miR-20b and miR-125a promote tumorigenesis in radioresistant esophageal carcinoma cells

Radiation therapy is an effective method in the management of esophageal cancer. MicroRNAs (miRNAs) have been reported to play an important role in tumorigenesis. However, the roles of specific miRNAs in radioresistant esophageal cancer remain to be investigated. In present study, the relative expression level of miR-20b-5p and miR-125a-5p were evaluated by quantitative Real-time polymerase chain reaction. Cell counting Kit-8 assay, wound-healing assay, transwell assay were used to assess cell proliferation, cell migration and cell invasion. TUNEL and Annexin V-FITC assays were applied to evaluate cell apoptosis. Dual-luciferase reporter gene assay was conducted to identify direct targets of miRNAs. The protein expression level was assessed by Western blot. The results indicated that miR-20b-5p was increased in radioresistant KYSE-150R cells compared with KYSE-150 cells, whereas miR-125a-5p was downregulated. MiR-20b-5p upregulation promoted cell proliferation, migration, invasion, and the EMT process, and decreased apoptosis by negatively regulating PTEN. MiR-125a-5p inhibited cell proliferation, migration, invasion, the EMT process and it induced apoptosis by negatively regulating IL6R. These data indicate that miR-20b-5p and miR-125a-5p promote tumorigenesis in radioresistant KYSE-150R cells and have the potential to be used as novel therapeutic targets for the treatment of esophageal cancer.


INTRODUCTION
Esophageal cancer (EC) is characterized by difficulties in early detection, a considerable decline in quality of life and poor prognosis. Radiotherapy is an important part of multidisciplinary treatment of EC. Chemoradiotherapy is recommended as a reasonable standard treatment option for unresectable esophageal squamous cell cancer patients [1]. Although today's three-dimensional conformal radiotherapy, intensitymodulated conformal radiotherapy increase the accuracy of radiotherapy, which improves the curative effect of EC patients. Acquired or innate radioresistance usually occurs, which is one of the major reasons for treatment failure and relapse [2]. The mechanisms of resistance to radiotherapy have not been fully understood. Previous studies indicated it may be caused by microenvironmental hypoxia, abnormal intrinsic DNA damage response activity, mutations of oncogenes or tumor suppressors or the altering of signaling pathways [3].
MicroRNAs (miRNA) are small non coding RNAs, which cannot encode any protein. However, they can AGING regulate gene expression by degrading messenger RNAs (mRNA) [4]. Accumulating evidence has indicated that miRNAs play a pivotal role in cancer formation and progression [5]. MiRNAs were also relevant to radioresistance in several cancers, such as nasopharyngeal carcinoma, laryngeal carcinoma and lung cancer [6][7][8]. For example, miR-20b is upregulated in several cancers, serves as an oncogene and is correlated with poor prognosis [9,10]. MiR-125a is reduced in various cancer cells, which prevents cancer cell proliferation and induces apoptosis [11][12][13]. However, whether miR-20b and miR-125a affect tumorigenesis in radioresistant EC cells remains unknown.
In our previous reports, we had identified 35 miRNAs that were differentially expressed in radiation resistant EC cells, including miR-20b and miR-125a [14]. Here, we used KYSE-150R cells, which are verified radioresistance cell lines of EC, as a cellular model to assess the regulartory effect of two miRNAs on tumorigenesis of EC.

MiR-20b-5p was upregulated, and miR-125a-5p was downregulate in radioresistant EC cells KYSE-150R
The results of qRT-PCR analyses showed that expression levels of miR-20b-5p were significantly upregulated in radioresistant EC KYSE-150R cells compared to those in the control EC KYSE-150 cells ( Figure 1A). While the expression of miR-125a-5p was declined in KYSE-150R cells ( Figure 1B). To dissect the biological function of miRNA, we planned to alter the expression of miR-20b-5p and miR-125a-5p in KYSE-150R cells and then investigate the cellular process after the change.

