MiR-126 promotes esophageal squamous cell carcinoma via inhibition of apoptosis and autophagy

MiRNA-126 (miR-126) has been shown to be involved in various malignancies as well as other biological processes. However, currently, its role in esophageal squamous cell carcinoma (ESCC) is not well understood. The present study is focused on the mechanisms that underlie the effect of miR-126 on cell survival and death (apoptosis and autophagy) in ESCC cells. MiR-126 expression was found to be enhanced in ESCC cells and tissues. Downregulation of miR-126 suppressed cell survival, and TUNEL staining indicated that miR-126 inhibition promoted ESCC cell death. In addition, the production of LC3B and p62 proteins, two autophagy signals, was reduced following miR-126 inhibition. A dual luciferase reporter assay demonstrated that the STAT3 3’-UTR is a direct target of miR-126. Furthermore, STAT3 knock-down rescued the effects on autophagy and apoptosis caused by miR-126 inhibition in ESCC cells. The results of this study may provide some insight into the molecular and biological mechanisms underlying ESCC generation and contribute to the development of novel therapeutic approaches for ESCC.


INTRODUCTION
Esophageal cancer (EC) is a fatal disorder typified by a poor clinical outcome owing, in part, to the fact that many patients with EC receive their diagnosis during the late stages of the illness [1,2]. Although remarkable progress has been made in diagnostic methods and adjuvant chemotherapy, the five-year survival rate of patients with EC remains below 30% [3]. Previous studies have indicated that complex epigenetic and genetic changes may result in the progression of esophageal squamous cell carcinoma (ESCC) [4,5]. However, a proper understanding of the mechanisms underlying such changes is currently lacking.
MicroRNAs (miRNAs) are a series of conserved small RNAs comprising 20-22 nucleotides; they modulate gene expression by binding to the 3'-UTR of the target mRNA, leading to mRNA degeneration and translational suppression [6]. Current evidence suggests that miRNAs may critically influence various biological processes, including cell death. For example, miR-34a is suppressed in malignancies as well as in multiple cellular processes such as cell proliferation, differentiation, and death [7][8][9].
MiR-126 has been shown to modulate metastasis as well as proliferation in multiple malignancies via the inhibition of gene expression. For example, miR-126 promotes sensitivity to drugs that counteract malignancy in nonsmall-cell lung cancer (NSCLC) by targeting vascular endothelial growth factor (VEGF) A [10]. MiR-126 represses proliferation and triggers cell death in hepatocellular carcinoma (HCC), at least in part, by acting on Sox2 [11]. The expression of miR-126 is often significantly suppressed in tissues from patients with gastric cancer. Excessive miR-126 expression inhibits the invasion of malignant cells instead of affecting proliferation in vitro. Furthermore, excessive miR-126 noticeably reduces the concentrations of Crk protein, which has been recognized as a direct target of miR-126.
The aim of this study was to explore the impact of miR-126 on ESCC etiology and development. MiR-126 expression was found to be increased in specimens from patients with ESCC as well as in ESCC cell lines. Furthermore, miR-126 silencing impaired ESCC cell proliferation and ESCC generation both in vivo and in vitro. In addition, our results suggest that miR-126 may participate in ESCC development by suppressing cell death via the downregulation of STAT3.

MiR-126 was upregulated in ESCC tissues and cells
miR-126 expression in ESCC (n = 20) and esophageal specimens from healthy volunteers (n = 10) was investigated using qPCR. Expression levels in the ESCC malignant group were considerably higher than those in the normal group ( Figure 1A). Furthermore, miR-126 expression in 20 human ESCC and 20 adjacent normal tissue specimens was also evaluated. The results showed that miR-126 expression was enriched in the human ESCC specimens, indicating a higher level of miR-126 in ESCC tumors than in the surrounding normal esophageal tissues ( Figure 1B). In addition, miR-126 expression was higher in the two ESCC cell lines than in normal esophageal cells ( Figure 1C). The above findings indicate that ESCC tissues and cells express high levels of miR-126.

MiR-126 silencing inhibited ESCC tumorigenesis in vivo
To assess the role of miR-126 in xenograft ESCC generation, we subcutaneously injected nude BALB/c mice with TE13-miR-126i or TE13-NC cells and observed the animals every day for tumor formation. First, miR-126 expression was measured in esophageal tissues to confirm miR-126 knock-down in the miR-126-silenced mice ( Figure 2A). Mice were sacrificed 21 days after the injection, and their esophageal tissues  were removed and weighed. The results indicate that the average weight and volume of the miR-126-silenced tumors were much lower than those of the control group tumors, and the average neoplasm weight was also significantly less ( Figure 2B, 2C).

