INHBA Promotes Cell Proliferation and Metastasis of Breast Cancer through Wnt/β-catenin Signaling Pathway

Emerging evidences have demonstrated that inhibin subunit beta A (INHBA) is dysregulated and play critical roles in various cancers. With the development of sequencing technology, studies have discovered that INHBA is overexpressed in breast cancer tissues. However, the biological roles of INHBA in breast cancer are still far to clear. In the present study, Methods We analyzed the INHBA expression in the Cancer Genome Atlas (TCGA) database. Quantitative real-time polymerase chain reaction (qRT-PCR) was conducted to assess the expression of INHBA in breast cancer cell lines. Cell proliferation, invasion and epithelial-mesenchymal transition (EMT) were determined by using CCK-8, EdU, Transwell and western blot assays. Total RNA extraction utilizing TRIzol (Invitrogen). RNA quantication amplied on ABI Real-time PCR system The reaction follows: 94 ℃ 3 min, ℃ 30s, ℃ 40 s, ℃ 30 s, a total of 32 cycles, and nally at ℃ for 10min. GAPDH used for normalization and the relative expression level was calculated by the 2 −ΔΔCt The primers followed: INHBA: ACACAACAACTTTTGCTGCC-3′ (Forward), 5′-TCGTGTCACCACTGTCTTCTC-3′ (Reverse); (Forward), 5′-GTAGG TGGAGTCCCAGGCGTAG-3′ (Reverse); Vimentin: 5′-TCTGGATTCACTCCCTCTGGTT-3′ (Forward), 5′-ATCGTGATGCTGAGAAGTTTCGT-3′ (Reverse). GAPDH: 5′-GAAGGTGAAGGTCGGAGTC-3′ (Forward) and 5′- GAAGATGGTGATGGGATTTC − 3′ (Reverse).


Introduction
Breast cancer is one type of the most common malignant tumors in women worldwide (1,2). The incidence of breast cancer has increased annually in recent years. With the continuous improvement of diagnosis and treatment, including chemotherapy, radiotherapy and molecular targeted biomarkers in the last few decades, the mortality of breast cancer patients remains relatively high (3)(4)(5). Hence, it is urgent to elucidate the molecular mechanism underlying the initiation and development of breast cancer.
INHBA A is located at 7p14.1, which is a member of the transforming growth factor β (TGF-β) superfamily (6). Many studies have proved that INHBA serves important roles in in various cancers progression. Peng et al. discovered that INHBA knockdown inhibits nasopharyngeal carcinoma cell proliferation and invasion of SUNE1 (6). INHBA gene silencing has been reported to be could inhibit cell migration and invasion by TGF-β signaling pathway in gastric cancer (7). In addition, INHBA have been demonstrated that is a prognostic predictor for colon adenocarcinoma patients (8). More importantly, Wang et al. found that INHBA was upregulated in the breast cancer tissues (9). However, the function and potential mechanism of INHBA in breast cancer remains unclear.
In the present study, we also found that INHBA is simultaneously over-expressed in breast cancer tissues and cells. We then focused on exploring the functions of INHBA in the progression of breast cancer and demonstrated that inhibition of its expression could markedly attenuate the proliferation and epithelialmesenchymal transition (EMT) of breast cancer cells. Mechanistically, we elucidated the mechanism of INHBA in the breast cancer progression and indicated that it could regulate breast cancer cell invasion and EMT through Wnt/β-catenin signaling pathway. Our ndings illustrate a new target and underlying mechanism of breast cancer progression, which provide an effective target for the treatment and diagnosis of breast cancer, and extend the understanding mechanism-related functions of INHBA in Culture medium containing 20% FBS was supplemented into the lower chamber. Following 24 h incubation, the non-migrated and non-invading cells were removed. At last, cells were xed with 4% formaldehyde and stained using crystal violet. Cells were counted under a microscope (Olympus, Tokyo, Japan) at 200× magni cation. The number of cells was the average value from six representative elds. The migratory or invaded cells were counted and photographed under a light microscope (Nikon, Tokyo).

Western blot analysis
Total protein was extracted from cells using ice-cold RIPA lysis buffer (Beyotime Bitechnology, China).

Statistical analysis
Graphpad prism 8.0 (GraphPad, San Diego, CA, USA) was applied for statistical analysis. Data are presented as the mean ± standard deviation (SD). Comparisons between two groups were determined using Student's t-test. One-way ANOVA analysis followed by Turkey's poc host was used for multiple comparisons. P < .05 meant statistically signi cant.

INHBA expression was up-regulated in breast cancer
After browsing the Gene Expression Pro ling Interactive Analysis (GEPIA) database (http://gepia.cancerpku.cn/), we found that the INHBA expression level was signi cantly higher in BRCA tissues than normal tissues (Fig. 1A, B). Subsequently, the expression level of INHBA in BC cell lines and human normal epithelial cell line MCF-10A was examined through qRT-PCR analysis. Consistently, the results indicated that the expression level of INHBA was dramatically elevated in BC cell lines compared to MCF-10A cells, especially in MCF-7 cells that were used in the subsequent experiments (Fig. 1C).  Fig. 3A, B). Epithelial-to-mesenchymal transition (EMT) is a process by which epithelial cells acquire the characteristics of mesenchymal cells in tumor progression [12,13]. EMT related markers containing Ecadherin and N-cadherin were assessed. As shown in Fig. 3C, the E-cadherin protein expression level was increased while N-cadherin expression level was decreased when INHBA was knocking down. Whereas the E-cadherin protein expression levels in pc-INHBA group was lower, while N-cadherin expression level was higher than those in the control group.

