MicroRNA‐1275 induces radiosensitization in oesophageal cancer by regulating epithelial‐to‐mesenchymal transition via Wnt/β‐catenin pathway

Abstract Acquired radioresistance is one of the main obstacles for the anti‐tumour efficacy of radiotherapy in oesophageal cancer (EC). Recent studies have proposed microRNAs (miRNAs) as important participators in the development of radioresistance in various cancers. Here, we investigated the role of miR‐1275 in acquired radioresistance and epithelial‐mesenchymal transition (EMT) in EC. Firstly, a radioresistant cell line KYSE‐150R was established, with an interesting discovery was observed that miR‐1275 was down‐regulated in KYSE‐150R cells compared to the parental cells. Functionally, miR‐1275 inhibition elevated radioresistance in KYSE‐150 cells via promoting EMT, whereas enforced expression of miR‐1275 increased radiosensitivity in KYSE‐150R cells by inhibiting EMT. Mechanically, we demonstrated that miR‐1275 directly targeted WNT1 and therefore inactivated Wnt/β‐catenin signalling pathway in EC cells. Furthermore, WNT1 depletion countervailed the promoting effect of miR‐1275 suppression on KYSE‐150 cell radioresistance through hampering EMT, whereas WNT1 overexpression rescued miR‐1275 up‐regulation‐impaired EMT to reduce the sensitivity of KYSE‐150R cells to radiation. Collectively, our findings suggested that miR‐1275 suppressed EMT to encourage radiosensitivity in EC cells via targeting WNT1‐activated Wnt/β‐catenin signalling, providing a new therapeutic outlet for overcoming radioresistance of patients with EC.

150R was established, with an interesting discovery was observed that miR-1275 was down-regulated in KYSE-150R cells compared to the parental cells. Functionally, miR-1275 inhibition elevated radioresistance in KYSE-150 cells via promoting EMT, whereas enforced expression of miR-1275 increased radiosensitivity in KYSE-150R cells by inhibiting EMT. Mechanically, we demonstrated that miR-1275 directly targeted WNT1 and therefore inactivated Wnt/β-catenin signalling pathway in EC cells.
Furthermore, WNT1 depletion countervailed the promoting effect of miR-1275 suppression on KYSE-150 cell radioresistance through hampering EMT, whereas WNT1 overexpression rescued miR-1275 up-regulation-impaired EMT to reduce the sensitivity of KYSE-150R cells to radiation. Collectively, our findings suggested that miR-1275 suppressed EMT to encourage radiosensitivity in EC cells via targeting WNT1-activated Wnt/β-catenin signalling, providing a new therapeutic outlet for overcoming radioresistance of patients with EC.

K E Y W O R D S
epithelial-mesenchymal transition, miR-1275, oesophageal cancer, radioresistance, Wnt/βcatenin MicroRNAs (miRNAs) are a class of small non-coding RNAs that regulate gene expression at post-transcriptional level. 7 Recently, researchers have increasingly paid attention to the parts of miRNAs in modulating cellular biological events in human diseases including cancer. 8 Importantly, miRNAs have been lately demonstrated to be involved in the development of chemoresistance or radioresistance. For example, down-regulated miR-199a-3p in hepatocellular carcinoma results in the increased doxorubicin sensitivity. 9 miR-494-3p enhances radiosensitivity in human oral squamous carcinoma cells. 10 MiR-1275 is a newly recognized miRNA which plays a paradoxical role in different human cancers. For instance, miR-1275 hinders tumour growth in hepatocellular carcinoma by targeting the oncogenic IGF2BPs. 11 Conversely, miR-1275 promotes the progression in head and neck squamous cell carcinoma through up-regulating IGF-1R and CCR7. 12 Importantly, the down-regulation of miR-1275 in radioresistant EC cells has already been proven previously. 13 However, the precise function of miR-1275 in EC remains to be identified.
Epithelial-mesenchymal transition (EMT) is a process that primarily describes tissue remodelling and cell migration during embryonic development to shape future organisms. Over the years, this process has also been discovered in disease course, such as tumour progression. 14 A growing number of studies explained that EMT is largely implicated in radioresistance of numerous cancers due to the close association with the plastic phenotype of cancer stem cells (CSCs). 15,16 Recently, reports have lately suggested that ionizing radiation induces transforming growth factor beta (TGF-β), while TGF-β facilitates EMT in human mammary cells. 17,18 Intriguingly, EMT induced by ionizing radiation in turn promotes radiation resistance. 19 Also, mounting evidence has unearthed that miRNAs have important functions in regulating EMT, 20 such as miR-200 family, miR-194, miR-192 family and so on. [21][22][23] Nevertheless, the relationships among miR-1275, EMT and radioresistance in EC have never been probed yet.
Currently, we aimed to investigate the role and potential mechanism of miR-1275 in the acquired radioresistance of EC cells.

