Long Non-coding RNA RP11-480I12.5 Promotes the Proliferation, Migration, and Invasion of Breast Cancer Cells Through the miR-490-3p-AURKA-Wnt/β-Catenin Axis

Background: RP11-480I12. 5 is a newly identified long non-coding RNA (lncRNA) that has never been studied in breast cancer (BC). The biological function of RP11-480I12.5 in breast carcinoma and its underlying mechanism are still unknown. Methods: We scanned The Cancer Genome Atlas (TCGA) database and identified RP11-480I12.5 as one of the most dysregulated lncRNAs. The level of RP11-480I12.5 was assessed in BC tissue samples and BC cell lines. The prognostic value of RP11-480I12.5 expression was assessed using the Kaplan–Meier method. The biological influence of RP11-480I12.5 on BC cell lines was studied using proliferation and Transwell migration and invasion assays. Results: RP11-480I12.5 expression was upregulated in data from both the TCGA database and our own database. Moreover, Kaplan–Meier and Cox proportional hazard analyses indicated that high RP11-480I12.5 expression was related to poor overall survival. Moreover, RP11-480I12.5 promoted the proliferation, migration, and invasion of BC. RP11-480I12.5 promoted the expression of AURKA and the activation of the downstream Wnt/β-catenin pathway by sponging the microRNA (miRNA) miR-490-3p. Conclusion: Taken together, our results indicate that RP11-480I12.5 is associated with tumor progression in BCs. Our findings indicate that the lncRNA RP11-480I12.5 promotes the proliferation, migration, and invasion of BC cells through the miR-490-3p-AURKA-Wnt/β-catenin axis, which may serve as a therapeutic target in the future.


BACKGROUND
Breast cancer (BC) is one of the most common cancers worldwide and the second leading cause of cancer-related mortality in women (1). Although advances in therapy have been made, patients still suffer from metastasis and recurrence (2)(3)(4). Immunotherapeutic strategies, such as chimeric antigen receptor (CAR) T cells and immune checkpoint inhibitors (such as PDL1 inhibitors), have been applied, but in most patients, chemotherapy and radiotherapy are still the most common treatments (5). Hence, more therapeutic targets are urgently needed.
Long non-coding RNAs (lncRNAs) are involved in many physiological and pathological processes in humans, such as cell division and differentiation. LncRNAs are reported to be involved in the progression of multiple kinds of carcinomas, such as gastric cancer (6) and lung cancers (7). Many studies have shown that lncRNAs contribute to multiple processes in the progression of breast carcinoma, such as proliferation, angiogenesis, and tissue invasion. The lncRNA HOXA-AS2 regulates the expression of SCN3A by sponging miR-106a (8), the lncRNA PTCSC3 inhibits triple-negative BC cell proliferation by downregulating lncRNA H19 expression (9), and LINC00511 knockdown enhances paclitaxel cytotoxicity in BC by regulating the miR-29c/CDK6 axis (10).
In the present study, we scanned The Cancer Genome Atlas (TCGA) database and identified RP11-480I12.5 as exhibiting significantly upregulated expression in BC. We next detected the expression difference between normal tissue and BC tissue. Then, we uncovered the biological function of RP11-480I12.5 through a series of experiments. We conclude that RP11-480I12.5 promotes the proliferation, migration and invasion of BC through the miR-490-3p-AURKA-Wnt/β-catenin axis.

Tissue Samples
All human tissue samples were obtained from the surgical suite at the First Affiliated Hospital of Sun Yat-Sen University and the Department of Breast and Thyroid Surgery, Affiliated Hospital of Youjiang Medical University for Nationalities after confirmation by a pathologist. The tissue specimens were obtained with the patients' written consent under a protocol approved by the institution's Institutional Review Board. Among the 101 patients enrolled, 21 had triple-negative disease, 23 had Her2+ disease, while the others had the luminal subtype.

Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)
Total RNA was extracted using TRIzol reagent (Invitrogen, NY, USA) according to the manufacturer's instructions. qRT-PCR was performed using a SYBR green detection RT-PCR system (Takara, Japan). Actin was utilized for normalization. All samples were analyzed in triplicate. The 2 − Ct method was used to calculate the relative RNA expression. The probes are listed below: Forward primer: ATTGCATTGCCAATTTGA Reverse primer: GTCAAATAAAGTTTGGAAAAC.

Colony Formation Assay
Transfected BC cells were seeded in a 6-well-plate at a density of 500 cells/well. After 2 weeks of culture, cell colonies were fixed with 4% paraformaldehyde and stained with 1% crystal violet. The colonies were examined and counted under a microscope.

Lentiviral Production and Stable Cell Line Construction
Lentiviral vectors expressing shRNA and RP11-480I12.5 were cotransfected with the packaging vectors psPAX2 and pMD2G (Addgene) into HEK293FT cells using Lipofectamine 3000 to produce lentiviruses in accordance with the manufacturer's instructions. To establish stable cell lines, cells were transduced by using the above lentiviruses with polybrene (8 mg/ml, Sigma). After incubating for 72 h, the cells were selected with 2 mg/ml puromycin for 3 days.

