miR-145-5p Overexpression Inhibits the Proliferation, Migration and Invasion of Esophageal Carcinoma Cells by Targeting ABRACL

Background: The study aimed to investigate the regulatory relationship between miR-145-5p and ABRACL, and tried to clarify the mechanisms underlying the proliferation, migration and invasion of esophageal cancer (EC) cells. Methods: Gene expression data related to EC were accessed from TCGA database and the “edgeR” package was used to screen the differentially expressed miRNA (DEmiRNAs) and genes (DEGs). TargetScan, miRDB and miRTarBase databases were used to predict potential targets for the target miRNA miR-145-5p. qRT-PCR and Western blot were performed to assess the expression of miR-145-5p and ABRACL in EC cells. Dual-luciferase reporter assay was performed for verication of the targeting relationship between miR-145-5p and ABRACL. Functional experiments including colony formation assay, Transwell migration and invasion assays were used to detect the proliferation, migration and invasion abilities of EC cells. Results: The expression of miR-145-5p was signicantly decreased in EC, while ABRACL was remarkably increased. In addition, there was a negative correlation identied between miR-145-5p and ABRACL expression levels. Overexpressing miR-145-5p was able to suppress cell proliferation, migration and invasion, whereas silencing miR-145-5p posed an opposite effect. In the meantime, ABRACL was identied as a direct target of miR-145-5p by dual-luciferase reporter assay. Furthermore, miR-145-5p could inhibit the expression of ABRACL, in turn inhibiting the proliferation, migration and invasion of EC cells. Conclusion: miR-145-5p functions on the proliferation, migration and invasion of EC cells via targeting ABRACL, and it may be a novel therapeutic target for EC treatment. follow-up individual); (F-G) qRT-PCR and Western Blot show the relative mRNA and protein levels of ABRACL in EC cell lines Eca-109, EC9706, KYSE150, KYSE180, BIC-1 and in normal esophageal cell line HET-1A;


Background
Esophageal carcinoma (EC) is a common gastrointestinal tumor characterized by a high incidence around the world, and it is the sixth most common cause leading to cancer-related death (Maghsudlu and Farashahi Yazd, 2017). Lung cancer, gastric cancer, hepatocellular carcinoma and EC are the four most common cancers in China. Among them, EC is the most prevalent disease and its incidence gradually increases . As there are no typical clinical symptoms manifested in the early stage of EC, the illness of many patients has progressed to an advanced stage when they are initially diagnosed, and that is the main reason for a high mortality. Special local vascular structure and abundant lymphatic capillaries are favorable factors for migration of EC cells, which contributes to the fact that metastasis has developed when most people are con rmed with EC. Esophageal squamous cell carcinoma (ESCC) is the most common type of EC with a relative higher incidence in China (Bohanes et al., 2012, Kamangar et al., 2006. At present, the treatment of EC is mainly based on surgery combined with radiotherapy or chemotherapy. Such treatment can have a signi cant effect on the progress of illness, yet the overall prognosis is still poor. Therefore, identifying effective therapeutic targets is of great signi cance for the treatment of EC. MicroRNAs (miRNAs) are small non-coding RNAs that regulate translation or degradation of mRNA by binding to the 3'-UTR of their target mRNAs (Baer et al., 2016, Croce, 2009). MiRNAs play pivotal roles in tumors including EC by acting as oncogenes or tumor suppressor genes. For example, miR-124-3p directly targets the 3'-UTR region of BCAT1 in ESCC, and down-regulation of miR-124-3p is highly correlated with ESCC cell proliferation and migration (Zeng et al., 2019). In addition, miR-125b has been reported to be able to negatively regulate the expression of BCL-2-modifying factor (BMF) in ESCC by interacting with the 3'-UTR within BMF, and overexpressing miR-125b can signi cantly inhibit the growth of ESCC and induce apoptosis (Fan et al., 2018).
The human ABRACL (ABPA C-terminal like) gene locates on chromosome 6 and encodes a small protein of 81 amino acids, which can enhance actin activity and cell viability (Pang et al., 2010). ABRACL, an atypical winged-helix protein previously named as HSCP280, belongs to a new family of low molecular weight proteins and is only present in eukaryotes but absent in fungi, with highly conserved sequences across different species (Lin et al., 2011). A mouse study conducted by Stylianopoulou et al. found that HSCP280 is a nucleoprotein that inhibits neuronal differentiation in vitro when it is overexpressed in Neuro2a cells, suggesting its involvement in the regulation of neural progenitor cell proliferation (Stylianopoulou et al., 2016). Studies show that ABRACL is associated with tumorigenesis and proliferation (Caffarel et al., 2008, Ura et al., 2017. However, its role in EC has not been reported. The aim of this study was to investigate the expression of miR-145-5p in EC cells and its role in cell proliferation, migration and invasion, and also tried to clarify the underlying mechanisms.

