RBM8A Depletion Decreases the Cisplatin Resistance and Represses the Proliferation and Metastasis of Breast Cancer Cells via AKT/mTOR Pathway

Background Breast cancer (BC) is the most prevalent malignancy in women. This study is aimed to explore the role and regulatory mechanism of RNA-binding motif protein 8A (RBM8A) in BC. Methods We detected the expression of RBM8A in BC tissues and cell lines (MCF-7, MDA-MB-231, and MDA-MB-436), and explored the correlation of RBM8A expression with clinicopathological features in patients. The function of RBM8A deficiency in MCF-7 and MDA-MB-231 cells was determined using MTT, wound healing, and transwell assay. The effect of RBM8A suppression on the cisplatin (DDP) resistance in MCF-7 and MDA-MB-231 cells was also evaluated. Besides, western blotting was used to examine AKT/mTOR pathway-related proteins. The mouse model was constructed to confirm the effect of RBM8A on tumor growth. Results The expression of RBM8A was elevated in BC tissues and cell lines. RBM8A silencing restrained the malignant behaviors of MCF-7 and MDA-MB-231 cells, including viability, migration, and invasion, while promoting apoptosis. Silencing of RBM8A overcame resistance to DDP in MCF-7 and MDA-MB-231 cells. Furthermore, RBM8A suppression restrained the activation of the AKT/mTOR pathway in both MCF-7 and MDA-MB-231 cells. Feedback experiments revealed that SC79 treatment reversed the reduction effects of RBM8A knockdown on viability, DDP resistance, migration, and invasion of MDA-MB-231 cells. Moreover, the silencing of RBM8A inhibited the growth of tumor xenograft in vivo. Conclusions RBM8A knockdown may reduce DDP resistance in BC to repress the development of BC via the AKT/mTOR pathway, suggesting that RBM8A may serve as a new therapeutic target in BC.


Introduction
Breast cancer (BC) is one of the most frequently diagnosed malignancies among women across the globe [1]. e incidence and mortality of BC are increasing each year [2]. e incidence of BC rises with age until menopause, and BC is more aggressive in younger females [3]. Despite the therapeutic strategies for BC have been improved greatly, patients still suffer from some adverse effects such as poor outcomes and drug-resistance [4]. erefore, it is essential to explore the pathogenesis of BC and find a new and effective therapeutic target for BC treatment.
RNA-binding motif protein 8A (RBM8A) is an RNAbinding motif protein widely expressed in cells and involved in the regulation of cell proliferation, metastasis, apoptosis, and other biological functions [5,6]. In addition, it is involved in various crucial cell signaling pathways and plays an important role in tumorigenesis and development [7,8]. Importantly, abnormal expression of RBM8A is closely associated with a variety of malignancies. For instance, Liang et al. have reported that RBM8A overexpression can enhance the resistance to oxaliplatin in hepatocellular cancer [9]. RBM8A suppression retards the proliferation of mesothelioma cells [10]. In addition, gastric cancer patients with increased RBM8A expression have lower overall survival [11]. However, the function and underlying mechanism of RBM8A in BC is still unknown.
Abundant evidence has indicated that the AKT/mTOR pathway plays a pivotal role in different gynecological cancers via modulating cellular processes and drug resistance. Overexpression of Derlin1 has been shown to promote cell proliferation and migration and induce apoptosis by activating the AKT/mTOR pathway in cervical cancer [12]. e activation of the AKT/mTOR pathway has been reported to reduce cisplatin (DDP)-induced apoptosis, thereby leading to cisplatin resistance in ovarian cancer [13]. Notably, the AKT/mTOR pathway is also involved in the progression of BC. Wei et al. have reported that Magnoflorine retards BC growth and elevates the sensitivity of BC cells to doxorubicin (DOX) treatment by suppressing the AKT/mTOR pathway [14]. Inhibition of the AKT/mTOR pathway restrains the adipocyte-mediated proliferation and migration of BC cells [15]. In addition, Ren et al. have indicated that the activation of the AKT/mTOR pathway is involved in the proliferation, migration, and invasion of BC cells [16]. However, whether RBM8A interacts with the AKT/mTOR pathway in BC remains undefined.
In the current study, the expression of RBM8A was determined in BC and cell lines. e effects of RBM8A on the malignant behaviors and DDP resistance of BC cells were evaluated and further analyzed its expression level with clinicopathological features and prognosis. We then explored whether RBM8A regulates the AKT/mTOR pathway to affect BC progression.
is research may uncover the molecular mechanism of RBM8A in BC and provide a new target for BC treatment.

Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR).
Total RNA was extracted with TRIzol reagent (Invitrogen). cDNA was generated from RNA template using a reverse transcription kit (Invitrogen). qRT-PCR analysis was performed by SYBR ® Premix Ex Tap Reagent kit (TaKaRa, Dalian, China) [17]. β-actin was used for the normalization of RBM8A. e primer sequences are shown in Table 1. e relative expressions were calculated by using 2 −ΔΔCt method.

Flow Cytometry Analysis.
e apoptosis of MCF-7 and MDA-MB-231 cells was evaluated using the Annexin V-FITC apoptosis detection kit ( ermo Fisher Scientific) in accordance with the manufacturer's protocol.       with culture medium supplemented with 10% FBS. e invaded cells were dyed using crystal violet after 24 h incubation, and then, were counted under an inverted microscope (Olympus, Tokyo, Japan). For the measurement of migration, MCF-7 and MDA-MB-231 cells were seeded into the non-coated upper chamber, and the other steps were same as the above described.

Tumor Xenografts in Nude
Mice. Female BALB/c nude mice (6 weeks) were purchased from Esebio (Shanghai, China). All procedures were in accordance with the ethical standards of the institution and approved by the Animal Care and Use Committee of the Second Affiliated Hospital of Xi'an Medical University. MDA-MB-231 cells (5 × 10 6 cells) infected with lentivirus containing sh1-RBM8A or sh-NC were subcutaneously injected into the right axillary region of the mice (n � 6). e tumor volume was measured every five days and calculated with the formula: 1 / 2 LW 2 (L, length; W, width). After 30 days post-cell injection, mice were anesthetized and then sacrificed by cervical dislocation, and tumor weight was tested. In addition, the tumor xenograft was collected for determination of RBM8A protein expression.

Statistical
Analysis. Data were presented as mean-± standard deviation (SD) and analyzed by GraphPad Prism 8.0 statistical software (La Jolla, CA, USA). e differences between two groups (normally distributed) were assessed using Student's t-test. e differences among multiple groups were analyzed by one-way ANOVA followed by Tukey's multiple comparison test. Differences were considered statistically significant at P < 0.05.

RBM8A Expression Is Increased in BC Cells and Tissues.
As shown in Figure 1(a), RBM8A expression in BC tissues was higher than that in paired normal tissues (P < 0.01). We further assessed the correlation between RBM8A expression and clinicopathological features of 70 BC patients ( Table 2). Our results illustrated that the high and low expression of RBM8A showed significant differences in TNM stage (P < 0.01) and lymph node metastasis (LNM) (P < 0.05). Compared with MCF10A cells, RBM8A protein expression was extremely enhanced in MCF-7, MDA-MB-436, and MDA-MB-231 cells (P < 0.01, Figure 1(b)). Additionally, RBM8A expression in MCF-7, MDA-MB-436, and MDA-MB-231 cells was higher than that in MCF10A cells (P < 0.05, Figure 1(c)). Afterward, the transfection efficiency of sh-RBM8A was determined. As shown in Figures 1(d)-1(e), RBM8A expression was remarkably decreased in BC cell lines transfected with sh1-RBM8A (P < 0.01) or sh2-RBM8A (P < 0.05). Similar patterns were observed in the results of RBM8A protein level measured by western blotting (P < 0.05, Figures 1(f )-1(g)). erefore, sh1-RBM8A was chosen for the subsequent experiments due to the relatively high transfection efficiency.

