HAND2-AS1 Works as a ceRNA of miR-3118 to Suppress Proliferation and Migration in Breast Cancer by Upregulating PHLPP2

Large quantities of long noncoding RNAs (lncRNAs) have been verified to exert vital functions in the process of breast cancer (BC). lncRNA heart and neural crest derivatives expressed 2-antisense RNA 1 (HAND2-AS1) was reported to suppress the development of several cancers. However, its detailed function in BC remained unclear. In the current study, HAND2-AS1 was discovered to be low expressed in BC cell lines, and overexpression of HAND2-AS1 could repress proliferation, migration, and invasion but facilitate apoptosis in BC cells. Moreover, HAND2-AS1 was found to act as a sponge of miR-3118 which was detected to be upregulated in BC cell lines. miR-3118 depletion could constrict the progression of BC. HAND-AS1 hindered the course of BC by reducing the expression of miR-3118. Besides, PHLPP2 was treated as a downstream target of miR-3118 under the selection of RNA pull-down assays. HAND2-AS1 inhibited the process of BC by enhancing expression of PHLPP2. In summary, our study testified that HAND2-AS1 suppressed BC growth by targeting the miR-3118/PHLPP2 axis, indicating that HAND2-AS1 could be regarded as a potential target for BC treatment.


Introduction
Breast cancer (BC) is a common cancer with high morbidity among females globally [1]. It was a great threat to female health and brought much trouble and disturbance to work and life. Great efforts were spared, and progress was achieved in treatment of BC [2]. It is extremely crucial to make an early prognosis for BC. Thus, it is necessary to have a thorough understanding of the molecular mechanism of BC.
Substantial long noncoding RNAs (lncRNAs) were reported to bring about vital effects on the progression of cancers [3,4]. UCA1 strengthened resistance to tamoxifen in BC via activating the Pl3K/AKT signaling pathway [5].
Numerous essays acclaimed that the competing endogenous RNA (ceRNA) regulatory system played huge roles in regulating the mechanism of cancers [10,11]. In this system, lncRNAs functioned as sponges of microRNAs (miRNAs), and their downstream target mRNAs were released to code into proteins [12,13]. NEAT1 was identified as an oncogene in BC by targeting miR-448/ZEB1 [14]. LINC01116 is competitively bound to miR-145 to regulate the expression of ESR1 in BC [15]. In this study, we mainly studied the role of HAND2-AS1 in the ceRNA system. miRNAs were a class of RNAs which were around 18-22 nucleotides in length without the capacity of coding proteins [16]. Emerging studies indicated that aberrant expression of miRNAs acted as oncogene or tumor inhibitors in cancers. miR-590-5p was reported as a tumor inhibitor in BC by interacting with SOX2 [17]. miR-198 exerted inhibitory effect on BC progression via targeting CUB [18]. Nonetheless, how miR-3188 took part in the BC progression remained unclear.
The main purpose of our current study was to explore the role of HAND2-AS1 in BC cells.

2.1.
Overexpression of HAND2-AS1 Repressed Proliferation, Migration, and Invasion in BC Cell. To investigate the role of HAND2-AS1 in BC, we used RT-qPCR assays to determine the expression of HAND2-AS1 in BC cell lines (MCF-7, MDA-MB-231, SK-BR-3, and MDA-MB-453) and normal mammary epithelial cell (MCF-10A). The results showcased that HAND2-AS1 was at a low level of expression in BC cell lines in comparison with MCF-10A (Figure 1(a)). Then, pcDNA3.1/HAND2-AS1 was transfected into MCF-7 and SK-BR-3 cells. The outcomes disclosed that HAND2-AS1 expression was rose dramatically in comparison with the normal control (Figure 1(b)). To explore the influence of HAND2-AS1 on the development of BC, we conducted gain-of-function assays. The ability of proliferation assessed by CCK8 and colony formation assays was attenuated via overexpression of HAND2-AS1 (Figures 1(c) and 1(d)). The apoptosis rate was ascended by overexpression of HAND2-AS1 (Figure 1(e)). The capacities of migration and invasion were lessened by pcDNA3.1/HAND2-AS1 in transwell assays (Figures 1(f) and 1(g)). The proteins of the epithelial-mesenchymal transition (EMT) process were examined by western blot. The consequences depicted that E-cadherin protein was enhanced but N-cadherin, MMP2, Vimentin, and slug proteins were declined by overexpression of HAND2-AS1 (Figure 1(h)). In brief, HAND2-AS1 was low expressed in BC cell lines and overexpression of HAND2-AS1 inhibited the process of BC.