Statistical Analysis
All data analyses were performed with SPSS 20.0 statistical software. The χ 2 -test was used to analyze the relationships between categorical variables. Differences between groups were compared by Student's t-test. Cox regression and Kaplan-Meier methods were used to analyze overall survival (OS), and the cutoff value of RP11-480I12.5 in patients was the mean of the whole cohort in both the TCGA database and our in-house database. p < 0.05 was considered to indicate significant difference from the control.

RP11-480I12.5 Expression Is Upregulated in BC Tissue and Associated With a Poor Prognosis
We scanned the TCGA database and identified RP11-480I12.5 as one of the lncRNAs contributing to the progression of BC ( Figure 1A, p < 0.05). To further verify this hypothesis, we analyzed the expression differences between 50 paired normal tissue samples and tumor samples with a qRT-PCR assay. RP11-480I12.5 expression was significantly upregulated in the tumor tissue specimens (p < 0.001) ( Figure 1B, Figure S2). Moreover, we analyzed the correlation between RP11-480I12.5 and the OS of patients. The results for both the TCGA database (p = 0.025) ( Figure 1C) and our own database showed that RP11-480I12.5 expression was negatively associated with prognosis (p < 0.05) ( Figure 1D). Frontiers in Oncology | www.frontiersin.org Frontiers in Oncology | www.frontiersin.org

RP11-480I12.5 Promotes the Proliferation of BC Cells
We have suggested that RP11-480I12.5 expression is upregulated in BC and negatively associated with prognosis. To further uncover the biological function of RP11-480I12.5, we detected the expression pattern in BC cell lines. A BC cell line harbored a higher level of RP11-480I12.5 than the control normal cell line MCF-10A (p < 0.001) (Figure 2A). We  < 0.001). RP11-480I12.5 promotes proliferation in short-and long-term manners.

RP11-480I12.5 Promotes the Migration and Invasion of BC
Recurrence and metastasis are the most common factors contributing to patient death. We next analyzed the migratory and invasive abilities of the cell lines described above. The results showed that cells with higher levels of RP11-480I12.5 developed improved migratory and invasive abilities. A Transwell assay indicated that cells with a higher level of RP11-480I12.5 migrated more easily and moved through the semipermeable membrane more easily. In the invasion chamber assay, cells with overexpression degraded the matrix gel, invaded into the membrane, and migrated through the membrane more easily than the control cell lines (p < 0.001) (Figures 3A,B). Epithelialmesenchymal transition (EMT) is the most common change in cancer cells during migration and invasion. EMT markers are the most common markers reflecting the mesenchymal status of cancer cells. We previously showed that RP11-480I12.5 promoted the migration and invasion of BC cells. We next detected EMT markers and the proliferation marker PCNA. The results showed that cells with a higher level of RP11-480I12.5 tended to transition from epithelial cells to mesenchymal cells.
The expression of mesenchymal markers, such as N-cadherin, vimentin, snail, and slug, and PCNA increased, while that of E-cadherin decreased at both the RNA and protein levels ( Figures 4A,B, p < 0.05, Figure S1A).

RP11-480I12.5 Promotes the Expression of AURKA and the Activation of the Downstream Wnt/β-Catenin Pathway
To uncover the mechanism underlying the role of RP11-480I12.5 in BC, we detected the expression of AURKA in RP11-480I12.5knockdown and RP11-480I12.5-overexpressing cell lines and found that the expression of AURKA at both the protein and mRNA levels was increased in the cells with a higher level of RP11-480I12.5 and decreased in the cells with a lower level of RP11-480I12.5. The level of the downstream molecule βcatenin correlated with the level of AURKA (Figures 5A,B, p < 0.001, Figure S1B). We next examined the correlation between RP11-480I12.5 and AURKA in the TCGA database and our own database; RP11-480I12.5 expression was positively correlated with the level of AURKA in the data from both the TCGA database ( Figure 5C, R = 0.36, p < 0.001) and our in-house database ( Figure 5D, R = 0.59, p < 0.001). The results indicated that RP11-480I12.5 may exert its function through AURKA and the downstream Wnt/β-catenin pathway.

