NONHSAT021545/miR-330-3p/EREG: A Cooperative Axis in Breast Cancer Prognosis and Treatment

Lymphatic metastasis is the most common form in breast cancer (BC) progression. Previously, we observed that lnc045874, a most conservative homology of Homo Sapiens NONHSAT021545 (lnc021545), miR-330-3p, and EREG may have some effects in mouse hepatocarcinoma cell lines with different lymphatic metastasis potentials. Through data from TCGA and GEO database analysis, we speculated that miR-330-3p might be a tumor promoter, while EREG could be a tumor suppressor in BC. MiR-330-3p was upregulated, while lnc021545 and EREG were downregulated in 50 BC tissues. MiR-330-3p advanced the metastatic behaviors of BC cells, whereas lnc021545 and EREG resulted in the opposite effects. The three molecules’ expressions were correlated respectively and showed that miR-330-3p targeted lnc021545 and EREG to affect their expressions. Lnc021545/miR-330-3p axis affected BC metastasis by regulating EREG in epithelial-to-mesenchymal transition. In 50 BC patients, these three molecules and their cooperation are associated with aggressive tumor phenotypes, patient outcomes, and trastuzumab therapy. We finally discovered that lnc021545, miR-330-3p, and EREG formed a multi-gene co-regulation system that affected the metastasis of BC and the cooperation reflects the synergistic effects of the three molecules, recommending that their cooperation may provide a more accurate index for anti-metastasis therapeutic and prognostic evaluation of BC.


Introduction
In female malignancies, breast cancer (BC) is considered to be the most frequently diagnosed cancer [1]. For cancer patients, distal metastases and resistance to therapeutics are the leading causes of death [2]. Biologically, distant cancer metastasis involves a variety of complex mechanisms, such as EMT, migration, invasion, angiogenesis, and adhesion [3,4]. Interpreting these mechanisms can be beneficial for discovering new therapeutic regimens and markers.
Lymphatic metastasis is the most common form of BC progression [5]. In our previous research, the differentially expressed miRNAs, lncRNAs, and mRNAs in mouse hepatocarcinoma Hca-F (with high lymphatic metastasis potential) and Hca-P (with low lymphatic metastasis potential) cell lines were screened using a high-throughput RNA sequencing method. Among them, miR-330-3p, lnc045874, and EREG have shown significantly different expressions between the two cell lines. Murine lnc045874 is the most conservative homology of human NONHSAT021545 (lnc021545). Meanwhile, lnc021545 conserved same the binding site as miR-330-3p. TargetScan (http://www.targetscan.org accessed on 1 March 2020) analysis suggested that miR-330-3p has two combining sites with the 3 -untranslated region (3 -UTR) of EREG and one combining site with lnc045874. We aimed to determine whether these three molecules could synergistically affect the progression of BC by modulating the lymphatic metastasis and evaluated the clinical feature of BC patients.
The microRNAs (miRNAs) are a type of small, non-coding RNA composed of 18-24 nucleotides. The miRNAs can negatively modulate genes' expressions by binding their 3 -UTRs, which act as crucial effectors in the malignant behaviors of tumor cells [6]. MiR-330-3p is located on chromosome 19q12.32 [7] and is bound to the malignant behaviors of cancerous cells; It plays a moderator role in different cancers [8]. Long non-coding RNAs (lncRNAs) are a type of RNA transcript with a length longer than 200 nucleotides without protein-coding ability [9]. LncRNAs have been demonstrated to modulate the biological functions of mRNAs by combing miRNAs [10]. Lnc021545 (NONCODE ID: NONHSAT021545.2) is located on human chromosome 11 with a length of 3149 bases; the exact role of lnc021545 in BC remains elusive. Epiregulin (EREG) is located on chromosome 4q13.3 and is a member of the epidermal growth factor family [11]. EREG acts as a crucial effector in the modulation of angiogenesis [12], inflammation [13], drug resistance [14], and metastasis [15], whereas the effect of EREG in the origination and progression of BC remains unconfident.
The development of a disease is a complex process caused by the dysregulation of multiple genes' functions rather than by the dysregulation of a single or an isolated gene's functions [16]. With the identification of a mass of cancer-related genes and their functions, the studies that coordinated function of multi-genes influence the prognosis and treatment of cancer are increasingly emerging. In the research of BC, the expression levels of 21 genes associated with the biological characteristics of BC are detected via RT-PCR, and the recurrence score (RS) is obtained using an algorithm based on previous clinical studies [17]. The RS has been integrated into the National Comprehensive Cancer Network clinical guidelines for treatment and predictive prognosis of hormone-receptor-positive and HER-2-negative diseases [18]. By studying the effects of lnc021545, miR-330-3p, and EREG in BC, we will explore whether these three molecules' coordinated functions could affect BC's prognosis and treatment.
As part of this study, we established a new lnc021545-miR-330-3p-EREG axis regulatory network in BC. We found that miR-330-3p was upregulated, while lnc021545 and EREG were downregulated in BC patients' samples. Moreover, lnc021545 and EREG act as tumor suppressors in regulating the metastasis of BC cells, while miR-330-3p acts as a tumor promoter and showed opposite effects on BC cells. Furthermore, lnc021545/miR-330-3p affects the EMT process of BC cells by regulating EREG expression. Our study demonstrates lnc021545-miR-330-3p-EREG axis regulatory network in BC progression and provides a new clue to the treatment and prognosis of BC.

Tissue Samples and Cell Culture
Fifty pairs of BC tissues with their one-to-one corresponding adjacent non-tumor tissues were collected from patients who underwent surgical resection at the Second Affiliated Hospital of Dalian Medical University between 2015 and 2019. The study was approved by the Medical Ethics Committee of Dalian Medical University (approval number 2019-069). The informed consent forms were signed by all participants. After resection, the tissue specimens were stored at −80 • C until use. Human BC cell lines MCF-7 and T47D were obtained from the American Type Culture Collection. Both cell lines were cultured in RPMI 1640 (Gibco, New York, NY, USA) supplemented with 15% fetal bovine serum (FBS, TransGen, Beijing, China) and 100 U/mL penicillin/streptomycin (Gibco) at 37 • C with 5% CO 2 .