miR-3118 Depletion Inhibited BC Proliferation While
Facilitating Apoptosis in BC. To analyze the role of HAND2-AS1 in the ceRNA regulatory system, we performed FISH assays, and the outcomes displayed that HAND2-AS1 accumulated in cytoplasm (Figure 2(a)). Subsequently, with the help of starBase, we discovered several miRNAs with binding sites for HAND2-AS1. These miRNA expressions were evaluated in cells transfected with pcDNA3.1/-HAND2-AS1. The results disclosed that only miR-3118 expression was decreased distinctly in contrast with the control group (Figure 2(b)). The binding sequences were exhibited by bioinformatics (Figure 2(c)). RNA pull-down assays were carried out, and the outcomes delineated that miR-3118-WT with biotinylation could make HAND2-AS1 abun-dant but miR-3118-Mut with biotinylation could not ( Figure 2(d)). We tested the expression of miR-3118 increased prominently by miR-3118 mimics in comparison with the control group (Figure 2(e)). The activity of plasmid built with HAND2-AS1-WT was declined remarkably by miR-3118 mimics, but there were no evident changes in that set with HAND2-AS1-Mut (Figure 2(f)). miR-3118 expression was appraised in BC cell lines, and it was showed that miR-3118 expression was extremely high in BC cell lines (Figure 2(g)). The expression of miR-3118 was found to be diminished in cells transfected with miR-3118 inhibitor (Figure 2(h)). Afterwards, we explored the effect of miR-3118 on the BC progression. miR-3118 inhibitor was transfected into cells. The results showcased that miR-3118 depletion could inhibit proliferation (Figures 2(i) and 2(j)) but foster the apoptosis rate (Figure 2(k)). And the capacity of migration and invasion was weakened by miR-3118 inhibitor and so did the EMT process (Figures 2(l)-2(n)). In short, miR-3118 was a target of HAND2-AS1, and depletion of miR-3118 could inhibit the process of BC.

PHLPP2 Was a Downstream
Target of miR-3118. Then, we searched the starBase and found that several mRNAs had possibilities to bind to miR-3118. RNA pull-down assays were applied, and the results manifested that biotinylated miR-3118-WT only could make PHLPP2 abundant while no changes could be seen in other mRNAs (Figure 4(a)). The binding sites between miR-3118 and PHLPP2 were predicted by bioinformatics (Figure 4(b)). miR-3118 inhibitor was transfected into cells, and the expression of PHLPP2 and proteins were measured by RT-qPCR and western blot. The results depicted that both PHLPP2 expression and proteins were increased by miR-3118 inhibitor (Figure 4(c)). RIP assays were applied, and the results revealed that HAND2-AS1, PHLPP2, and miR-3118 were enriched in Ago2 antibody not in IgG antibody (Figure 4(d)). Luciferase reporter assays were conducted, and the results showed that miR-3118 mimics diminished the activity of plasmid constructed with PHLPP2-WT but not that set with PHLPP2-Mut. After transfecting pcDNA3.1/HAND2-AS1 into cells,       2.6. Overexpression of HAND2-AS1 Constricted the Growth of BC In Vivo. The effects of HAND2-AS1 on BC growth were verified in vivo. MCF-7 cells were transfected into pcDNA3.1/HAND2-AS1 and pcDNA3.1 vector. And the cells were injected into nude mice. After culture for 28 days, the process of tumor growth was recorded. The tumors transfected with pcDNA3.1/HAND2-AS1 grew slower than that with pcDNA3.1 vector (Figure 6(a)). The tumors were taken out, and both volume and weight of tumors with pcDNA3.1/-HAND2-AS1 were smaller than that with pcDNA3.1 vector (Figures 6(b) and 6(c)). Immunohistochemistry was conducted to examine the expression of Ki67. The results revealed that Ki67 expression was declined conspicuously by pcDNA3.1/HAND2-AS1 compared with the normal control ( Figure 6(d)). Collectively, overexpression of HAND2-AS1 could repress tumor growth in vivo.