3 Vector construction and cell transfection
MiR-145-5p mimic, miR-145-5p inhibitor, si-ABRACL and their negative controls purchased from GenePharma (Shanghai, China) were transiently transfected into EC cells by Lipofectamine 2000 (Thermo Fisher Scienti c, Inc.) and maintained in corresponding medium under the environment of 5% CO 2 at 37 °C. The lentiviral expression vector pLVX-IRES-neo (Clontech) was used to establish ABRACL overexpression vector oe-ABRACL, which was then used to infect cancer cells with the virus particle as the negative control. used to compare the difference in the relative expression of the target mRNA and miRNA between the control group and the experimental group. The experiment was repeated three times.

Western Blot
After transfection for 48 h, cells were washed 3 times with cold PBS. Whole cell lysate was added for cell lysate on ice for 10 min and the BCA protein assay kit (Thermo, USA) was applied for determination of protein concentration. A measure of 30 μg of protein samples were processed for separation by polyacrylamide gel electrophoresis (PAGE) at a constant voltage of 80 V for 35 min followed by 120 V for 45 min, and sequentially transferred to polyvinylidene uoride (PVDF) membranes (Amersham, USA).
After being blocked with 5% skim milk for 1 h at room temperature, the membranes were incubated with ABRACL rabbit polyclonal antibody (ab163694, 1:1000, abcam, Cambridge, UK) and GAPDH rabbit polyclonal antibody (ab9485, 1:2500, abcam, Cambridge, UK) overnight at 4 °C. The membranes were washed 3 times with PBST (Phosphate buffered saline containing 0.1% Tween-20) for 10 min each time. Subsequently, the membranes were incubated with horseradish peroxidase (HRP) -labeled secondary antibody goat anti-rabbit IgG H&L (ab6721, 1:3000, abcam, Cambridge, UK) for 1 h at room temperature. The membranes were washed 3 times with PBST buffer. An optical luminometer (GE, USA) was employed for visualization of protein bands, and the Image Pro Plus 6.0 (Media Cybernetics, USA) software was applied for further analysis.
1.6 Colony formation assay 24 h post transfection, cells were seeded at 5×10 2 cells/well in 6-well plates. After 10 days, the colonies of the viable cells were xed with 4% paraformaldehyde and then stained with 0.1% crystal violet. Stained colonies were observed and calculated. The experiment was repeated three times.

Transwell
Transwell migration assay: Cancer cells in logarithmic growth phase were starved for 24 h. On the following day, cells were digested, centrifuged and resuspended to a nal concentration of 2×10 5 cells/ml. A measure of 0.2 ml of cell suspension was added into the Transwell inserts, while 700 μl of precooled RPMI-1640 medium containing 10% FBS was added into the plates. Cells were cultured in an incubator containing 5% CO 2 at a temperature of 37 °C. After 24 h, the cells still in the inserts were wiped off with a wet cotton swab, and the cells migrated out of the inserts were xed using methanol for 30 min and then stained by 0.1% crystal violet for 20 min. Finally, cells were observed under an inverted microscope and ve elds of view were randomly selected for cell count.
Transwell invasion assay: 24-well Transwell chambers (8 μm in aperture, BD Biosciences) were used for Transwell invasion assay. Approximately 2×10 4 cells were plated in the upper chambers that were precoated with Matrigel matrix (Corning, Corning, NY). RPMI-1640 medium containing 10% FBS was lled into the lower chambers. The following procedures were as similar as the above migration assay.