Bioinformatic Analysis
The data of miRNA-seq were downloaded from The Cancer Genome Atlas (TCGA, https://tcgadata.nci.nih.gov/tcga accessed on 18 September 2022). We also queried the Gene Expression Omnibus (GEO, https://www.ncbi.nlh.gov/geo accessed on 20 September 2022) database for miRNA and mRNA profiling by arrays. We analyzed the expression data of 1103 BC tissues and 104 non-tumor tissues from the TCGA-BRCA dataset by employing R package "edgeR" to identify and normalize the differentially expressed miRNAs. GSE22216 and GSE40267 were utilized for survival analysis according to miR-330-3p expression changes. To evaluate the prognostic value of EREG, survival analysis was performed using the web tool KMplot (http://kmplot.com accessed on 26 September 2022). Gene set enrichment analysis (GSEA) was executed on the GSE3494 dataset to analyze the associated functional gene sets in BC pathogenesis.
In total, 3 × 10 5 MCF-7 and T47D cells were seeded per well and incubated at 37 • C with 5% CO 2 for 12 h. To downregulate lnc021545 in cells, 3.5 µL si-lnc021545-1 and 3.5 µL si-lnc021545-2 or 7 µL si-NC with a consistency of 20 µM and the supplementary of 6 µL Lipofectamine™ 2000 (Lipo2000, Invitrogen, Waltham, MA, USA) were mixed to form a transfection mixture and kept still for 20 min at room temperature (RT). Then, 5 µL miR-330-3p mimic, mimic NC, miR-330-3p inhibitor or inhibitor NC with a consistency of 20 µM, and the supplementary of 5 µL Lipo2000 were mixed to form a transfection mixture to overexpress and downregulate miR-330-3p in cells. An amount of 1 µg PCDH-EREG or 1 µg PCDH was mixed with 50 µL serum-free medium, 5 µL Lipo2000 was mixed with 45 µL serum-free medium, and the above two mixtures were mixed in order to establish the cells of EREG over-expression and placed for 20 min at RT. 2.5 µL si-EREG-1, si-EREG-2, and si-EREG-3 or 7.5 µL si-NC with a consistency of 20 µM. A supplement of 6 µL Lipo2000 was mixed and placed for 20 min at RT as the transfection mixture to EREG downregulation. Each of the transfection mixtures were added into each group of MCF-7 or T47D cells. Then, the cells were cultured at 37 • C under 5% CO 2 for 24 h or 48 h for further experiments.

Cell Proliferation Assay
The proliferation abilities of different group of MCF-7 and T47D cells were measured via MTT assay. Each group was seeded with a density of 3 × 10 3 cells/well in a 96-well plate (NEST, Wuxi, China). MTT reagent was added to each well in 200 µL increments at 24 h, 48 h, 72 h, 96 h, and 120 h. After 4 h, the MTT reagent was substituted with 150 µL dimethyl sulfoxide (Sigma, St. Louis, MO, USA). The absorbance of 492 nm was detected using a plate reader (Eppendorf, Hamburg, Germany) for the quantification of cell viability.

Cell Motility Assay
The motility ability of MCF-7 cells was measured by wound-healing experiments. In total, 6 × 10 5 of each group of MCF-7 cells were seeded into a 6-well plate (NEST) at 37 • C with 5% CO 2 and cultured to 85-90% confluence. Then, a 200 µL sterilized tip drew vertical scratches on the monolayer cell surface. Detached and floated cells were washed with PBS, then cultured at 37 • C for 24 h. The widths of scratch wounds were obtained under a microscope (Olympus, Tokyo, Japan) at 100× magnification at 0 h, 24 h, and assessed using Image J software (version 2.3.0).

Cell Migration and Invasion Assay
The migration and invasion of cells were tested via transwell chamber assay. For migration assay, 1 × 10 4 transfected MCF-7 or T47D cells in 200 µL RPMI 1640 with serum-free media were seeded in the upper chamber of the transwell. For invasion assay, the chambers were layered in an extracellular matrix (Sigma). The upper chamber was assembled into the plate wells, and the bottom chamber was filled with 600 µL RPMI 1640 with 20% FBS and cultured for 48 h at 37 • C with 5% CO 2 . The migrative or invasive cells through the membrane were fixed for 30 min in absolute methanol and stained in 0.5% crystal violet stain solution (Solarbio, Beijing, China) for 1 h at RT, then imaged and counted with the microscope (Olympus) at 100× magnification.

Western Blotting (WB) Assay
RIPA lysis buffer was used to extract the total proteins of transfected BC cells. The quantitation of protein was evaluated using Bradford assay [19]. After, the sample of protein (30 µg) was separated using 10% sodium dodecyl sulphate-polyacrylamide gels and transferred onto nitrocellulose membrane (PALL, Port Washington, NY, USA), followed by a blocking at RT with 5% skim milk for 2 h. Then, the membranes were incubated with the following primary antibodies: EREG (Abcam, Cambridge, MA, USA), E-cadherin (ProteinTech, Wuhan, China), N-cadherin (ProteinTech), vimentin (ProteinTech), Snail (ProteinTech), Slug (ProteinTech), and GAPDH (ProteinTech) overnight at 4 • C. After washing three times with TBST, the membranes were incubated in the secondary antibody (ProteinTech) at RT for 3 h and washed three times with TBST. The blots were developed by electrochemiluminescence (Advansta, San Jose, CA, USA) and imaged with the ChemiDoc MP imaging system (Bio-Rad, Hercules, CA, USA).

Luciferase Activity Assay
Wild-type (WT), mutant (MUT) lnc021545 3 -untranslated region (UTR), and EREG 3 -UTR were amplified and cloned into the luciferase reporter vector pmirGLO (Promega, Madison, WI, USA) to create pmirGLO lnc021545, pmirGLO EREG WT, and MUT plasmids. Then, 1 × 10 5 MCF-7 cells were co-transfected with 5 ng each of the corresponding plasmids and 20 pmol each of miR-330-3p mimics. A dual-luciferase reporter assay system (Promega) was used in this assay. Each well of luminometer plates was loaded with lysates and the firefly luciferase activity was detected through an EnSpire multifunctional microplate reader (PerkinElmer, Waltham, MA, USA). Each well of the luminometer plates was again loaded with 100 µL Stop&Glo reagent, and renilla luciferase activity was detected. The results were represented as normalized firefly luciferase activity/renilla luciferase activity values.