MiR-126 silencing reduced cell viability and induced cell death in ESCC cells via apoptosis and autophagy
The role of miR-126 in ESCC cell proliferation and viability was analyzed by transfecting TE13 and Eca109 cells with a miR-126 inhibitor. This resulted in a notable reduction in the miR-126 levels in the cells ( Figure 3A, 3B). Likewise, the results of a soft agar CFA showed that aberrant miR-126 expression caused an obvious reduction in the colony numbers formed by TE13 and Eca109 cells, whereas the NCinhibitor had no effect ( Figure 3C, 3D). The results of the MTT assay indicated that TE13 and Eca109 cell proliferation was noticeably inhibited at 1, 2, and 3 d after transfection with the miR-126 inhibitor ( Figure 3E, 3F). AGING miR-126 silencing attenuated TE13 and Eca109 cell proliferation and viability; we speculated that this may be due to the stimulation of apoptosis or autophagy in the cells. TUNEL staining ( Figure 4A, 4B) and flow cytometry ( Figure 4C, 4D) did indeed confirm that a higher level of apoptosis occurred in PC cells treated with the miR-126 inhibitor than in normal cells or cells transfected with the NC-inhibitor.
The effect of miR-126 inhibition on autophagy in ESCC cells was also explored. IFA was used to detect LC3B protein expression after miR-126 silencing; the results show LC3B (green) accumulation in the cells treated with the miR-126 inhibitor ( Figure 5A, 5B). Autophagy was enhanced in the two ESCC cell lines following miR-126 inhibition, as indicated by the augmented LC3B biosynthesis and processing and p62 degradation (two main indicators of autophagy) ( Figure  5C, 5D). Furthermore, qPCR was used to assess p62 transcription; the results revealed a positive correlation with miR-126 concentrations ( Figure 5E, 5F). These results demonstrate that miR-126 may inhibit ESCC cell autophagy, which could help promote cell proliferation.

MiR-126 targeted the 3'-UTR of JAK1
STAT3 has been reported to be associated with apoptosis and autophagy in various cell types [17][18][19][20][21]. Therefore, we analyzed STAT3 expression in ESCC cells; we observed higher expression in ESCC tumors than in normal esophageal cells, at both the mRNA and protein level ( Figure 6A, 6B). Furthermore, bioinformatics prediction suggests that miR-126 may target the 3'-UTR of STAT3. A possible direct link between miR-126 and the STAT3 3'-UTR was examined using a DLRA ( Figure 6C). The results indicate that luciferase activity was 70% lower after transfection of the miR-126 mimic than in the control groups ( Figure 6D). Apoptotic rate is displayed in the lower right panel. Results are displayed as the average ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001.

AGING
The effect of miR-126 inhibition on STAT3 expression in TE13 and Eca109 cells was determined using qPCR and WB. The expression levels of STAT3 mRNA and protein were found to be upregulated following transfection of the miR-126 inhibitor ( Figure 6E-6H). These findings demonstrate that STAT3 expression is increased after miR-126 silencing and suggest that miR-126 may target the STAT3 3'-UTR.

STAT3 knock-down attenuated the effect of miR-126 silencing on ESCC cell viability
To determine whether STAT3 knock-down inhibits the effect of miR-126 on ESCC cell viability, including apoptosis and autophagy, we silenced STAT3 transcription in TE13 and Eca109 cells following treatment with the miR-126 inhibitor. qPCR and WB were used to assess the changes in STAT3 expression ( Figure 7A-7D). An MTT assay was also used to evaluate the effect on cell proliferation ( Figure 7E, 7F). To assess the role of STAT3 in ESCC cell apoptosis, we performed a TUNEL assay on TE13 and Eca109 cells that were treated with the miR-126 inhibitor. FC results showed that aberrant STAT3 expression caused an obvious reduction in the number of apoptotic ESCC cells ( Figure 8A, 8B).
In addition, STAT3 silencing led to a decrease in autophagy, as indicated by the attenuated LC3B biosynthesis and processing and p62 stabilization ( Figure 8C, 8D). qPCR was used to detect p62 transcription. The results showed an inverse correlation with STAT3 expression (Figure 8E, 8F). These findings indicate that STAT3 knock-down may restore the reduction in proliferation caused by miR-126 inhibitioninduced apoptosis and autophagy.