RBM8A Knockdown Reduces Viability and DDP Resistance of BC Cells.
e effect of RBM8A knockdown on cell viability was then explored. As illustrated in Figure 2  e Breast Journal was also inhibited in the sh1-RBM8A group compared with the sh-NC group, suggesting that transfection of sh1-RBM8A enhanced the sensibility of BC cells to DDP (P < 0.01, Figure 2(b)). Additionally, DDP-resistant BC cells were transfected with sh1-RBM8A or sh-NC to further investigate the function of RBM8A in DDP-resistant BC. As demonstrated in Figure 2(c), RBM8A knockdown dramatically reduced the half-maximal inhibitory concentration (IC50) of DDP (P < 0.05).

Silencing of RBM8A Inhibits the Proliferation and
Promotes the Apoptosis of BC Cells. e direct impact of RBM8A on the proliferation and apoptosis of MCF-7 and MDA-MB-231 cells was also investigated. As shown in Figure 3(a), the EdU-positive cells in the sh1-RBM8A group were significantly reduced relative to the sh-NC group (P < 0.01), which confirmed that RBM8A silencing inhibited the proliferation of BC cells. Furthermore, we revealed that the apoptosis rate of BC cells transfected with sh1-RBM8A was remarkably elevated compared to those transfected with sh-NC (P < 0.05, Figure 3(b)).

RBM8A Knockdown Suppresses Migration and Invasion of BC Cells.
e metastasis of BC cells was measured to further assess the role of RBM8A in BC. Wound-healing assay confirmed that RBM8A silencing markedly suppressed the migration of MCF-7 and MDA-MB-231 cells (P < 0.01, Figure 4(a)). Transwell assay also confirmed the inhibitory effect of RBM8A silencing for BC cell migration (P < 0.01, Figure 4(b)). Additionally, transwell assay also   e Breast Journal revealed that RBM8A knockdown could significantly repress the invasion of MCF-7 and MDA-MB-231 cells (P < 0.01, Figure 4(c)).

RBM8A Silencing Represses the Viability, DDP Resistance, and Metastasis of BC Cells via Inhibition of the AKT/mTOR
Pathway. We added the AKT activator SC79 to MDA-MB-231 cells to further verify whether RBM8A regulated the AKT/mTOR pathway in BC. As expected, the addition of SC79 reversed the inhibitory effects of RBM8A silencing on AKT/mTOR pathway-related proteins (P < 0.01, Figure 6