MiR-20b-5p increases cell proliferation, migration, invasion, and EMT process, and attenuates cell apoptosis
The results of comparison between KYSE-150 and KYSE-150R cells, indicated that latter exhibited a significant increase in cell proliferation, migration and invasion (Figures 2A-2C, 3A-3C). As miR-20b-5p was upregulated in KYSE-150R cells, we explored the miR-20b-5p function in cellular process. The results showed that overexpression of miR-20b-5p promoted cell proliferation, migration, and invasion. In contrast, downregulation of miR-20b-5p suppressed these cell activities in both KYSE-150 and KYSE-150R cells ( Figure  2A-2C). Supporting Information Supplementary Figure 1A, 1B showed the transfection efficiency. TUNEL assay results revealed a significant decrease in the percentage of apoptotic cells in KYSE-150R cells compared to that in KYSE-150 cells ( Figures  2D, 3D). In both cell lines, a reduction of apoptosis was found in miR-20b-5p overexpressing cells, whereas an enhancement of apoptosis was found in cells transfected with miR-20b-5p inhibitors ( Figure 2D). Epithelial to mesenchymal transition (EMT) refers to a biological process during which epithelial cells lose cell junctions and adhesion and transit to an invasive mesenchymal phenotype [15]. Our previous research demonstrated that radioresistant EC cells were transformed to a AGING mesenchymal state [16]. In this study, we found that in both cell lines, overexpression of miR-20b-5p downregulated the E-cadherin protein, an epithelial marker, and upregulated N-cadherin and Vimentin proteins, which are mesenchymal markers ( Figure 2E). These data indicated that miR-20b-5p promoted the EMT process in EC cells.

MiR-125a-5p decreases cell proliferation, migration, invasion, and EMT process, and increases cell apoptosis
Since the expression of miR-125a-5p was lower in KYSE-150R cells ( Figure 1B), we then explored the effect of miR-125a on the biological function of EC cells. We found that overexpression of miR-125a-5p inhibited cell proliferation, migration and invasion, whereas miR-125a-5p inhibitor exhibited a reverse effect in both KYSE-150 and KYSE-150R cells ( Figure  3A-3C). The transfection efficiency was shown in Supporting Information Supplementary Figure 1C, 1D.
The results of TUNEL assays demonstrated that cell apoptosis was accelerated by transfection with miR-125a-5p mimics and suppressed by transfection with miR-125a-5p inhibitors ( Figure 3D). As for EMT process, in both cell lines, overexpression of miR-125a-5p increased E-cadherin protein expression levels, and reduced N-cadherin and Vimentin protein expression levels compared to those in the control group ( Figure  3E). These results suggested that miR-125a-5p inhibited EMT in EC cells.

PTEN is a downstream target of miR-20b-5p, while IL6R is a downstream target of miR-125a-5p
Based on the miRNA database TargetScan and miRanda, the predicted targets of miR-20b and miR-125a are PTEN and IL6R, respectively (Supporting Information Supplementary Figure 2A, 2B). Therefore, we constructed PTEN and IL6R reporter vectors to perform dual-luciferase reporter assays. In KYSE-150R cells, miR-20b-5p mimics significantly inhibited PTEN luciferase activity ( Figure 4A). However, a mutation in the binding site of miR-20b-5p in PTEN 3'-UTR abrogated the effects of miR-20b-5p on the luciferase activity. The co-transfection of miR-125a-5p mimic and IL6R WT 3'-UTR remarkably reduced the luciferase activity in KYSE-150R cells. The phenomenon was not observed after miR-125a-5p mimic and IL6R Mut 3'-UTR co-transfection ( Figure 4B). Western blot as well as RT-PCR analysis proved that overexpression of miR-20b-5p and miR-125a-5p could negatively regulate the expression of PTEN and IL6R at both mRNA and protein levels ( Figure 4C-4F), respectively. Previous studies discovered that PTEN/Akt signaling pathway and IL-6R/JAK/STAT pathway are associated with carcinoma tumorigenesis [17,18]. Our results showed that miR-20b-5p mimics did not change the protein level of Akt and ERK in KYSE-150 cells, but remarkably increased p-Akt and p-ERK expression compared with miR-20b mimics-NC (Supporting Information Supplementary Figure 3). MiR-125a-5p mimics significantly down-regulated p-STAT3 and p-JAK2 expression compared with miR-125a-5p mimics-NC (Supporting Information Supplementary Figure 4). These data verified that miR-20b-5p and miR-125a-5p directly target PTEN and IL6R in EC, respectively.

Restored PTEN expression could reverse the changes caused by miR-20b-5p overexpression in EC cells
To clarify whether miR-20b-5p increased cellular growth and migration controlled by PTEN, both cell lines were co-transfected with miR-20b-5p mimics and PTEN. Efficient overexpression of PTEN was achieved as shown in Supporting Information Supplementary Figure 5A. As expected, co-transfection with PTEN reduced cell proliferation ( Figure 5A), migration ( Figure 5B) and invasion abilities ( Figure 5C) (all of which were increased by miR-20b-5p mimics), and increased cell apoptosis ( Figure 5D). Western blot analysis showed that in KYSE-150R cells, transfection of PTEN decreased the EMT process ( Figure 5E). Based on these data, we proved that miR-20b-5p increased KYSE-150R cell growth and metastasis by targeting PTEN.

