 0.05 were considered as statistically significant.