Quantitative Reverse Transcription PCR (qRT-PCR) Assay
Total RNA was obtained by Trizol TM reagent (Invitrogen). Then, 1 µg of total RNA was reversely transcribed into high-quality cDNA with PrimeScript TM 1st cDNA Synthesis (Takara, Kyoto, Japan). qRT-PCR was performed using the FastStart Universal SYBR Green Master (Roche, Basel, Switzerland) on a StepOne TM Real-Time PCR System (ABI, Los Angeles, CA, USA). The relative expression levels of EREG and lnc021545 were quantified using β-actin as the internal reference, while the relative expression levels of miR-330-3p were analyzed against U6 reference. The relative expression levels of RNA were calculated using the 2 −∆∆CT method. The primer sequences are listed in Table S2.

Immunohistochemistry (IHC) Assay
The 2.5 µm paraffin slices were dewaxed by xylene, rehydrated with gradient ethanol, covered with 3% H 2 O 2 for 10 min, washed with PBS three times, and incubated with goat anti-epiregulin polyclonal antibody (Invitrogen), rabbit anti estrogen receptor (ER) polyclonal antibody (Invitrogen), rabbit anti progesterone receptor (PR) monoclonal antibody (Invitrogen), or rabbit anti Ki-67 monoclonal antibody (Invitrogen) overnight at 4 • C. The slices were then nurtured with a biotin-streptavidin horseradish peroxidase detection system (ZSGB-BIO, Beijing, China) for 2 h and stained by DAB at RT. The slices were counterstained with hematoxylin, dehydrated with gradient ethanol, clarified with xylene, scanned with an Aperio GT450 (Leica, Wetzlar, Germany) at 200× magnification, and evaluated with an Aperio ImageScope (version 12.4.3.5008). The protein expression in the selected region was reflected by H-Score [20].

Fluorescence In Situ Hybridization (FISH)
The 3 µm paraffin slices were heated for 30 min at 56 • C. The slices were deparaffinized for 10 min with xylene and tapped off xylene for 5 s in 100% ethanol, repeated twice. The slides were placed into gradient ethanol and deionized water, then into the boiled pretreatment solution for 15 min. The slices were washed with 2 × SSC solution, incubated with proteinase K solution for 15 min, washed with 2 × SSC solution for 1 min, and dehydrated with each gradient ethanol for 3 min at RT. The specimen area was dropped to a 10 µL probe mix. The coverslip was sealed with rubber cement, and the slices were hybridized at 47 • C overnight. The slices were cleaned with 2 × SSC solution and dehydrated in each gradient ethanol for 3 min. Following air-drying, the slides were counterstained with 10 µL DAPI counterstain. After 15 min, the slides were scanned with fluorescence microscope (Olympus) at 100× magnification.

Amplification-Refractory Mutation System PCR (ARMS-PCR) Assay
A standard pathology methodology was used to select the tumor tissue with more than 30% tumor cells for DNA extraction. Fifty BC patients' DNA extractions were carried out from FFPE tumor tissue according to the instructions of the DNA extraction kit (AmoyDx, Xiamen, China). The qualities of DNA extractions were assessed by NanoDrop (Thermo Fisher, Waltham, MA, USA). Then, 5 ng sample DNA was added into each PCR reaction mix tube of PIK3CA mutations (AmoyDx). The PIK3CA mutations were detected on a 7500 Real-Time PCR System (ABI). The PIK3CA mutations were analyzed using the "Results Interpretation" analysis module. were used for data statistical analyses. The differences and statistical significance between the two groups of data were assessed using Student's t-test. The relation between any two molecules in BC tissues was assessed using the Pearson correlation coefficient. p value < 0.05 was regarded as significant.

Evaluation of the Effect of miR-330-3p and EREG in BC
To evaluate the role of miR-330-3p in BC, we performed an analysis of differential expression levels based on the TCGA-BRCA dataset. It showed that the expression of miR-330-3p was up-regulated in BC tissues compared to the normal controls ( Figure 1A, p = 8.5 × 10 −8 ). We further analyzed the association of miR-330-3p expression with the invasive disease-free survival (iDFS) of BC patients based on the GSE22216 dataset. Patients with low miR-330-3p expression showed better survival probability than patients with high miR-330-3p expression ( Figure 1B, p = 0.0089). In addition, we pooled the BC samples from GSE22216 and GSE40267 into one metadata cohort including 265 patients who had undergone 10-year follow-up. The batch effect was removed using R package "sva". We also stratified the iDFS by differential expression of miR-330-3p, resulting in a more significant survival analysis ( Figure 1C, p < 0.0001). The data imply that the expression level of miR-330-3p might be a poor prognostic marker in BC. stratified the iDFS by differential expression of miR-330-3p, resulting in a more significan survival analysis ( Figure 1C, p < 0.0001). The data imply that the expression level of miR 330-3p might be a poor prognostic marker in BC. To explore the effect of EREG in BC, we performed survival analysis using KMplo We found that patients with high expression of EREG showed higher overall survival (OS rates than patients with low expression of EREG from analyzing the E-MATB-365 (Figur 1D, p = 0.038), GSE65194 ( Figure 1E, p = 0.00056), GSE2990 ( Figure 1F, p = 0.011), an GSE1456 ( Figure 1G, p = 0.02) datasets. The results suggest that EREG expression migh be a positive prognostic marker in BC. The above results demonstrate miR-330-3p as tumor promoter in BC and EREG as a tumor suppressor in BC.
MTT assay was used to assess the role of miR-330-3p on the proliferation in MCFand T47D cells. Compared to their corresponding NC group cells, both the overexpressio and downregulation of miR-330-3p had no effect on the proliferation of MCF-7 and T47 cells (p > 0.05, Figure 2B). This also suggests that the influence of miR-330-3p on the ma lignant behavior of BC without affecting its proliferation.
A wound healing assay was used to measure the effect of miR-330-3p on the motilit of BC cells ( Figure 2C). The scratch closure distance of MCF-7-miR-330-3p-mimic cells wa measured as 48.3 ± 5.5 µm, which was 50.1% (p = 0.0052) more than that of MCF-7-mimi NC cells. The scratch closure distance of MCF-7-miR-330-3p-inhibitor cells was 12.8 ± 2 µm, which was 48.0% (p = 0.0047) less than that of MCF-7-inhibitor-NC cells. Our dat reveal that miR-330-3p overexpression promotes the motility of MCF-7 cells, while miR To explore the effect of EREG in BC, we performed survival analysis using KMplot. We found that patients with high expression of EREG showed higher overall survival (OS) rates than patients with low expression of EREG from analyzing the E-MATB-365 ( Figure 1D, p = 0.038), GSE65194 ( Figure 1E, p = 0.00056), GSE2990 ( Figure 1F, p = 0.011), and GSE1456 ( Figure 1G, p = 0.02) datasets. The results suggest that EREG expression might be a positive prognostic marker in BC. The above results demonstrate miR-330-3p as a tumor promoter in BC and EREG as a tumor suppressor in BC.
MTT assay was used to assess the role of miR-330-3p on the proliferation in MCF-7 and T47D cells. Compared to their corresponding NC group cells, both the overexpression and downregulation of miR-330-3p had no effect on the proliferation of MCF-7 and T47D cells (p > 0.05, Figure 2B). This also suggests that the influence of miR-330-3p on the malignant behavior of BC without affecting its proliferation.