Discussion
BC is a complex and heterogeneous disease [18]. Aberrations in gene expression are linked to BC pathogenesis. At present, the main treatment strategy for BC is surgery, supplemented by chemotherapy [19]. e increasing incidence and drug resistance of BC have put enormous pressure on clinical treatment [20]. erefore, it is necessary to find new BC markers to improve the diagnosis, treatment, and prognosis of BC. In this work, we showed that overexpressed RBM8A promoted tumor cell growth in BC. In addition, RBM8A knockout in BC enhanced DDP sensitivity of BC cells.
Recently, increasing studies have pointed out that RBM8A exerts multiple biological functions. Aberrant expression of RBM8A has been detected in various malignancies, including hepatocellular carcinoma (HCC) and glioblastoma (GBM) [21,22]. Abnormal expression of RBM8A is associated with carcinogenesis. RBM8A promotes tumor cell migration and invasion in HCC by activating the EMT signaling pathway in vitro [21]. Furthermore, it has been reported that RBM8A is upregulated in GBM tissues, and its high expression is correlated with poor prognosis, while knockdown of RBM8A inhibits GBM progression and invasion [22]. Similar to the previous research, RBM8A expression was significantly increased in BC in the present study. RBM8A deletion suppressed BC cell viability, invasion, and migration, and promoted apoptosis. In addition, the high and low expression of RBM8A showed significant differences in TNM stage and LNM of BC. Similarly, LV et al. have reported that RBM8A expression is increased in gastric carcinoma tissues, and the level of RBM8A is correlated with tumor size, LNM, TNM stage, and distant metastasis in gastric cancer [11]. erefore, we speculated that RBM8A may be an oncogene existing in BC to promote the progression of BC, which could be a promising biomarker and therapeutic target in the diagnosis and treatment of BC.
It is well known that DDP resistance is a critical problem in BC treatment, and the resistance to DDP in BC is generally evaluated by comparison of IC50. e repressed IC50 means the attenuated effect on the resistance to DDP of tumor cells [23]. Studies have reported that RBM8A is involved in the regulation of oxaliplatin resistance in HCC [9]. Based on previous research, we speculated that RBM8A may be an effective target to repress the development of BC at the cellular level. In the current study, RBM8A downregulation not only decreased the cell viability as the concentrations of DPP are increased but also repressed the IC50 of DDP in MCF-7/DDP and MDA-MB-231/DDP cells. erefore, we suggested that RBM8A knockdown may provide a therapeutic approach to enhance the sensitivity of BCs to DDP. To further explore the function of RBM8A in BC, the tumor xenograft model was established. As expected, animal experiments showed that RBM8A inhibition retarded the growth of tumor xenograft in mice. Collectively, we believed that the silencing of RBM8A may inhibit BC tumorigenesis via repression of the growth and metastasis of BC cells.
Studies have reported that the Akt/mTOR pathway plays a crucial role in essential cellular activities, such as cell proliferation, growth, and metabolism and found that it is commonly activated in human cancers [24]. e AKT/ mTOR pathway is closely associated with BC tumorigenesis [25]. In this study, RBM8A knockdown suppressed the AKT/mTOR pathway activity in BC cells. e results indicated that RBM8A may interact with the AKT/mTOR pathway. To further confirm this conclusion, SC79 (an activator of AKT) was added to MDA-MB-231 to perform feedback experiments. We demonstrated that SC79 reversed the inhibiting effects of RBM8A knockdown on BC cell viability, invasion, and migration. erefore, we suggested that RBM8A knockdown may retard the progression of BC via repression of the AKT/mTOR pathway. In addition, a growing number of studies have reported that the AKT/ mTOR pathway is also a pivotal pathway involved in drug resistance regulation of BC [26]. For instance, Li et al. have indicated that LncRNA HOTAIR silencing attenuates the resistance of BC cells to DOX by inhibiting the AKT/mTOR pathway [27], suggesting that the AKT/mTOR pathway may be a key pathway to reverse DOX-resistance in BC. Studies have demonstrated that activation of the AKT/mTOR pathway enhances BC resistance to Adriamycin and promotes cancer development [28]. Moreover, Zong et al. have shown that the AKT/mTOR pathway is involved in cisplatin resistance induced by aerobic glycolysis in BC cells [29]. Consistent with previous studies, in the present study, SC79 reversed the inhibitory effect of RBM8A knockdown on DDP resistance in BC, pointing out that RBM8A knockdown may alleviate the resistance of BC cells to DDP by constraining AKT/mTOR pathway. In conclusion, RBM8A expression was up-regulated in BC tissues and cells. RBM8A may regulate proliferation, apoptosis, migration, and invasion, as well as promote DDP resistance, thereby affecting BC progression. Furthermore, RBM8A silencing decreased the DDP resistance in breast cancer cells by inhibiting the AKT/mTOR pathway. Our findings indicated that RBM8A has the potential to become a new therapeutic target for the treatment of BC.

Data Availability
All data generated or analyzed during this study are included within the published article.

Conflicts of Interest
e authors declare that they have no conflicts of interest.