Restored IL6R expression could reverse the changes caused by miR-125a-5p overexpression in EC cells
Efficient overexpression of IL6R was achieved as shown in Supporting Information Supplementary Figure  5B. Co-transfection with miR-125a-5p mimics and IL6R in both cell lines improved cell proliferation ( Figure 6A), migration ( Figure 6B) and invasion abilities ( Figure 6C), and decreased cell apoptosis ( Figure 6D) that was altered by miR-125a-5p mimic. Western blot analysis showed that transfection of KYSE-150R cells with IL6R, promoted the EMT process ( Figure 6E). Thus, our results suggested that miR-125a-5p decreased KYSE-150R cell growth and metastasis by targeting the IL6R.

DISCUSSION
Radiation therapy has been suggested as an efficient treatment in patients with EC. Enhancement of tumor response to radiotherapy and reduction of radioresistance are crucial for improving the treatment outcome of EC. MiRNAs have been recognized for their role in mediation of the radiation resistance in tumors [19].

AGING
In current study, we firstly verified that the expression level of miR-20b-5p was markedly elevated in KYSE-150R cells compared to that in KYSE-150 cells. MiR-20b has been verified to increase cell aggressiveness via regulation of PTEN expression in EC and prostate cancer [20,21]. Consistent with these studies, we observed increased cell proliferation, invasion, and migration in cells overexpressing miR-20b-5p. Apoptosis is one of the mechanisms of tumor death induced by radiotherapy. Apoptosis resistance is one of the characteristics of tumors and leads to cancer progression [22]. EMT has also been verified to be relevant to tumor invasion and metastasis [13]. Recent reports have indicated that EMT was also associated with therapy resistance [23]. Several miRNAs have been demonstrated to affect the EMT process in cancers [24]. MiR-21 was reported to improve transformation of EMT in drug-resistant lung adenocarcinoma cancer cells [25]. Our findings suggested that miR-20b-5p inhibited cell apoptosis and promoted EMT process in EC cells.
Moreover, we found miR-20b-5p exerted its function on KYSE-150R cells by targeting PTEN, which is among the most frequently transformed tumor-suppressor genes in human cancers. PTEN has been reported to inhibit oncogenic PI3K activation, and subsequently block the PI3K/Akt signaling pathway, which is known to induce tumor cell progression [26]. Wu et al. reported that miR-21 promoted EC cell proliferation, migration through the PTEN/PI3K/AKT signaling pathway. Loss of PTEN leads to a continuous activation of the signal pathway, and therefore, fail to control cell growth [17]. Kohnoh et al. demonstrated that unphosphorylated PTEN could inhibit hypoxia-induced EMT in lung cancer cells [27]. Low expression of PTEN has also been reported correlates with poor clinical outcomes of various human cancers [28], as well as plays a role in resistance of anti-tumor treatment. Chen et al. demonstrated that miR-21-5p conferred doxorubicin resistance in gastric cancer cells by targeting PTEN [29]. Wu et al. verified that miR-222 conferred radioresistance in nasopharyngeal carcinoma through modulating PTEN expression [30]. Thus, all these results supported the important role of PTEN in cancer cell development. Results of current study indicate miR-20b-5p exerts its function partly through PTEN /PI3K/Akt signaling pathway.
Our study also found that there was a low expression level of miR-125a-5p in radioresistant KYSE-150R cells. Zhang et al. reported that miR-125a suppressed bladder cancer cell proliferation by inducing cell cycle arrest and cell apoptosis. Cell cycle became stagnant at the G0/G1 phase when miR-125a-5p was overexpressed [31]. In cervical cancer, cell invasion and tumor metastasis has been reported to be regulated by miR-125a-5p as it targets STAT3 [32]. Current study results showed that miR-125a-5p weakened cell proliferation, migration, invasion, and the EMT process. In addition, miR-125a-5p also increased apoptosis via IL6R, an oncogene which encodes a subunit of the interleukin 6 (IL6) receptor complex [33]. Combining IL6 and IL6R leads to the activation of several signal transduction pathways including the JAK/STAT, PI3K/Akt and MAPK pathways [34]. High levels of IL6 in several cancer cells, such as prostate cancer, ovarian cancer, and renal cell carcinoma, resulted in poor prognosis in cancer patients [35][36][37]. Overexpression of IL6R has been demonstrated to correlate with the increase in proliferative activity and the inhibition of apoptosis [38]. Rokavec et al. reported that the activation of IL-6R/STAT3/miR-34a loop was not only necessary for invasion and metastasis of colorectal cancer cells, but also associated with lymph node metastasis and distant metastasis in patient samples. Silencing IL6R expression suppressed tumor growth, migration and angiogenesis, as well as enhanced the antitumor activity in several cancer cells [39]. In pancreatic ductal adenocarcinoma, application of IL6R blocking antibodies through inhibiting IL6 signaling could shift the tumor microenvironment from a chemoresistant state to a chemosensitive state, which was accompanied with reduced STAT3 activation [40]. Results of current study indicate that miR-125a-5p exerts its function via IL6R to activate JAK-STAT3 signaling pathway. Combined with the previous study, our data suggested that miR-125a-5p served as an important tumor suppressor gene in EC cells.