Inhba Knockdown Inhibits
INHBA facilitated breast cancer cell invasion and EMT through activation of the Wnt/β-catenin signaling pathway Wnt/β-catenin signaling pathway plays an essential role in cancer progression, and we speculated that INHBA facilitated breast cancer progression through Wnt/β-catenin signaling pathway. To test our hypothesis and investigate the possible mechanism of INHBA in regulating the invasion and EMT of breast cancer, we investigated the expression of the related mRNA and proteins in Wnt/β-catenin pathway. We constructed INHBA plasmid to force expression of INHBA in MCF-7 cells (Fig. 4A), and treated with XAV-939 (Wnt/β-catenin signaling inhibitor). Compared with the control group, ectopic of INHBA signi cantly increases β-catenin and GSK-3β mRNA expression levels and can be reversed by XAV-939 treatment (Fig. 4A). As shown in Fig. 4B, the overexpression of INHBA group showed an up-regulation in the protein expression levels of β-catenin and phosphorylation levels of GSK-3β. Meanwhile, these effects were reversed by XAV-939 treatment.
To investigate whether INHBA plays its role in promoting tumor progression through Wnt/β-catenin signaling pathway, rescue experiments were performed. Followed functional analysis indicated that overexpression of INHBA facilitated the proliferative capacity of MCF-7 cells, while were partly suppressed by XAV-939 treatment compared with control group (Fig. 5A). In addition, the migration and invasion abilities of MCF-7 cells transfected with pc-INHBA was higher than those in the control group. These effects were reversed in MCF-7 cells treated with XAV-939 ( Fig. 5B-D).

Discussion
In recent years, with the development of molecular biology, individualized cancer management has developed rapidly. Breast cancer has become one of the most challenging malignant tumors in woman. Interest in nding useful biomarkers for diagnosis and treatment of breast cancer has been accumulating. Increasing data indicated that aberrant INHBA expression has serve important roles in almost all cell biological behaviors, including growth, cell cycle, apoptosis, differentiation, apoptosis and metastasis (10)(11)(12)(13)(14). However, little is known about the function of INHBA in breast cancer. The purpose of our study was to explore the role of INHBA in breast cancer.
By performing a series of bioinformatics analyses, we found that INHBA was notably up-regulated in breast cancer tissues. According to the clinical evidence, we deduced that INHBA may affect breast cancer cell functions. Based on the results of qRT-PCR, INHBA expression was higher in breast cancer cells than that in normal epithelial cells. In vitro experiments were performed to evaluate the role of INHBA in cell proliferation and invasion of breast cancer by using the CCK-8, EdU and transwell assays with MCF-7 cells exhibiting overexpression or knockdown of INHBA. As expected, we found that knocking down of INHBA could inhibit cell proliferation, migration and invasion, indicating that INHBA downexpression exerted a signi cant inhibitory role in these biological processes.
Tumor metastasis involves many tumor processes and EMT is a key initial step for tumor cells to acquire the potential of metastasis and invasion (15,16). EMT is an evolutionary process in which cells lose epithelial properties and acquire mesenchymal properties. E-cadherin and Vimentin as the epithelial biomarkers, have been proved to play an important role in tumor metastasis (17)(18)(19)(20). For example, Zhang et al. found that GRIM-19 could inhibit colorectal cancer cell invasion and EMT by inactivation of STAT3/HIF-1α signaling axis (21). REC8 have been proved to inhibit gastric cancer cell EMT by downregulating EGR1 expression (22). In the present study, the results of qRT-PCR and western blot assays demonstrated that knockdown of INHBA markedly repressed the expression level of mesenchymal marker N-cadherin, while the expression level of epithelial marker E-cadherin was increased.
The role of Wnt/β-catenin signaling played in the growth and metastasis of tumor has been investigated in many researches (23)(24)(25). Above these ndings led us to consider the potential association of INHBA with the Wnt/β-catenin pathway. The results indicated that ectopic of INHBA expression activated Wnt/βcatenin pathway with the change of β-catenin and GSK-3β. In addition, disruption of Wnt/β-catenin signaling by XAV-939 treatment reversed the stimulative effect of INHBA overexpression on breast cancer cell proliferation, migration and invasion.
In summary, our results demonstrated that INHBA was signi cantly stimulated the proliferation, migration, invasion and EMT of breast cancer by activating Wnt/β-catenin pathway. These ndings indicated that INHBA may be a potential candidate predictive factor in breast cancer, which may contribute to the diagnosis and treatment of breast cancer.

Declarations
Contributions of all authors MJ conceived and designed the study. TX drafted the manuscript and analyzed the data. HY performed the experiments.

Con ict of interest
The authors state that there is no con ict of interest.
Ethics approval and consent to participate Not applicable.

Consent for publication
Not applicable.

Funding
Not applicable.  INHBA facilitated breast cancer cell invasion and EMT through activation of the Wnt/β-catenin signaling pathway. A. Wnt/β-catenin signaling pathway associated mRNA and protein expressions were assessed using qRT-PCR and western blot assays.