Lnc021545 Knockdown Promotes the Motility, Migration, and Invasion of BC Cells
In MCF-7 and T47D cells, the lnc021545 expressions were downregulated by lnc021545 transient transfection. Compared with MCF-7-si-NC and T47D-si-NC ce lnc021545 expression was reduced by 62.3% (p = 0.0003) and 85.7% (p = 0.0005) in MCF si-lnc021545 and T47D-si-lnc021545 cells ( Figure 3A), respectively. MTT assay was measured the effect of lnc021545 on the proliferation of BC ce Compared to the si-NC cells, the knockdown of lnc021545 showed no influence on t proliferation abilities of MCF-7 and T47D cells at 24 h, 48 h, 72 h, 96 h, and 120 h (p > 0. Figure 3B). This indicates that lnc021545 affects BC progression without affecting its p liferative capacities.
Comparative cell motility was demonstrated from cells' movement via wound he ing assay based on scratch closure distance. As shown in Figure 3C, a relatively high scratch closure ratio was observed for si-lnc021545-transfected MCF-7 cells 24 h af wound scratches. The scratch closure distance of MCF-7-si-lnc021545 cells was measur as 54.0 ± 1.7 µm, which was 48.4% (p = 0.0009) more than that of MCF-7-si-NC cells. T indicates that lnc021545 downregulation might increase the motility of MCF-7 cells.
Boyden transwell assays were executed to investigate the effects of lnc021545 dow regulation on the migration and invasion of MCF-7 and T47D cells. Lnc021545 knockdow increased their migration and invasion capacities ( Figure 3D,E). The numbers of migrat MTT assay was measured the effect of lnc021545 on the proliferation of BC cells. Compared to the si-NC cells, the knockdown of lnc021545 showed no influence on the proliferation abilities of MCF-7 and T47D cells at 24 h, 48 h, 72 h, 96 h, and 120 h (p > 0.05, Figure 3B). This indicates that lnc021545 affects BC progression without affecting its proliferative capacities.
Comparative cell motility was demonstrated from cells' movement via wound healing assay based on scratch closure distance. As shown in Figure 3C, a relatively higher scratch closure ratio was observed for si-lnc021545-transfected MCF-7 cells 24 h after wound scratches. The scratch closure distance of MCF-7-si-lnc021545 cells was measured as 54.0 ± 1.7 µm, which was 48.4% (p = 0.0009) more than that of MCF-7-si-NC cells. This indicates that lnc021545 downregulation might increase the motility of MCF-7 cells.

EREG Influences the Motility, Migration, and Invasion of BC Cells
To explore the role of EREG in MCF-7 and T47D cells, si-NC, si-EREG, PCDH, or PCDH-EREG were transfected into them to downregulate and overexpress EREG. The WB assays revealed that the expression of EREG was reduced by 63.9% (p = 0.0023) in MCF-7 cells and 73.3% (p = 0.0019) in T47D cells and increased by 162.7% (p = 0.0177) in MCF-7 cells and 146.0% (p = 0.0166) in T47D cells compared to their respective NC cells ( Figure 4A).
The effect of EREG on the proliferations of MCF-7 and T47D cells were assessed via MTT assay. The expression changes of EREG expression had no effect on the proliferations of either cell at 24 h, 48 h, 72 h, 96 h, and 120 h (p > 0.05, Figure 4B). These data suggest that EREG involves in BC malignancy without affecting BC cells' proliferation.
As shown in Figure 4C, the scratch closure distance of MCF-7-si-EREG cells was 41.8 ± 2.3 µm, which was 46.7% (p = 0.0008) more than that of MCF-7-si-NC cells. Compared to the PCDH group, the scratch closure distance of MCF-7-PCDH-EREG cells was 9.4 ± 0.6 µm with a reduction of 59.3% (p = 0.0015). Therefore, the downregulation of EREG promotes the motility of MCF-7 cells, while EREG overexpression showed the opposite effect. Hence, EREG negatively affects the motility of BC cells.
The influences of EREG expression on the migration and invasion in MCF-7 and T47D cells were studied via transwell assays. EREG downregulation increased the migration and invasion of both cells ( Figure 4D

