LncRNA BDNF‐AS inhibits proliferation, migration, invasion and EMT in oesophageal cancer cells by targeting miR‐214

Abstract This study was aimed at exploring the effect of lncRNA BDNF‐AS on cell proliferation, migration, invasion and epithelial‐to‐mesenchymal transition (EMT) of oesophageal cancer (EC) cells. The expression of BDNF‐AS and miR‐214 in tissue samples and cells was measured by qRT‐PCR. The targeted relationship between BDNF‐AS and miR‐214 was analysed by dual‐luciferase reporter assay. After cell transfection, the cell proliferation activity was assessed by MTS method, while the migrating and invading abilities were evaluated by transwell assay. LncRNA BDNF‐AS was remarkably down‐regulated, while miR‐214 was up‐regulated in EC tissues and cells in comparison with normal tissues and cells. Overexpression of BDNF‐AS significantly inhibited the abilities of cell proliferation, migration and invasion as well as the EMT processes of EC cells. The bioinformatics analysis and luciferase assay indicated that BDNF‐AS could be directly bound by miR‐214. Furthermore, overexpression of miR‐214 and BDNF‐AS exerted suppressive influence on EC cell multiplication, migration, invasion and EMT processes. LncRNA BDNF‐AS restrained cell proliferation, migration, invasion and EMT processes in EC cells by targeting miR‐214.

epigenetically regulate coding DNAs to exert specific biological functions in human diseases. 5 The epithelial mesenchymal transition (EMT) is a crucial biological process for the migration and invasion of tumour cells derived from epithelial cells. At present, the study on the mechanisms related to the EMT in the EC has become a major focus of research considering that most patients have local infiltration and distant metastasis at diagnosis. 6 LncRNAs have been involved in multiple processes related to the EMT. 7 For instance, Li et al found that lncRNA ATB, which was induced by TGF-b, stimulated EMT through silencing miR-200s and facilitated multiplication by balancing IL-11 mRNA in hepatocellular carcinoma, thus promoting cancer metastasis. 8 Moreover, lncRNA H19 promotes cell invasion and migration in pancreatic ductal adenocarcinoma by increasing HMGA2-mediated EMT through antagonizing let-7. 9 LncRNA of brain-derived neurotrophic factor antisense (BDNF-AS) is a natural non-coding antisense of neuronal transcriptional factor BDNF. 10 BDNF-AS was discovered to act as an adverse regulator of BDNF and has profound impacts in neuronal system. 11 Although an increasing number of studies have revealed that BDNF could act as a cardinal oncogenic factor in human cancers, whether BDNF-AS could also act as a functional regulator or biomarker in human cancer remains little known. Thus, in our study, we investigated the expression, prognostic potential and functional mechanisms of BDNF-AS in EC.
MicroRNAs (miRNAs) are short non-coding single-stranded RNA molecules, which could regulate gene expression through base pairing with the 3 0 -untranslated region (3 0 -UTR) of target mRNAs, resulting in post-transcriptional suppression or mRNA degradation. 12 Growing findings have proved that microRNAs may play a critical role in carcinogenesis. 13 Accumulating studies have certified that both up-and down-regulation expression of miR-214 can be causative for the progress of various malignant tumours, including pancreatic, hepatoblastoma, hepatocellular, lung, breast and cervical. 14,15 However, the role of miR-214 in oesophageal cancer progression and the molecular mechanisms, in particular, EMT and subsequently invasion and metastasis, remains to be further investigated.
In this study, we present that BDNF-AS inhibits the capacity of cell proliferation, invading and migrating in EC by regulating the EMT. We

| Tissue samples and cell lines
Fifty-four pairs of surgical primary EC tissues, corresponding adjacent normal tissues and non-tumour samples, were obtained directly after surgical resection between the years of 2014 and 2016 at the First Affiliated Hospital of Zhengzhou University, China (Table S1). All biopsy specimens were directly frozen at À196°C (liquid nitrogen) and stored at À70°C before further treating. Ethical approval was ratified by the local Ethics Committee, and all patients provided written informed consent for the utilization of tissue samples in research.