Discussion
In this study, we mainly analyzed HAND2-AS1 and discovered that it is downregulated in BC cell lines, which was in accordance with previous reports [19,20]. In addition, HAND2-AS1 could suppress proliferation, migration, and invasion but accelerated death in BC cells, which was consistent with the findings in the previous study about the HAND2-AS1 role in cancers [20,21]. In addition, there has not been research involving the effect of HAND2-AS1 in a breast cancer animal model. Therefore, we conducted experiments in vivo to disclose the influence of HAND2-AS1 upregulation on the progression of BC malignancy. The tumor volume in the mouse group with pcDNA3.1/-HAND2-AS1 was smaller than that in the normal group. So was the tumor weight, supporting that HAND2-AS1 works as an inhibitor in breast cancer.
Previous studies demonstrated that lncRNAs sponged miRNAs to regulate mRNA expression and thus the progression of cancers in the ceRNA regulatory system [22,23]. Specifically, HAND2-AS1 was reported to sponge miR-340-5p to upregulate BCL2L11, inducing cell apoptosis in BC [24]. In this research, we discovered HAND2-AS1 targeted and inhibited miR-3118 in BC cell lines. Also, miR-3118 depletion repressed proliferative, invasive, and migratory capacities and promoted apoptosis of BC cells. The rescue assays demonstrated that HAND2-AS1 could hinder the process of BC by suppressing miR-3118 expression.
PHLPP2 was unveiled as a tumor suppressor in multiple cancers acting as an inhibitor of AKT and inducing apoptosis of cancer cells [25,26]. On the other hand, microRNAs, lncRNAs, or other exogenous molecules interact with PHLPP2, thus modulating the cellular functions through the AKT pathway in tumors and diseases [27][28][29][30]. In our   10 BioMed Research International study, we selected out PHLPP2 by RNA pull-down assays from several mRNAs targeting miR-3118. Then, we knocked down the PHLPP2 in BC cells and discovered that inhibition of PHLPP2 greatly reversed the inhibitory impact on cell functions brought by HAND2-AS1. Taken together, this study showed that HAND2-AS1 sponges and inhibits miR-3118, curbing cell proliferation, migration, and invasion and inducing apoptosis of BC cells. Besides, miR-3118 suppressed PHLPP2 by targeting it while silencing PHLPP2 could alleviate the impact of HAND2-AS1 in BC. Therefore, we presented that HAND2-AS1 competed binding to miR-3188 with PHLPP2 and curbed the progression of breast cancer in vitro by elevating PHLPP2. In addition, in vivo assays also verified the suppressive effect of HAND2-AS1 in BC. However, there is one shortcoming in this present study. The subcutaneous tumor models are not sufficient for understanding of pathogenesis of breast cancer. Therefore, further tumor models are required to validate the role of HAND2-AS1 in the progression of breast cancer. To sum up, our studies confirmed that HAND2-AS1 could inhibit BC proliferation, migration, and invasion and induce apoptosis as a ceRNA of miR-3188 to elevate PHLPP2 expression, which poses a potential that HAND2-AS1/miR-3188/PHLPP2 might be a therapeutic axis for breast cancer in the future.