Lnc021545 Acts as a Sponge for miR-330-3p in BC Cells
To confirm the binding between miR-330-3p and lnc021545 in BC, we first used bioinformatic analysis to predict the potential binding of the 1264-1269 GCUUUG sequence at site of lnc021545 with the CGAAAC sequence of miR-330-3p ( Figure 5A). To prove this point, we subcloned wild-type (WT lnc021545) and mutant-type (MUT lnc021545) miR-330-3p binding sites into the dual-luciferase reporters. As expected, co-transfection of miR-330-3p mimics substantially decreased the relative luciferase activity of the WT lnc021545 in MCF-7 cells (42.3%, p = 0.0017) but showed no effect on that of the MUT lnc021545 ( Figure 5A), which suggests the direct binding of miR-330-3p with lnc021545.
To further verify the negative correlation between miR-330-3p and lnc021545, we analyzed the relationship between miR-330-3p and lnc021545 in BC patients' tissues. Our previous results revealed that in 50 paired BC patients' samples, miR-330-3p expression was notably increased and lnc021545 expression was the opposite, and we observed a significantly negative association between miR-330-3p expression and lnc021545 expression (R2 = 0.4880, p < 0.0001, Figure 5E). Lnc021545 deregulation is involved in BC carcinogenesis by reversely mediating the expression of miR-330-3p.

miR-330-3p Expression Is Negatively Correlated with EREG Expression in BC
To demonstrate that miR-330-3p binds to EREG in BC, we first predicted that the GCUUUG at 3,803-3,808 site of EREG-3'-UTR might complementarily bind to the CGAAAC sequence of miR-330-3p ( Figure 6A). To verify whether EREG was a target gene of miR-330-3p in BC, through the dual-luciferase reporter assay, we found that miR-330-3p mimic remarkably decreased the relative luciferase activity of the WT EREG (32.7%, p

miR-330-3p Expression Is Negatively Correlated with EREG Expression in BC
To demonstrate that miR-330-3p binds to EREG in BC, we first predicted that the GCUUUG at 3,803-3,808 site of EREG-3 -UTR might complementarily bind to the CGAAAC sequence of miR-330-3p ( Figure 6A). To verify whether EREG was a target gene of miR-330-3p in BC, through the dual-luciferase reporter assay, we found that miR-330-3p mimic remarkably decreased the relative luciferase activity of the WT EREG (32.7%, p = 0.002) in MCF-7 cells but did not affect that of the MUT EREG ( Figure 6A). These data provide evidence that miR-330-3p can directly bind to EREG-3 -UTR.
The negative correlation of EREG expression alteration with miR-330-3p differential expression was greater in 50 paired BC patients' tissues (R 2 = 0.2014, p = 0.0011, Figure 6E). Furthermore, we investigated the EREG expression profiles in the above-mentioned tissues by IHC. In the previous experiments, these patients have already been detected for lnc021545 expression and miR-330-3p expression. MiR-330-3p and EREG differential expression changes were allocated to low and high expression groups based on the median H-score of EREG protein expression ( Figure 6F,G). The expression alteration of miR-330-3p showed an apparent negative correlation with EREG protein expression alteration (R 2 = 0.2768, p < 0.0001, Figure 6H). Altogether, our data declare that EREG is a directly downstream goal of miR-330-3p via binding to its 3 -UTR to negatively mediate EREG expression in BC. = 0.002) in MCF-7 cells but did not affect that of the MUT EREG ( Figure 6A). These data provide evidence that miR-330-3p can directly bind to EREG-3'-UTR. The changes in EREG expression following transfection with miR-330-3p mimic or inhibitor in MCF-7 and T47D cells were checked using qRT-PCR and WB assays. The results as shown in Figure 6B-D demonstrated that overexpression of miR-330-3p downregulated the mRNA and protein levels of EREG in MCF-7 (59.1%, p = 0.0009, 54.7%, p = 0.0005) and T47D (65.5%, p = 0.0005, 49.8%, p = 0.0110) cells. Accordingly, miR-330-3p knockdowns increased endogenous EREG expression. The EREG mRNA and protein levels were increased in MCF-7 (124.8%, p = 0.0242, 63.3%, p = 0.0114) and T47D (190.7%, p = 0.0249, 98.8%, p = 0.0016) cells ( Figure 6B-D). The data indicated that miR-330-3p reversely mediates EREG expression by targeting the latter's 3-UTR in BC cells.
The negative correlation of EREG expression alteration with miR-330-3p differential expression was greater in 50 paired BC patients' tissues (R 2 = 0.2014, p = 0.0011, Figure 6E). Furthermore, we investigated the EREG expression profiles in the above-mentioned tissues by IHC. In the previous experiments, these patients have already been detected for lnc021545 expression and miR-330-3p expression. MiR-330-3p and EREG differential expression changes were allocated to low and high expression groups based on the median
To further confirm the correlations between lnc021545 and EREG, we analyzed the relationship between lnc021545 and EREG expression level changes in BC tissues. We observed that lnc021545 had a positive relationship with EREG mRNA expression (R 2 = 0.1572, p = 0.0044, Figure 7C) and the protein expression level of EREG (R 2 = 0.2754, p < 0.0001, Figure 7D).
To clarify the mechanism of lnc021545-miR-330-3p-EREG axis plays in BC metastasis, we recognized the extraordinarily regulated functional gene sets via single-gene GSEA. It showed a significantly negative correlation between EREG (NES = −2.455, FDR = 0.0007) and EMT ( Figure 7H Figure 7I). These data indicate that the lnc021545-miR-330-3p axis mediated EMT process to inhibit the metastasis of BC cells via specifically suppressing EREG expression, and it verified that the effect of the lnc021545-miR-330-3p axis in repressing the metastasis of BC primarily depends on the EREG to influence the changes of EMT markers.