Statistical analysis
All data were processed using SPSS 22.0 statistical software, and the measurement data were expressed in the form of mean ± standard deviation. Student's t test and one-way analysis of variance (ANOVA) were applied for analyzing the comparisons between two groups and among multiple groups. P<0.05 indicates statistically signi cant difference.
A total of 158 differentially expressed miRNAs (DEmiRNAs) were screened via differential analysis using the "edgeR" package (Fig. 1A). Among the DEmiRNAs, miR-145-5p was found to be signi cantly decreased in EC tumor tissues (n=153) relative to that in the normal tissues (n=11) in the TCGA database (Fig. 1B). In addition, EC cell lines Eca-109, EC9706, KYSE150, KYSE180 and BIC-1 and normal esophageal cell line HET-1A were collected for examination of miR-145-5p expression. qRT-PCR was performed and it was showed that compared with HET-1A, the expression of miR-145-5p was signi cantly reduced in EC cells especially in Eca-109 (Fig. 1C). Thus, Eca-109 cell line was chosen for subsequent experiments for further analysis.

miR-145-5p inhibits the proliferation, migration and invasion of EC cells
A series of functional experiments were conducted for identifying the mechanism by which miR-145-5p regulates EC cell biological behaviors. Mimic NC, miR-145-5p mimic, inhibitor NC and miR-145-5p inhibitor were transfected into Eca-109 cells. Colony formation assay suggested that the number of cell colonies was greatly decreased in the miR-145-5p mimic group relative to that in the mimic NC group, but signi cantly increased in the miR-145-5p inhibitor group in comparison with that in the inhibitor NC group ( Fig. 2A). Similarly, cell migration and invasion abilities were both reduced in the miR-145-5p mimic group but enhanced in the miR-145-5p inhibitor group relative to those in the NC groups, as judged by Transwell migration and invasion assays ( Fig. 2B-C). Overall, all results indicated that high expression of miR-145-5p could inhibit the proliferation, migration and invasion of EC cells.

ABRACL is a direct target of miR-145-5p
Differential analysis showed that there were 3,224 differentially expressed mRNAs (DEmRNAs) identi ed in the TCGA-ESCA dataset (Fig. 3A). TargetScan, miRDB and miRTarBase databases were applied to predict the target mRNAs of miR-145-5p, which were then mapped into a Venn diagram with the upregulated DEmRNAs in the TCGA-ESCA dataset, resulting in 10 overlapping mRNAs (Fig. 3B). Among the 10 mRNAs, four genes including TGFBR2, ABRACL, ZBTB47 and MASTL were detected to be signi cantly associated with the prognosis of EC patients as revealed by survival analysis based on the clinical samples (n=204) obtained from the TCGA-ESCA dataset. Meanwhile, ABRACL was shown to be mostly elevated in EC tissue samples (n=176) relative to that in the normal tissue samples (n=13) (Fig. 3C). Additionally, Pearson correlation analysis suggested that there was a negative correlation between the expression levels of miR-145-5p and ABRACL (Fig. 3D). In order to validate whether ABRACL is associated with the survival of patients with EC, a total of 114 samples (including 57 sample with low ABRACL and 57 samples with high ABRACL) and corresponding complete clinical data were accessed from TCGA database and then used for survival analysis (Fig. 3E). Results showed that high expression of ABRACL was able to predict poor prognosis. In the meantime, the expression of ABRACL was test in EC cell lines. Compared with HET-1A, the expression of ABRACL in EC cell lines was remarkably elevated (Fig. 3F-G).
To further understand the regulatory effect of miR-145-5p on ABRACL, the expression of ABRACL was detected in cells with miR-145-5p overexpression and the results showed that overexpression of miR-145-5p signi cantly decreased the expression of ABRACL (Fig. 3H-I). Additionally, TargetScan database was applied and showed that there were potential binding sites of miR-145-5p on ABRACL 3'-UTR (Fig. 3J). Dual-luciferase reporter assay was conducted for further veri cation and indicated that overexpression of miR-145-5p signi cantly decreased the luciferase activity in Eca-109 co-transfected with ABRACL-WT but had no effect in cells containing ABRACL-MUT. Collectively, these results supported the notion that ABRACL was a direct target of miR-145-5p.