| Cell culture
The cell line of human oesophageal squamous carcinoma cell line KYSE-150 was purchased from the American Type Culture Collection and the radioresistant subtype KYSE-150R cells were established as described. 24 Then, both the KYSE-150 and KYSE-150R cells were grown in RPMI-1640 (Gibco, Life Technologies Inc) supplemented with 10% foetal bovine serum (FBS; HyClone, South Logan), 100 U/mL penicillin and 100 μg/mL streptomycin (Invitrogen) and stored in a humidified atmosphere at 37°C, containing 5% CO 2 .

| Establishment of radioresistant cell line KYSE-150R
The radioresistant KYSE-150R cell line was developed as previously described. 24 Briefly, 1 × 10 6 of KYSE-150 cells suspended in 100 μL culture medium were seeded in the culture flasks and cultured for 2 days, followed by irradiation treatment with 1 Gy of X-ray at a dose rate of 100 cGy/min through a high energy linear accelerator (Varian 600C, USA). After irradiation, the culture medium was immediately renewed and the cells were further incubated until reached approximately 90% confluence. Subsequently, cells were subcultured into new flasks after trypsinization and collection, followed by irradiation again (second fraction) once they reached nearly 50% confluence.
One and a half months later, the radioresistant cell populations were obtained when treated with a total doses of 21 Gy after repeated above procedures (1 Gy 3 times, 2 Gy 3 times and 4 Gy 3 times) for 12 times, which were proved to maintain a radioresistant phenotype for at least 5 months of 50 passages. And such irradiated cells were not used in the experiments until at least 1 month after the last exposure.
At the meantime, the parental KYSE-150 cells were also passaged and cultured under the same conditions except irradiation all the time.

| Cell transfection
Cell transfection was performed using Lipofectamine 2000 (Invitrogen) in light of the manufacturer's instructions. The miR-1275 inhibitor and negative control (NC inhibitor) oligonucleotides, the miR-1275 mimic and the negative control (NC mimic) oligonucleotides, and the small interfering RNA (siRNA) oligonucleotides targeting WNT1 mRNA (si-WNT1) and the negative control (si-NC) were obtained from GenePharma Corporation. Meanwhile, the full-length WNT1 cDNA was subcloned into pcDNA3.1 vector (Invitrogen) for WNT1 overexpression (pcDNA3.1/WNT1) with the empty vector as negative control. After planted and grown for 1 day in 6-well plates, KYSE-150 and KYSE-150R cells were transfected appropriately with the plasmids obtained above. Transfection for 48 hours later, the cells were collected and maintained for following use. The transfection efficiency was confirmed by quantitative real-time PCR.

| Quantitative real-time PCR (qRT-PCR)
Based on the manufacturer's instructions, qRT-PCR analyses were

| Irradiation and colony formation assays
Cells were processed with X irradiation at indicated doses (0, 2, 4, 6 and 8 Gy

| Wound-healing assay
Wound-healing assay was also performed to assess cell migration in vitro. The cells were seeded and cultured in 6-well plates until 80% confluence. Afterwards, a wound among the cells was created artificially using the tip of a sterile 200-μL pipette tube. Then, cells were washed and further grown in medium with 2% FBS. The wound closure was observed regularly and imaged under a microscope 36 hours later. The migration rate was determined as the ratio of healed wound to the wound line. All the experiments were conducted in triplicate.

| RNA pull-down assay
First of all, the biotin-labelled RNAs including Bio-miR-1275-WT, Bio-miR-1275-Mut and negative control (Bio-NC) were obtained with Biotin RNA Labelling Mix (Roche). Meanwhile, cells were processed in a lysis buffer (Gibco) and cell lysates were incubated with the biotinylated RNAs overnight. Subsequently, the magnetic beads with streptavidin (Gibco) were added and co-incubated for 48 hours.
After being washed, the bonding RNAs in the beads were eluted down and then detected by qRT-PCR analysis. All the experiments were repeated for three times.

| RNA immunoprecipitation (RIP) assay
RIP assay was performed to verify the interaction between WNT1

| In vivo xenograft experiment
In All the experiments were repeated for three times.

| TUNEL assay
The paraffin-embedded sections were applied to assess the apop-

| Immunohistochemical (IHC) staining
Immunohistochemistry (IHC) assay was conducted on the paraffinembedded sections obtained in in vivo experiments. In brief, the sections were incubated with primary antibodies against Ki67 (1:100; Roche) and then stained by a universal DAB detection kit (Roche).
Then, the images of stained sections were obtained using a light microscope, followed by the counting of the Ki67-positive cells in five random fields.