The Clinical Feature and Cooperative Effects of lnc021545, miR-330-3p, and EREG in BC Patients
The relative expressions of miR-330-3p, lnc021545, and EREG were already measured in 50 BC patients' tissues using the qRT-PCR assay mentioned above. Moreover, we underwent 7 years of follow-up for 50 patients and stratified clinical pathological features from them through expressions of miR-330-3p, lnc021545, and EREG. Fifty patients were allocated to different expression levels according to the median of the three molecules expressions in BC tissues to establish the connection of clinicopathologic features and prognosis of BC. Fifty patients were allocated to different expression levels according to the H-score medians of ER, PR, and Ki-67 protein expression by IHC ( Figure 8A-F), and the three molecules are all expressed in the nucleus. The median of the H-scores of ER, PR, and Ki-67 respectively 137.1, 125.0, and 88.1, respectively. Fifty BC patients were allocated to the groups of HER-2 amplification and HER-2 un-amplification based on the results of FISH assay ( Figure 8G,H). Accordingly, 15 out of 50 BC patients had HER-2 amplification, while the remaining 35 patients had HER-2 un-amplification. prognosis of BC. Fifty patients were allocated to different expression levels according to the H-score medians of ER, PR, and Ki-67 protein expression by IHC ( Figure 8A-F), and the three molecules are all expressed in the nucleus. The median of the H-scores of ER, PR, and Ki-67 respectively 137.1, 125.0, and 88.1, respectively. Fifty BC patients were allocated to the groups of HER-2 amplification and HER-2 un-amplification based on the results of FISH assay ( Figure 8G,H). Accordingly, 15 out of 50 BC patients had HER-2 amplification, while the remaining 35 patients had HER-2 un-amplification. Lnc021545 was significantly downregulated in 50 BC patients' cancerous tissues (p < 0.0001, Figure 8I). We found that low expression of lnc021545 was closely associated with some clinical features (Table 1)-for example, tumor size (p = 0.0025), advanced TNM stage (p = 0.003), lymph node metastasis (p = 0.01), and HER-2 amplification (p = 0.012). The results indicated that the reduced expression of lnc021545 is detected in BC and may be involved in the progression and trastuzumab therapy of BC. In these clinical data, we also found that lnc021545 was not related to age; position; vascular invasion; nerve Lnc021545 was significantly downregulated in 50 BC patients' cancerous tissues (p < 0.0001, Figure 8I). We found that low expression of lnc021545 was closely associated with some clinical features (Table 1)-for example, tumor size (p = 0.0025), advanced TNM stage (p = 0.003), lymph node metastasis (p = 0.01), and HER-2 amplification (p = 0.012). The results indicated that the reduced expression of lnc021545 is detected in BC and may be involved in the progression and trastuzumab therapy of BC. In these clinical data, we also found that lnc021545 was not related to age; position; vascular invasion; nerve invasion; the expression of ER, PR, or Ki-67; or the mutant of PIK3CA (p > 0.05). To investigate lnc021545's prognostic value, we analyzed the effects of lnc021545 status on iDFS in 50 BC patients. The iDFS of cases with high lnc021545 expression were better than those of cases with low lnc021545 expression (p = 0.0085, Figure 8L). These results implicate that lnc021545 plays a suppressor role in BC.
MiR-330-3p expression was markedly increased in BC tumorous tissues (p < 0.0001, Figure 8J). MiR-330-3p was also closely associated with some clinical features (Table 1). High expression of miR-330-3p was related to the tumor size (p < 0.0001), advanced TNM stage (p < 0.0001), lymph node metastasis (p = 0.001), pathological stage (p = 0.012), and HER-2 amplification (p = 0.001). MiR-330-3p overexpression was positively associated with the progression and trastuzumab therapy of BC. Moreover, high miR-330-3p expression EREG expression was significantly reduced in 50 cancerous tissues (p < 0.0001, Figure 8K). As shown in Table 1, low EREG expression was associated with the tumor size (p < 0.0001), advanced TNM stage (p = 0.003), lymph node metastasis (p = 0.045), HER-2 amplification (p = 0.001), and ER expression (p = 0.023). Our data reveal that EREG normal expression may play a positive effect in inhibiting the progression and trastuzumab therapy in BC. Moreover, high EREG expression had a longer iDFS than a low EREG expression (p = 0.0099, Figure 8N) in BC patients and a better prognostic. The above results suggest that EREG might be a good prognostic factor of BC by inhibiting BC metastasis.
To explore whether lnc021545, miR-330-3p, and EREG work together to regulate the malignancy of BC, a cooperative ratio introduced by us as the sum of the relative expressions of lnc021545 and EREG divided by the relative expression of miR-330-3p was applied to address the cooperative prediction utilization of the three molecules with certain clinicopathologic features of BC. Interestingly, the introduction of this parameter led us to notice a low cooperative ratio was associated with tumor size (p = 0.05), advanced TNM stage (p = 0.008), lymph node metastasis (p = 0.035), and HER-2 amplification (p = 0.021), as shown in Table 1. The data declare that the cooperative ratio can be used to speculate on the progression and trastuzumab therapy of BC. A lower cooperative ratio resulted in a poorer iDFS (p < 0.0001, Figure 8O) and a shorter survival duration in month of iDFS ( Figure 8P, R 2 = 0.2049, p = 0.001) for BC patients. In our study, we observed that 10 deaths all occurred at a low cooperative ratio, a low expression of lnc021545, or a high expression of miR-330-3p, while only 6 deaths occurred at a low expression of EREG. More interestingly, recurrence occurred in all the patients cataloged with low cooperative ratios, while the patients without recurrence were either characterized with low lnc021545 expression, with high miR-330-3p expression, or with low EREG expression, which suggests that the integrated comparative utilization of low expressions of lnc021545 and EREG and high expression of miR-330-3p more accurately assess the prognosis of patients than any single factor.