DISCUSSION
Tumorigenesis is a synergistic process involving the activation of multiple oncogenes, tumor inhibitor genes, and epigenetic alterations, all of which lead to tumor formation. ESCC has been widely studied, but the underlying mechanism is still unclear. In the present study, miR-126 was found to be upregulated in two different ESCC cell lines (TE13 and Eca109). miR-126 inhibition suppressed viability and proliferation in these cells, while inducing apoptosis and autophagy. In vivo experiments showed that miR-126 knock-down inhibited ESCC tumor growth in mice and inhibited the expression of STAT3, an essential oncogene and therapeutic target. Further investigation revealed that miR-126 may directly bind to the STAT3 3'-UTR and abrogate its expression. STAT3 inhibition may also rescue ESCC from the effects of miR-126 inhibition. These observations strongly suggest that miR-126 may suppress ESCC tumorigenesis by modulating cell viability and migration, by acting on STAT3. 3'-UTR following transfection with the miR-126 mimic. Luciferase activity was standardized to β-galactosidase. Treatment with the miR-126 mimic dramatically reduced the relative luciferase activity in the WT 3'-UTR. WB (E, F) and qPCR (G, H) were used to measure STAT3 protein and mRNA expression following transfection with miR-126 or NC inhibitors. Results are displayed as the average ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001.

AGING
MiRNAs are considered to influence biological processes by regulating target genes. Several studies have shown that miR-126 targets include ADAM9 [22], LRP6, PIK3R2 [23], CXCR4 [24], and SLC7A5 [25], all of which participate in the development of diverse tumor types. In the current work, we used a bioinformatics analysis to predict that miR-126 may target a conserved STAT3 site. Previous DLRA results showed that STAT3 acts directly on miR-126 in ESCC and plays a role in many diverse biological processes [26], whereas phosphorylated STAT3 regulates a number of different genes, including Bcl-xL, Bcl-2, VEGF, and MMPs [26][27][28][29]. Previous research has shown that the STAT3 pathway may be related to different aspects of autophagy [30]. A complex set of pathways regulates autophagy, and their interaction with other stressresponse pathways, such as the STAT3 pathway, could influence cell survival or death [31]. In the present work, STAT3 was found to be augmented after miR-126 inhibition, and knock-down of STAT3 in ESCC cell lines restored the miR-126-induced changes in the cells. These observations suggest that miR-126 may have a prominent role in ESCC tumorigenesis and may be regulated by STAT3.

AGING
In summary, this research provides strong evidence that miR-126 may have a multi-functional effect on oncogenes, as suggested by the major changes observed in the ESCC cell lines and ESCC tumorigenesis in the mouse model. Nevertheless, we are unable to exclude other factors that miR-126 may target, in addition to STAT3, and that contribute to ESCC pathogenesis. In future studies, an improved HITS-CLIP and MiR-Trap method, described by Baigude et al. [32], could be used to identify the targets of biotin-marked miR-126 in ESCC cells. This may provide a more exact and elaborate interpretation of the role of miR-126 in ESCC progression.  , D) WB was used to examine LC3B and p62 protein levels in cells after transfection. (E, F) qPCR was used to measure p62 mRNA expression in TE13 and Eca109 cells after different treatments. Results are displayed as the average ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001.

Tissue specimens
Twenty patients with esophageal illness were enrolled in the Nursing Department, Affiliated Hospital of Jining Medical University (Jining, China). Patients who had undergone chemotherapy, radiotherapy, or other esophageal operations before the esophagectomy were excluded from the study. Twenty pairs of ESCC and matching healthy esophageal tissues (five centimeters from the malignant lesions according to the NCCN guidelines for EC) were acquired. Patients who received postoperative pathological diagnoses of both intraepithelial neoplasia (IEN) and ESCC formed the ESCC group, whereas those who were diagnosed with IEN only formed the IEN group. All specimens were snapfrozen in liquid N2 and stored at -80°C. The study was approved by the local ethics committee, and all participants provided written informed consent.