| Statistical analyses
The data were analysed using GraphPad Prism 6 and then showed as the mean ± standard deviation (SD). Differences were evaluated by a two-tailed unpaired Student's t test between two groups or a one-way analysis of variance among at least three groups. Statistical significance was identified as P < .05.

| MiR-1275 was significantly down-regulated in radioresistant EC cells
To evaluate the effect of miR-1275 on the sensitivity of EC cells to radiotherapy, we established a radioresistant EC cell line KYSE-150R which showed limited response to irradiation treatment as described. 24 Seen from Figure 1A, the radioresistant KYSE-150R cells were morphologically changed into spindle shapes (more elongated) with weakened adhesion and strengthened motility. In addition, the survival fraction of KYSE-150R cells was identified to be noticeably higher than the parental KYSE-150 cells ( Figure 1B) and the SER for KYSE150R cells was 0.811, corroborating that KYSE-150R cells became less sensitive to irradiation treatment.
Finally, we detected the expression of miR-1275 in KYSE-150R and its parental cells. As a result, the level of miR-1275 was dramatically reduced in KYSE-150R cells in contrast to that in KYSE-150 cells ( Figure 1C). Collectively, we speculated that the expression level of miR-1275 might be strongly associated with the radiosensitivity of EC cells.

| MiR-1275 elevated radiosensitivity of EC cells by inhibiting EMT
To specifically probe the role of miR-1275 in the development of radioresistance in EC cells, loss-and gain-of-function assays were, respectively, conducted in KYSE-150 cells and KYSE-150R cells.  Figure 2C). Based on the above results, miR-1275 was greatly indicated to contribute to the sensitivity of EC cells to radiation exposure.
Previously, a report revealed that more than one-third of EC patients cannot be cured by CCRT treatment because of distant metastasis. 25 Besides, epithelial-mesenchymal transition (EMT) is a hallmark of tumour metastasis 26 and a process largely related to cancer radioresistance. 27 Thus, we assumed that miR-1275 influenced the radiosensitivity of EC cells through effecting EMT.
Firstly, we assessed whether miR-1275 had an impact on EC cell migration that can be enhanced during EMT. 26

| MiR-1275 directly targeted WNT1 and inactivated Wnt/β-catenin pathway
In depth, we intended to figure out the probable targets that participated in the mechanism by which miR-1275 modulated radiosensitivity and EMT of EC cells. And the RNA22 v2 (https ://cm.jeffe rson.edu/rna22/ Inter activ e/) predicted that WNT1, an activator of Wnt/β-catenin signalling, was one of the potential downstream targets of miR-1275. Additionally, we observed that the protein levels of β-catenin and its targets including c-myc and Slug were all aggrandized in radioresistant KYSE-150R cells in comparison with the parental cells ( Figure 3A). Also, miR-1275 down-regulation notably activated Wnt/β-catenin signalling with boosted level of WNT1 and p-GSK-3β so as to reduce the level of p-β-catenin and enhance β-catenin level; however, up-regulation of miR-1275 gave rise to observable inactivation of this pathway ( Figure 3B), suggesting that miR-1275 was a negative regulator of Wnt/β-catenin signalling.
Afterwards, we made a further investigation into whether miR-1275 affected Wnt/β-catenin pathway through a WNT1mediated manner. Obviously, it was confirmed that WNT1 was directly targeted by miR-1275 because only the luciferase activity of WNT1-WT was strikingly strengthened under miR-1275 inhibition but pronouncedly diminished upon miR-1275 overexpression ( Figure 3C,D). Moreover, the following RNA pull-down assay and RIP assay further validated the interaction between miR-1275 and WNT1 in a RNA-induced silencing complex (RISC) (Figure 3E-G).
Furthermore, we observed that WNT1 was negatively regulated by miR-1275 in EC cells ( Figure 3H). Intriguingly, it was discovered that miR-1275 mainly abolished the level of nuclear β-catenin ( Figure 3I).
More importantly, the TOP/FOP ratio was sharply prompted in face of miR-1275 down-regulation but greatly abated in response to miR-1275 up-regulation ( Figure 3J). Jointly, these findings demonstrated that miR-1275 inactivated Wnt/β-catenin signalling pathway in EC by targeting WNT1.

| The role of miR-1275 in EC radiosensitivity was dependent on WNT1
Next, we studied whether WNT1 was implicated in miR-1275-affected EC radiosensitivity. Firstly, the expression of WNT1 was  Figure 4A. Besides, miR-1275 inhibition-stimulated WNT1 protein was normalized under WNT1 knockdown, whereas the protein level of WNT1 attenuated by miR-1275 mimic was recovered upon WNT1 overexpression ( Figure 4B). Also, the activity of Wnt/β-catenin signalling influenced by miR-1275 inhibition or stimulation was reversed, respectively, after WNT1 knockdown or overexpression ( Figure 4C). Furthermore, WNT1 silence distinctly  Figure 4E).
Hence, we concluded that the radiosensitizing effect of miR-1275 in EC was dependent on WNT1-activated Wnt/β-catenin pathway.