Discussion
BC has become a significant health problem in modern society [1]. In the past few years, the metastasis and poor prognosis of BC have presented a great challenge despite the progression of chemotherapy, endocrine therapy, and HER-2-targeted therapy [21].
Lymphatic metastasis is the most important mode by which BC spreads, mainly including axillary lymphatic metastasis and parasternal lymphatic metastasis [5]. The management of lymphatic metastasis has become an important link in the treatment of BC. Therefore, the research on lymphatic metastasis of BC has become an important part of the diagnosis and treatment of BC. In previous research, the high-throughput genetic sequencing assays using two murine hepatocarcinoma cell lines with different lymphatic metastasis potentials from our group (unpublished) prompted us to believe that miR-330-3p, lnc021545, and EREG might also form a regulation mechanism in human cancers. Through analyzing the TCGA-BRCA dataset, we noticed that miR-330-3p was overexpressed in BC tumorous tissues. Patients with low expression of miR-330-3p showed longer iDFS probabilities than those with high expression of miR-330-3p. We used KMplot to analyze the association between EREG expression and BC patients' OS. We found that patients with high EREG expression showed better OS probabilities than the patients with low EREG expression. As a novel lncRNA, profile of lnc021545 in BC is unavailable. Herein, its role in BC was not analyzed. From the analysis of the database, it can be seen that miR-330-3p might be a tumor promoter and that EREG might be a tumor suppressor in BC. We herein investigated their mutual relationship and their correlation together with their function mechanism and potential clinical practice in BC metastasis.
MiRNAs play important roles in the progressions and treatments of multiple tumors [22]. Earlier research has confirmed that miR-330-3p acted as a tumor promoter in multiple cancers. miR-330-3p acted as a tumor promoter in multiple cancers. It accelerated the migration and EMT of NSCLC cells via GGRIA3-TGFβ1 [23]. In hepatocellular carcinoma, miR-330-3p enhanced cancer cells' viability, migration, invasion, and apoptosis resistance by binding to BTG1 [24]. MiR-330-3p targeted the CCBE1 and reduced its expression to promote the metastasis of BC [25]. MiRNAs have multiple target genes and affect the progression of diseases by regulating the interaction of multiple genes, even in the same disease. For example, miR-200 activates the EMT by targeting HIPK1 or HDAC2 to promote the metastasis of BC [26,27]. Our study aims to demonstrate not only the role of miR-330-3p in BC metastasis but also its correlations with lnc021545 and EREG together with their axial regulation mechanism in BC metastasis.
LncRNAs are a type of new-style biomarkers and potential therapeutic targets in mediating BC progression and metastasis through regulating gene expression [28,29]. Currently, the research on lnc021545 in disease is limited. We found that lnc021545 knockdown promoted the metastatic abilities of BC cells rather than altering their proliferations. As a member of the EGR family, EREG can bind to each of EGFR and ErbB-4 through ligandinduced heterodimerization, which affects the progression of diseases through activating signaling pathways downstream of ErbB1/ErbB [30]. EREG has largely been researched in the prognosis and drug resistance of BC [31], but little is known about its action mechanism in BC progression. In BC cells, EREG also reduced the tendency of metastasis but had no effect on the proliferation. Therefore, lnc021545 and EREG deficiency promotes the progression of BC by increasing the metastatic behaviors of BC cells.
We discovered that the changes in the three molecules' expression levels consistently conferred greater metastasis of BC cells rather than altering proliferation. We also found this phenomenon in BC tumorous samples; there was no correlation between any of the three molecules and the expression of Ki-67, which is a marker of cell proliferation and reflects the degree of proliferation in BC. Consequently, these data suggest that the three molecules regulate BC patients' outcomes and treatment through mediating the metastasis of BC cells.
In recent studies, the association between miRNAs and lncRNA has been the most popular as lncRNAs can bind to miRNAs to antagonize miRNA-induced activity [32]. We found that lnc021545 sponges miR-330-3p in BC cells. Bioinformatic analysis and dual-luciferase reporter assay demonstrated that lnc021545 targeted miR-330-3p to inhibit the latter's expression. Our study also provided the relationship between lnc021545 and miR-330-3p in BC cells. Lnc021545 knockdown resulted in miR-330-3p upregulation, and the change in lnc021545 or miR-330-3p expression resulted in the corresponding change in miR-330-3p or lnc021545 expression in BC cells. MiR-330-3p and lnc021545 expression with a negative relationship was shown in the clinical data of 50 BC patients. By binding to each other to reversely regulate the expression of the other one, lnc021545 and miR-330-3p are functionalized in the metastasis of BC.
In order to prove that lnc021545, miR-330-3p, and EREG conform to this regulatory mode to modulate the metastasis of BC, we proved that lnc021545 could combine with miR-330-3p to affect BC's metastasis in previous experiments, and we still need to prove that miR-330-3p and EREG could combine and interact with each other. Therefore, we proved that miR-330-3p could directly bound to 3 -UTR of EREG by using the online target gene prediction tool and the dual-luciferase reporters' assay. Meanwhile, EREG expression levels was remarkably reversely regulated by the expression level change of miR-330-3p upregulated in BC cells. Further analysis confirmed that the mRNA and protein expression of EREG had a significant negative correlation with miR-330-3p in BC patients. We further showed that EREG mRNA and protein levels had an obviously positive association with lnc021545. Subsequently, EREG expression was remarkably suppressed in si-lnc021545 transfected cells. Our data demonstrate that lnc021545, miR-330-3p, and EREG are closely correlated in regulating the BC's metastasis.
Cumulative evidence has pointed out that lncRNAs could modulate the biological activity of mRNA via hindering the miRNA from binding to its target mRNAs [33]. Although we failed to overexpress lnc021545 in BC cells, its knockdown in BC cells still clearly led us to detect the increase of miR-330-3p expression and the decrease of EREG expression. The overexpression or knockdown of miR-330-3p level led to reduced or increased expressions of lnc021545 and EREG in BC cells. miR-330-3p was consistently inversely related to lnc021545 and EREG in BC tissues. EREG was positively related to lnc021545 in BC tissues. Meanwhile, we also confirmed that miR-330-3p had direct bindings between lnc021545 and EREG. In summary, lnc021545, miR-330-3p, and EREG form a negative feedback regulation pathway in promoting BC. Nevertheless, the functions and biological mechanism of the lnc021545-miR-330-3p-EREG axis in the metastasis of BC are unclear.