| Quantitative real-time PCR (qRT-PCR)
Total RNA was extracted from biopsy specimens or in vitro EC cell lines with the TRIzol reagent (Invitrogen, USA) following the manufacturer's protocol. RNA quantification was carried out using a NanoDrop TM 3000 Spectrophotometer (Thermo Fisher Scientific, USA), and complementary DNA (cDNA) was generated using the First Strand cDNA Synthesis kit (Thermo, USA). QRT-PCR was conducted using a Platinum SYBR Super Mix (Stratagene, USA) according to the manufacturer's protocol. The brief reaction was as follows: 95°C for 5 minutes, then 40 cycles of 95°C for 15 seconds followed by 60°C for 3 seconds and finally 72°C for 30 seconds.
The change in transcript abundance was calculated using the mastercycler sample analysis software. The endogenous control for miR-214 was U6 RNA. Others tested in this study were standardized to GAPDH. Primers are reported in Table 1.

| MTS assay
Logarithmic growth phase cells were suspended and inoculated in 96-well plate with the density of 3000 cells/well in 100 lL medium. Each experiment was conducted in triplicate. Matrigel chambers were incubated at 37°C, the volume fraction of 5% CO 2 incubator for 24 hours. Migration assays were performed in a similar mode using chambers without the Matrigel coating. After incubation, the cells on the upper membrane surface were wiped away using a cotton swab and the cells from the bottom membrane surface were fixed with methanol, stained with 0.1% crystal violet.

| Transwell migration and invasion assay
Then, five areas were randomly chosen in each chamber.

| Western blot
OE19 and OE33 cells were collected and lysed with SDS lysis buffer (Promega, USA). After determining the concentration of protein with the BCA assay, the equal amount of protein samples was separated by 10% SDS-PAGE gel (BD, USA) and then transferred to PVDF membranes. After being disposed with 5% skimmed milk, these membranes were then blocked at room temperature for 1 hour followed by being washed 10 minutes for three times. For primary antibody incubation, PVDF membranes were treated with a rabbit antibody against N-cadherin, vimentin, E-cadherin and GADPH (1:200, Proteintech, USA) for all night at 4°C. After primary antibody treatment, membranes were washed 10 minutes for three times.
Afterwards, membranes were incubated with the secondary horseradish peroxidase-conjugated antibody (ROCKLAND, USA) for 1 hour at room temperature avoiding light. Membranes were washed once more 10 minutes for three times. The blots were assessed by an enhanced chemiluminescence (Amersham Biosciences, USA) according to the manufacturer's protocol. Experiments were carried out at least 3 times. The luciferase activities from pGL3-control-derived plasmids were standardized to renilla luciferase activity from pRL-SV40 by the luciferase assay system (Promega, USA). After post-transfection for 48 hours, luciferase activity was evaluated in the harvested cells using the Multimode Detector reporter assay system (Beckman Coulter, WI, USA).

| Statistical analysis
Data were expressed as the mean AE SD (standard deviation). All data were calculated using SPSS 21.0 (SPSS, USA). The comparison between two independent groups was tested by adopting the Student's t test. The comparison of mean standard between multiple groups was carried out using one-way ANOVA method. The SNK-q test was used for intragroup comparison. Chi-square test was used to estimate the clinicopathological features of EC. P-values <.05 were deemed statistically significant.

| Expression levels of BDNF-AS and miR-214 in EC tissue samples and cell lines
QRT-PCR was used to measure BDNF-AS and miR-214 expression levels in clinical samples and cell lines, which were normalized to U6.
Lower expression of BDNF-AS was observed in the EC tissues and the corresponding adjacent normal tissues than the non-tumour tissues (P < .05, Figure 1A). In addition, qRT-PCR was utilized to gauge | 3731 Figure 1D). The OE19 and OE33 cell lines were used for subsequent experiments for their most significant differences.

| Overexpression of BDNF-AS restrained cell growth, migration and invasion of EC cells
The growth curves of EC cells in untransfected group (Control), negative control group (NC) and BDNF-AS transfection group (BDNF-AS) were drawn at absorbance of 492 nm measured by ELISA (Figure 2A and OE33 cells (P < .05, Figure 2C,D). Similarly, invasion of OE19 and OE33 cells was reduced following overexpression of BDNF-AS (P < .05, Figure 2E,F). Furthermore, no significant change was found out between untransfected control group and NC group (P > .05).
Overall, these results demonstrate that overexpression of BDNF-AS could suppress the migration and invasive ability of EC cells in vitro.