Real-Time qPCR (RT-qPCR).
Total cellular RNAs were extracted from cell samples via the TRIzol reagent (Invitrogen) to synthesize cDNA with the PrimeScript RT reagent kit (Takara Biotechnology, Tokyo, Japan). qPCR was developed using SYBR Green PCR Master Mix (Invitrogen) on a Bio-Rad IQ5 thermocycler (Bio-Rad Laboratories, Inc., Hercules, CA, USA). RNA levels of HAND2-AS1, miR-3118, and PHLPP2 in all groups were normalized to U6 or GAPDH using the 2 -ΔΔCT method. . BC cells in 96-well plates (5 × 10 3 cells/well) were incubated with CCK8 solution (Beyotime, Shanghai, China) in a 37°C, 5% CO 2 incubator for 48 h. Cell viability in different groups was monitored by assessing the optical density by a microplate reader at absorbance of 450 nm.

Colony Formation Assay. Clonogenic cells of MCF-7 and
SK-BR-3 were incubated in 6-well plates (1 × 10 3 cells/well), following 14 days of incubation. Cells were stained with 0.5% crystal violet in 4% paraformaldehyde, and colonies were counted in all groups as a reference to the cell proliferation capability.

Western
Blotting. The western blotting method was used to observe the relative protein expression in cells of all groups. Protein samples from MCF-7 and SK-BR-3 cells were separated by electrophoresis on 10% SDS polyacrylamide gels and transferred onto PVDF membranes which were then blocked with 5% skimmed milk at room temperature for 2 h. The incubation with primary antibodies against E-cadherin, N-cadherin, MMP2, Vimentin, slug, PHLPP2, and GAPDH, along with the corresponding secondary antibodies (all from Abcam, Cambridge, MA, USA), was performed prior to analysis of the enhanced chemiluminescence reagent (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

FISH Assay.
To investigate the localization of HAND1-AS1 in cells, we resorted to the FISH method. For the FISH assay, the HAND2-AS1 RNA probe was produced by Ribo-Bio. After treating MCF-7 and SK-BR-3 cells with Hoechst, stained cells were photographed by a laser scanning confocal microscope (ZEISS, Jena, Germany).
4.11. RNA Immunoprecipitation (RIP). In light of the guidebook of Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore, Bedford, MA, USA), 1 × 10 7 BC cells in RIP lysis buffer were subjected to immunoprecipitation with antibodies against Ago2 or control IgG (Millipore). After adding beads, the precipitates were assayed by RT-qPCR. The RIP method was used to assess the RNA enrichment in cells.

4.12.
In Vivo Assay. 6 weeks old of male nude mice were procured from the National Laboratory Animal Center (Beijing, China) and maintained in SPF animal laboratory, with the approval of the Animal Research Ethics Committee of Tianjin Medical University Cancer Institute and Hospital. MCF-7 cells transfected with pcDNA 3.1/HAND2-AS1 or pcDNA 3.1 were injected to mice subcutaneously for 28 days to form in vivo models of breast cancer, with tumor volume recorded every 4 days. The tumor samples from killed mice were weighed for further analysis.
4.13. Immunohistochemistry (IHC). The tumor tissue samples from in vivo study were fixed by 4% paraformaldehyde and embedded in paraffin. After cutting, the sections of 4 μm thick were subjected to IHC using anti-Ki67 (Santa Cruz Biotechnology), through which we evaluated the proliferation marker Ki67 in tumor tissues from the pcDNA 3.1/HAND2-AS1 group with the pcDNA 3.1 group as a control.
4.14. Statistical Analysis. All assays included three biological repeats. Data were processed by Student's test to compare between two groups, and ANOVA (one-way) was adopted when three or more groups were included in data in Graph-Pad PRISM 6 (GraphPad, San Diego, CA, USA), with p < 0:05 as the cutoff value.

Data Availability
The data used to support the findings of this study are available from the corresponding authors upon request.

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