RP11-480I12.5 Promotes Proliferation, Migration, Invasion, and EMT in an AURKA-Dependent Manner
For further verification, we established a rescue cell line, where we re-expressed AURKA in an RP11-480I12.5-knockdown cell line and knocked down AURKA expression in an RP11-480I12.5-overexpressing cell line. We next performed a series of experiments and found that proliferation and invasion were restored in the AURKA re-expressing cell line and abolished in the AURKA-knockdown cell line (Figures 6A-C, p < 0.001), indicating that RP11-480I12.5 exerts its function in an AURKAdependent manner. We next detected EMT markers and PCNA, and the results showed that the EMT markers were completely restored at both the RNA and protein levels in the rescue cell line (Figures 7A,B, p < 0.05, Figure S1C), indicating that RP11-480I12.5 promotes EMT in an AURKA-dependent manner. Thus, we have proven that RP11-480I12.5 promotes proliferation, migration, and invasion through AURKA and the downstream Wnt/β-catenin pathway.
To verify this hypothesis, we detected the miR-490-3p level in the RP11-480I12.5-knockdown and RP11-480I12.5-overexpressing cell lines. Cells with a higher level of RP11-480I12.5 had lower levels of miR-490-3p ( Figure 8B, p < 0.001). We next transfected a miR-490-3p inhibitor into the RP11-480I12.5-knockdown cell line and a miR-490-3p mimic into the RP11-480I12.5overexpressing cell line. We then detected the expression level of AURKA and found that the level of AURKA in the rescue cell line was completely restored, indicating that RP11-480I12.5 exerts its function through miR-490-3p (Figures 8C,D, p < 0.001, Figure S1D). MiRNAs are well-known non-coding RNAs engaged in the posttranscriptional regulation of gene expression. We next predicted the binding site of miRNA-490-3p in the AURKA pre-mRNA and found that miR-490-3p could bind to the 3 ′ -untranslated region (UTR) of the pre-mRNA of AURKA ( Figure 8E). We constructed a dual-luciferase reporter system, transfected wild-type AURKA (AURKA WT) or AURKA with a 3 ′ -UTR mutation into the HEK293 cell line, and examined the relative luciferase activity. The results showed that the luciferase activity of the AURKA mutant was significantly increased compared with that of AURKA WT, indicating that miR-490-3p directly binds to the 3 ′ -UTR of AURKA and promotes its degradation (Figure 8F, p < 0.001). Thus, we demonstrate that the lncRNA RP11-480I12.5 promotes the proliferation, migration, and invasion of BC cells through the miR-490-3p-AURKA-Wnt/β-catenin axis (Figure 9).

DISCUSSION
Breast carcinoma is the most common malignancy in women worldwide. Although many advancements have been made, many patients still suffer from recurrence and metastasis.
Recent studies have shown that many lncRNAs participate Keshavarz, M and Asadi, M H found that the lncRNA ES1 controlled the proliferation of BC cells by regulating the Oct4/Sox2/miR-302 axis. Our study identified a new lncRNA, RP11-480I12.5, that had not been studied previously in BC and found that RP11-480I12.5 could promote proliferation, migration, and invasion, indicating that this lncRNA may be a therapeutic target.
MiR-490-3p is a well-studied miRNA that is involved in the progression of tumorigenesis. MiRNA-mediated posttranscriptional regulation is one of the most important types of epigenetic regulation. MiR-490-3p can be sponged by many kinds of lncRNAs and targets multiple pre-mRNAs. Mu et al. showed that the lncRNA CCAT1 upregulates TGFbetaR1 expression by sponging miR-490-3p to promote TGFbeta1induced EMT in ovarian cancer cells (13). The lncRNA TP73-AS1 suppresses triple-negative BC cell vasculogenic mimicry by targeting the miR-490-3p/TWIST1 axis (14). CircSLC3A2 functions as an oncogenic factor in hepatocellular carcinoma by sponging miR-490-3p and regulating PPM1F  expression (15). However, the role of miR-490-3p has rarely been studied. Zhang showed that miR-490-3p inhibited growth and invasion in triple-negative BC, but the underlying mechanism is still unknown. Our study showed that the lncRNA RP11-480I12.5 could sponge miR-490-3p, which inhibited its function.
The AURKA kinase is a serine/threonine kinase that is distributed in many kinds of cellular organisms and is essential for mitosis and cytokinesis. AURKA is reported to be involved in the progression of many kinds of cancers, such as gastric cancer and colon cancers (16,17). AURKA has been reported to promote cell proliferation through AKT and the Wnt/βcatenin pathway (18). AURKA was also reported to contribute to the stemness (19) and chemoresistance (20) in BC cells. Combination of targeting AURKA and p21 was recently reported to be efficient for BC (21). The Wnt/β-catenin pathway is important in maintaining stemness and is overactivated in many cancers.

CONCLUSION
Taken together, our results indicate that RP11-480I12.5 is associated with tumor progression in BCs. Our findings indicate that the lncRNA RP11-480I12.5 promotes the proliferation, migration, and invasion of BC cells through the miR-490-3p-AURKA-Wnt/β-catenin axis, which may serve as a therapeutic target in the future.

DATA AVAILABILITY STATEMENT
All datasets generated for this study are included in the article/Supplementary Material.

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by the ethics committee of Affiliated Hospital of Youjiang Medical University for Nationalities, the ethics committee of Eastern Hospital of the First Affiliated Hospital of Sun Yat-sen University, the ethics committee of the First Affiliated Hospital of Sun Yatsen and the ethics committee of Guangdong general hospital with the 1964 helsinki declaration and its later amendments or comparable ethical standards. The patients provided written informed consent to participate in this study.

AUTHOR CONTRIBUTIONS
JL, GL, and CY designed the research. All authors were engaged into the performance of experiments and data analysis. All authors contributed to the article and approved the submitted version.