| LncRNA BDNF-AS directly bound to miR-214
To explore interaction between lncRNA BDNF-AS and miR-214, we

| Effects of cotransfection of BDNF-AS and miR-214 on proliferation, migration and invasion of OE19 and OE33 cells
Results of MTS assay showed that transfection of BDNF-AS could obstruct the cell growth of EC cells, and the transfection of miR-214 mimic promoted the proliferation activity of EC cells in vitro (P < .05).
In addition, no significant change is found in proliferation activity after cotransfection of BDNF-AS and miR-214 mimic (P > .05, Figure 5A

| Effects of cotransfection of BDNF-AS and miR-214 on expression of EMT-related factors in EC cells
To explore how lncRNA BDNF-AS and miR-214 regulated the EMT in EC, the expression levels of EMT-related mRNA and protein were, respectively, measured by qRT-PCR and Western blot assays. Overexpression of miR-214 EC cells down-regulated E-cadherin mRNA and protein expression and up-regulated N-cadherin and vimentin expression in contrast to the NC group (P < .05). Nevertheless, there were no noteworthy differences between BDNF-AS + miR-214 mimic cotransfection group and the NC group (P > .05, Figure 6).

| DISCUSSION
Human oesophageal cancer (EC) ranks ninth of occurrence and sixth as the leading cause of cancer mortality, which occurs around the world in a varying geographic distribution and influences male more than female. 1 In spite of the development of miscellaneous therapies, the prognosis of the patient with EC remains poor, indicating the limitations of conventional treatment, which motivates us to investigate an innovative treatment for ECs. 16 Current risky therapeutic strategies may be improved due to the recognition of new prognostic biological markers for EC, which helps ameliorate unfavourable clinicopathological characteristics and shorter survival for patients. 3 In this study, we first described that lncRNA BDNF-  17 Over the past few years, some studies have revealed that 18% of the protein-coding genes that produce lncRNAs are associated with cancers, whereas for all human protein-coding genes, the ratio is only 9%. 18 Because of their great significance in the gene expression regulation, lncRNAs are confirmed to be associated with diversified cellular functions including cell proliferation, apoptosis, migration, invasion and differentiation and existed in various physiological and pathological processes. 19 Thus, it is of paramount importance to identify and investigate the cancer-associated lncRNAs, which helps better understand the roles of lncRNAs in cancer progression and develop novel therapeutic targets. In this study, several key observations we made were related with lncRNAs in EC. Firstly, we focused on, a widely expressed but previously unstudied in EC lncRNA, BDNF-AS, as being down-regulated in EC tissues and cell lines. Then, we discovered that overexpression of BDNF-AS exerted inhibitory influence on EC cell proliferation, migration and invasion in vitro. In addition, we also paid our attention to the EMT process, which was closely associated with tumour metastasis.
EMT is the process which usually occurs in the period of the embryonic development and organogenesis. 20 However, abnormal activation of the EMT facilitates the pathogenesis of tumour, thus inducing metastasis. 21 LncRNAs play an important role in inducing the EMT in various types of tumour. 22 Our study indicated that are associated with the EMT. 23,24 In this assay, we explored the effects of overexpression of BDNF-AS on the EMT process.
According to the results of qRT-PCR and Western blot, the expression quality of E-cadherin was up-regulated, while the expression of N-cadherin and vimentin was down-regulated with the treatment of BDNF-AS overexpression, indicating the inhibition role of BDNF-AS in the EMT process.
MicroRNAs (miRNAs) were discovered to be dysregulated in certain human cancers including EC. 25 Growing evidence has confirmed that miRNAs can play a vital role in the regulation of development, differentiation, proliferation, metastasis and apoptosis. 26

| CONCLUSION
In summary, our study focuses on the mechanism of EC progression, including cell proliferation, invasion and metastasis. Pivotal evidence observed in our study could support the hypothesis that the overexpression of the antioncogenic lncRNA BDNF-AS is relevant to both clinic and function in the progression of EC, which inhibits cell proliferation, migration, invasion and the EMT process. In addition, the increased expression of miR-214 has been associated with poor prognosis. Further analysis demonstrates BDNF-AS functions as a miR-214 sponge to facilitate cell proliferation, migration and invasion in the EC. Taken together, all these findings suggested that BDNF-AS has essential effects on the pathogenesis of EC. Understanding the specific role of BDNF-AS in the development and progression of the EC will provide a novel diagnostic marker and efficacious therapeutic strategies for EC treatment.

CONFLI CT OF INTEREST
The authors confirm that there is no conflict of interests.

ETHICAL APPROVAL
This study was approved by the ethical committee of the First Affiliated Hospital of Zhengzhou University, and all participants signed the informed consent.

INFORMED CONSENT
Informed consent has been obtained from each patient or subject after full explanation of the purpose and nature of all procedures used.