Cell culture and reagents
Human esophageal squamous cancer cell line KYSE-150 was purchased from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). Our department has already established the radioresistant cell line KYSE-150R using a gradient dose irradiation treatment [17]. Cells were cultured in RPMI-1640 medium (Gibco, Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco) and 1% penicillin/streptomycin solution (Gibco) in an atmosphere of 5% CO2 at 37° C. The cell lines were subcultured every 2-3 days following digestion at room temperature with 0.5 mL trypsin/EDTA per well (Sigma-Aldrich Ltd, UK).

Cell viability analysis
Cell proliferation was determined using Cell Counting Kit-8 (Beyotime Biotechnology, Shanghai, China). Cells were seeded into 96-well plates at a density of 3×10 3 cells per well. At 24, 36 and 48 hours post transfection, 20 μL of CCK-8 solution was added to each well. After a 4-hour incubation, the absorbance values were determined at 450 nm.

Cell migration assay
Wound-healing assay was used to assess cell migration ability. EC cells, after transfection treatment, were seeded in 24-well plates and then cultured for 24 h until 90% confluence was obtained. In each well, a scratch in a confluent monolayer was created using a 100 μL pipette tip and cultured for 24 h. The cells were photographed at 0 and 24 h under a Nikon Eclipse TE2000-U Inverted Microscope (Nikon, Tokyo, Japan).

Cell invasion assay
Cell invasion ability was examined using a transwell chamber (Corning, NY, USA). After coating the upper chamber with Matrigel, 3 × 10 4 transfected cells in serum-free DMEM medium were transferred to the upper chamber. In the lower chamber, 500 μL of the complete medium with 10% FBS was added. After 24 h of incubation, a cotton swab was used to wipe noninvading cells. The invaded cells were fixed with 4% paraformaldehyde, stained with 1% crystal violet solution (Sigma, St. Louis, MO, USA) and counted in five randomly selected fields under a light microscope.

Cell apoptosis
Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay TUNEL assay was performed using the Cell Death Detection Kit (Roche, Mannheim, Germany) according to the manufacturer's instructions. Briefly, 1 × 10 5 cells were seeded in a six-well plate 48 h post-transfection, then washed with PBS and fixed with the buffer provided in the kit. DAPI was used to label cell nuclei for 10 minutes. Under the fluorescence microscope, cells with a dark green fluorescence were defined as apoptotic cells.

Annexin V-FITC assay
Annexin V-FITC assay was used to detect apoptosis. The transfected cells were seeded in six-well plates at a density of 1 × 10 5 per well 48 h post-transfection. After washing with PBS, 10 µL of Annexin V-FITC (ab14085, Abcam, Cambridge, MA, USA) and 5 μL of propidium iodide were added to each well and mixed gently. After 15-minutes, 300 μL of the binding buffer was added and cell apoptosis was detected using FACSCalibur TM (BD Biosciences, San Jose, CA, USA).

Dual-luciferase reporter gene assay
A wild-type 3'-UTR and a mutant 3'-UTR of PTEN and IL6R were amplified from KYSE-150 cells using PCR, and cloned into a pGL3-Basic vector. Cells were cotransfected with the miRNAs, firefly luciferase reporter vector and renilla luciferase control vector using Lipofectamine 2000 according to the manufacturer's instructions. All cells were incubated for 48 hours and assayed using a luciferase reporter assay system (Promega, Madison, WI, USA). Renilla luciferase activity was regarded as an internal control.

Statistical analysis
SPSS version 20.0 statistical software (SPSS, Chicago, IL, USA) was used for statistical analyses. Experimental data were calculated as mean ± standard deviation of at least three independent assays. The Student t-test was conducted to compare the difference between the two groups. P < 0.05 denoted statistical significance. Supplementary Figure 4. miR-125a-5p regulates the JAK-STAT3 signaling pathway by targeting IL6R. The protein expression of IL6R, p-STAT3 and p-JAK2 in KYSE-150R cells co-transfected with miR-con/miR-125a and pcDNA3.1/pCMS-IL6R was measured by western blot.