miR-145-5p regulates ABRACL to affect the proliferation, migration and invasion of EC cells
To further explore the regulatory mechanism by which miR-145-5p targets ABRACL to mediate cell biological behaviors in EC, rescue experiments were performed in Eca-109 cells. Firstly, we silenced ABRACL in Eca-109 cells and found that the number of cell colonies was signi cantly reduced (Fig. 4A).
In previous experiments, we veri ed that low expression of miR-145-5p could promote cell proliferation. Therefore, we simultaneously transfected si-ABRACL and miR-145-5p inhibitor into Eca-109 cells, nding that the number of cell colonies was signi cantly lower than that of the miR-145-5p inhibitor group (Fig.  4A), and similar ndings were obtained in cell migration and invasion as detected by Transwell migration and invasion assays (Fig. 4B-C). It was suggestive that miR-145-5p affected the physiological activities of cancer cells by regulating the expression of ABRACL.

Discussion
MiRNAs compose a large family widely present in eukaryotic cells and some of them harbor a targeting relationship with mRNAs. Genomic analysis shows that there are over 5,300 human genes that miRNAs target, accounting for 30% of all human genes, and their expression shows an intimate correlation with cancers (Calin et al., 2004, Lewis et al., 2005. Studies have revealed that there are many miRNAs altered between EC patients and healthy people. miR-145 has been reported to be signi cantly down-regulated in ESCC tissue and cell lines and play an important role in inhibiting cancer cell proliferation, migration and metastasis. For example, miR-145 can directly target the 3'-UTR of PLCE1, and the down-regulation of PLCE1 induces cell apoptosis as well as enhances the sensitivity of tumor cells to chemotherapeutic drugs (Cui et al., 2016). Meanwhile, miR-145 is able to inhibit the growth of ESCC cells by targeting c-Myc (Wang et al., 2013). In addition, there is a study showing that miR-145 can target and interact with connective tissue growth factor (CTGF) to affect EC cell proliferation, migration, invasion and epithelialmesenchymal transition (EMT) process (Han et al., 2016). In our study, we also observed that the proliferation, migration and invasion of EC cells were affected signi cantly after miR-145-5p was overexpressed or inhibited, and high miR-145-5p negatively worked in EC progression. To further analyze the mechanism by which miR-145-5p regulates epithelial cell activities in EC, target prediction was performed and it was found that ABRACL was a potential target of miR-145-5p. Additionally, survival analysis revealed that there was a close relationship between ABRACL and the prognosis of EC patients, and dual-luciferase reporter gene assay further validated that miR-31-5p could target and bind with ABRACL to suppress its expression. Therefore, we speculated that miR-31-5p mediated EC progression probably via targeting the expression of ABRACL.
ABRACL, a winged-helix protein, plays an important role in a variety of developmental processes. Comparative expression analysis of HSPC280 with Dlx2, cyclinD2 and Lhx6 revealed that HSPC280 is restricted in the proliferating cell population in the subventricular zone within ganglionic eminences, with a pattern similar to that of cyclinD2 (Stylianopoulou et al., 2016). In addition, ABRACL has been detected to be up-regulated in gastric cancer tissue compared to that in normal gastric tissue, which is associated with poor prognosis. Meanwhile, enrichment analysis revealed that ABRACL is mostly enriched in some signaling pathways, such as cell cycle, TNFR pathway, proteasome degradation, and mitochondrial pathway (Wang et al., 2019). Studies have found that ABRACL bears a similar structure to ABD2 and may be a transcriptional synergistic activator (Fogl et al., 2012, Galkin et al., 2008, Lin et al., 2011) that is closely related to cell cycle (Baumann et al., 2018). In this study, dual-luciferase reporter assay demonstrated that ABRACL was a direct target of and negatively regulated by miR-145-5p. Meanwhile, functional experiments con rmed that silencing ABRACL could inhibit the proliferation, migration and invasion of cancer cells. Rescue experiments suggested that the effect of miR-145-5p silence on cell proliferation, migration and invasion could be attenuated upon ABRACL knockdown. These results suggest that miR-145-5p regulates the cellular functions of EC cells by targeting ABRACL.
In conclusion, our study rst proposes that miR-145-5p targets ABRACL to inhibit the proliferation, migration and invasion of EC cells, which helps to provide a novel therapeutic target for future EC treatment.

Declarations
Ethics approval and consent to participate Not applicable.

Consent for publication
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Availability of data and materials
The data and materials in the current study are available from the corresponding author on reasonable request.