EMT has been demonstrated as an essential driver of carcinoma metastasis [34][35][36]. Growing evidence has proved that miRNAs regulated the EMT by interacting with certain targeted mRNAs to impact carcinoma metastasis. miR-508-3p hindered the EMT by targeting ZEB1 to inhibit cell invasion in triple-negative breast cancer [37]. Noyan et al. demonstrated that miR-770-5p targeted DNMT3A to suppress the EMT and the invasion of triple-negative breast cancer cells [38]. In earlier research, we confirmed EREG was one of the downstream target genes of miR-330-3p. Specifically, we confirmed that miR-330-3p could reduce endogenous EREG protein expression to promote and restore the metastasis of BC cells. Their mechanisms for the changes in metastasis of BC were elusive. Single-gene GSEA database analysis indicated EREG had a negative correlation with EMT. We consistently detected that miR-330-3p overexpression increased the expressions of mesenchymal molecules vimentin and N-cadherin, repressors of the epithelial molecules Snail and Slug, but decreased the expression of the epithelial molecule E-cadherin in MCF-7 cells via directly targeting EREG. These data confirm that lnc021545 affects the metastasis of BC by inhibiting miR-330-3p regulation on EREG expression through EMT process (Figure 9). gle-gene GSEA database analysis indicated EREG had a negative correlation with EMT. We consistently detected that miR-330-3p overexpression increased the expressions of mesenchymal molecules vimentin and N-cadherin, repressors of the epithelial molecules Snail and Slug, but decreased the expression of the epithelial molecule E-cadherin in MCF-7 cells via directly targeting EREG. These data confirm that lnc021545 affects the metastasis of BC by inhibiting miR-330-3p regulation on EREG expression through EMT process ( Figure 9). After we verified the mechanism by which the three molecules affect the metastasis of BC, we further observed the clinical effect of the three molecules on the therapeutic outcome and prognosis of BC patients. In the process of individualized treatment of BC, the expression of ER and PR, amplification of HER-2, and mutation of PIK3CA are some treatment-related indicators [39,40]. ER and PR expression levels correlate with endocrine therapy in BC and are the indexes used to develop therapeutic strategy and its efficacy [39]. The detection of HER-2 amplification is very crucial for the prognosis and treatment of BC; the amplification of the HER-2 gene suggests that BC patients have a high degree of malignancy and poor prognosis, which is suitable for targeted therapy [39]. Mutations of PIK3CA are one of the most common oncogenic mutations that occur in BC patients who may benefit from the treatments with PI3K inhibitors [39].
In 50 BC patients, we observed that miR-330-3p expression was elevated in cancerous tissues and related to tumor size, TNM stage, lymph node metastasis, pathological stage, and HER-2 amplification while lnc021545 and EREG were decreased in BC tissues and exerted the opposite effects of miR-330-3p in the BC patient cohorts. We also established that high expression of miR-330-3p had a shorter iDFS and high expression of lnc021545 and EREG were conducive to lengthening BC patients' iDFS. All of these suggest that miR-330-3p overexpression or lnc021545 or EREG downregulation contribute to the Figure 9. A schematic action mechanism of the lnc021545-miR-330-3p-EREG axis in the BC' malignancy.
After we verified the mechanism by which the three molecules affect the metastasis of BC, we further observed the clinical effect of the three molecules on the therapeutic outcome and prognosis of BC patients. In the process of individualized treatment of BC, the expression of ER and PR, amplification of HER-2, and mutation of PIK3CA are some treatment-related indicators [39,40]. ER and PR expression levels correlate with endocrine therapy in BC and are the indexes used to develop therapeutic strategy and its efficacy [39]. The detection of HER-2 amplification is very crucial for the prognosis and treatment of BC; the amplification of the HER-2 gene suggests that BC patients have a high degree of malignancy and poor prognosis, which is suitable for targeted therapy [39]. Mutations of PIK3CA are one of the most common oncogenic mutations that occur in BC patients who may benefit from the treatments with PI3K inhibitors [39].
In 50 BC patients, we observed that miR-330-3p expression was elevated in cancerous tissues and related to tumor size, TNM stage, lymph node metastasis, pathological stage, and HER-2 amplification while lnc021545 and EREG were decreased in BC tissues and exerted the opposite effects of miR-330-3p in the BC patient cohorts. We also established that high expression of miR-330-3p had a shorter iDFS and high expression of lnc021545 and EREG were conducive to lengthening BC patients' iDFS. All of these suggest that miR-330-3p overexpression or lnc021545 or EREG downregulation contribute to the progression of BC and a poor prognosis while raising the chances for BC patients to benefit from trastuzumab therapy.
In summary, we found that miR-330-3p, lnc021545, and EREG had links to the prognosis and treatment of BC, but there were some differences among the three molecules. The process of tumor requires the cooperation of multiple genes [16]. Therefore, we further analyzed the above results and tried to find an expressive mode to more accurately reflect the synergistic effect of the three molecules on the progression and treatment of BC. We established the expressive mode of the cooperative ratio according to the relationship and functions of the three molecules. Interestingly, we found that the patients with a low cooperative ratio had a more aggressive malignancy in BC patients, and the cooperative ratio was correlated with the size of the tumor, pathological TNM stage, lymphatic metastasis, pathological stage, and HER-2 amplification. It was concluded that the cooperative ratio had a good directivity for the malignant evaluation and clinical treatment of BC. More interestingly, the high cooperative ratio restrains the progression of BC and reduces the chance of trastuzumab therapy. Moreover, we also observed that a high cooperative ratio was conducive to lengthening BC patients' iDFS, and the cooperative ratio was correlated with the months of iDFS. The results of these two parts showed that the cooperation ratio was closely tried with BC patients' prognosis. Importantly, we found that the cooperative ratio of the three molecules was more comprehensive than each single molecule in the assessment of the patient's malignant grade, recurrence, and death. Current work indicates that the roles and synergistic cooperation of lnc021545, miR-330-3p, and ERGE in regulating the malignant behaviors of BC cells as well as in evaluating the progression, therapeutic treatment, and prognosis of BC patients.

Conclusions
Lnc021545 inhibits miR-330-3p expression to modulate EREG expression, thereby affecting BC metastasis through mediating EMT. Our results highlight the coordinated functions of lnc021545, miR-330-3p, and EREG in BC progression, metastasis, and therapy. The lnc021545-miR-330-3p-EREG axis may act as a pivotal role in BC and even in other cancers. The combinational utilization of the three molecules may serve as a more accurate indicator for the carcinogenesis and a more precise target for the anti-metastasis therapeutic and prognosis of BC.
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/jcm12072478/s1, Table S1: Sequences of si-RNAs for EREG and lnc021545 knockdown; Table S2: Sequences of primers and oligonucleotides.  Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Written informed consent has been obtained from the patients to publish this paper.

Data Availability Statement:
The raw data supporting the conclusions of this article will be made available by the authors without undue reservation.