In vivo tumorigenesis experiment
Male nude BALB/c nu/nu mice aged 6 weeks or older, lacking a thymus, were used for tumorigenicity evaluation. To examine the impact of miR-126 exhaustion on malignancy generation, we transplanted TE13-miR-126i or TE13-NC cells subcutaneously into the dorsal flank of recipient mice (1 × 10 7 cells/ml). The volume of the malignancy was assessed every 5 days. Mice were sacrificed after 30 days, and biopsies were carried out. The weight of the malignancies was estimated. Malignancy volume was determined according to the equation: V=A × B 2 /2, where A represents the maximal diameter and B represents the A-perpendicular diameter. Five mice were included in each group. All procedures complied with the institutional guidelines.

Transfection
Hsa-miR-126 mimic, miRNA-126 inhibitor, and their corresponding negative controls (NC-mimic, NCinhibitor) were purchased from RiboBio (Guangzhou, China). The mimics and NC-mimics consisted of incomplete complementary double strands, whereas the inhibitors and NC-inhibitors consisted of a single strand. ESCC cells were plated overnight. Transient transfections were performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Cells were cultured for 24 or 48 h before being analyzed.

Western blotting (WB)
RIPA buffer (pH 8.0), including 150 mM NaCl, 0.1 % SDS, 50 mM Tris-HCl, and 1% NP-40, was added to a protease inhibitor cocktail (Roche Applied Science) and used to obtain whole cell lysates. Protein was quantified using a bicinchoninic acid protein quantitation kit and isolated via SDS-PAGE. Proteins were transferred onto a 0.450-µm PVDF membrane (Millipore, MA, USA), blocked with 5% BSA for 60 min at 25°C, and incubated with primary antibodies overnight at 4°C. Subsequently, blots were incubated with secondary antibodies for 60 min at 25°C. Immunoreactivity was determined using a Maximum Sensitivity Kit (Thermo, MA, USA). Imaging was performed using a Blot Scanner.

RNA isolation and qPCR
Total RNA was isolated using Trizol reagent (Invitrogen CA, USA). Transcription was determined via PCR (Roche, Germany) with SYBR Green, and GADPH served as an internal reference. qPCR cycles were as follows: denaturation for 30 s at 95°C, then 30 cycles of 15 s at 95°C and 60°C for 30 s, and extension at 72°C for 30 s. The 2 -ΔΔCT method was used to calculate target values by standardizing to the internal reference (average of controls).

MTT assay
Cell viability was evaluated using an MTT assay. VSMCs were supplemented with MTT (0.02 ml, 0.5 g/l) and DMSO (0.15 ml); then, the supernatant was removed, and the mixture was rotated for 10 min to incorporate the formazan dye. A490 was measured using an Infinite M200 microplate reader (Tecan, Männedorf, Switzerland).

Colony formation assay (CFA)
Cells were transfected with the appropriate treatments and then resuspended in DMEM with FBS (10%) and top agar (0.4%, 8 mm) for 2 d before being moved to a 12-well plate containing bottom agar (0.5%, 0.5 ml). Two weeks later, three areas of each plate were randomly selected for colony quantification.

TUNEL staining
To assess changes in apoptosis, we subjected the cells to a TUNEL assay (Roche, Basel, Switzerland) and counterstained them with DAPI (1:5000, Beyotime, AGING China) to visualize the nuclei. Apoptosis was evaluated by counting the TUNEL-positive cells using an SP8 confocal microscope (Leica, Japan).

Cell death evaluation
Cell death was evaluated using an annexin V-FITC/PI apoptosis detection kit (BD Pharmingen™). Twenty microliters of binding buffer was added to the cell suspension after transfection. Cells were incubated with 5 µl propidium iodide (PI) and 10 µl annexin V-FITC for 20 min in the dark. Flow cytometry (FC) was used to quantify the positive cells.

Dual luciferase reporter assay (DLRA)
A 3'-UTR luciferase reporter assay was used to verify targets of miR-126 using wild type and mutant STAT3 3'-UTR. Constructs containing firefly luciferase and Renilla luciferase were prepared and used to calibrate fluorescence (Luc) and reporter fluorescence (Rluc), respectively. Cells were cultured for 24 h with vectors or miRNA mimics as appropriate. Luciferase activity was evaluated via the DLRA system.

Immunofluorescence assay (IFA)
The human prostate cancer cell line PC-3 was treated with GFP-LC3B expression constructs (Addgene). Images were acquired using a microscope, and GFPpositive puncta from 10 distinct areas were evaluated.

Statistical analysis
Data are expressed as the average ± SD. A two-tailed ttest or one-way ANOVA was applied to assess intergroup differences. P < 0.05 indicates statistical significance.