| miR-1275 had a suppressive impact on EMT in EC cells by a WNT1-mediated way
Subsequently, we explored the impact of miR-1275/WNT1 axis on the EMT process in EC cells. As demonstrated in Figure  was mediated by WNT1 ( Figure 5D). By and large, miR-1275 enhanced radiosensitivity in EC cells due to its impairment on EMT via a WNT1-mediated pathway.

| MiR-1275 confined tumour growth and prompted EC rdiosensitivity in vivo
To further confirm the part of miR-1275 in EC tumour growth and radiosensitivity, the in vivo experiments were carried out through inoculating the KYSE-150 cells with or without miR-1275 inhibition into naked mice (five mice in each group). Seemingly, miR-1275 inhibition markedly accelerated while IR exposure obviously restrained the growth of tumours; more importantly, the tumours derived from miR-1275-silenced cells were still bigger and heavier than those originated from control cells, despite both were treated with IR ( Figure 6A-C). In addition, cell apoptosis was abrogated under miR-1275 suppression but encouraged by IR treatment, and inhibition of miR-1275 had a mitigatory effect on the apoptosis of EC cells facing exposure ( Figure 6D). Conversely, the Ki67 staining in KYSE-150 cells represented as an absolutely opposite phenomenon to above results when under the same conditions ( Figure 6E).
Furthermore, we validated that EMT was enhanced by miR-1275 silence but obstructed under IR treatment, with a promoted EMT in irradiated KYSE-150 cells in response to miR-1275 suppression ( Figure 6F), indicating that miR-1275 stimulated EC radiosensitivity by inhibiting EMT in vivo. On the whole, miR-1275 exerted an inhibitory function in EC cell growth and it strengthened the sensitivity of tumours to radiation by suppressing EMT in vivo.

| D ISCUSS I ON
Over the past decades, the emerging role of ncRNAs, including miRNAs, have been reported in the development of EC radioresistance. [28][29][30][31] In this study, we proved that miR-1275, which has been revealed to exert paradoxical functions in different cancer types, 12 Next, we attempted to explore the in-depth mechanism whereby miR-1275 inhibited EMT and therefore enhanced radiosensitivity.
Wnt/β-catenin signalling pathway is a well-recognized oncogenic pathway that is involved in a multitude of biological processes, including cell proliferation, differentiation, migration, genetic stability and apoptosis, 36 and the aberrant regulation of this pathway usually led to tumorigenesis of multiple cancers. 37 Furtherly, recent researches have highlighted the participation of Wnt/β-catenin pathway not only in cancer progression 37,38 but also in the development of radiation resistance in numerous carcinomas. 39,40 Also, the interplay between miRNAs and Wnt/β-catenin pathway in regulating EMT has also been indicated before. 41 Presently, we discovered this pathway also mediated the influence of miR-1275 on EC cell radioresistance.
Fortunately, WNT1, a key molecule involved in Wnt/β-catenin pathway, 42 was predicted and verified as the direct target of miR-1275 for the first time in this study, so that miR-1275 negatively regulated Wnt/β-catenin signalling in EC cells by targeting WNT1. Collectively, we proved that miR-1275 affected EC radiosensitivity by targeting WNT1-activated Wnt/β-catenin pathway, and similar mechanism was previously unearthed in the influence of miR-301a on the migration and radiosensitivity of EC cells. 43 More importantly, the rescue of WNT1 on miR-1275-regulated EC radiosensitivity was mediated by the enhancement of WNT1-regulated Wnt/β-catenin signalling per se on EMT, 44,45 thus indicating the strong association among miR-1275, EMT and WNT1 in regulating the radiosensitization of EC cells.
In current study, we disclosed unsurprisingly that miR-1275 reduced EC cell radioresistance through impairing EMT via inactivating Wnt/β-catenin pathway by targeting WNT1, indicating a new effective target to overcome radioresistance of EC patients. However, to intensify the clinical significance of miR-1275 as a therapeutic target for radioresistant EC patients, the effect of reagents or drugs that result in miR-1275 up-regulation needs to be further assayed in advanced animals and even human in the future.

ACK N OWLED G EM ENT
Thanks all individuals participated.

CO N FLI C T O F I NTE R E S T
None.

DATA AVA I L A B I L I T Y S TAT E M E N T
Research data and material are not shared.