Reciprocal activation between STAT3 and miR-181b regulates the proliferation of esophageal cancer stem-like cells via the CYLD pathway

According to previous studies [19, 21, 37], esophageal cancer cell lines Eca109 and Eca9706 were cultured in serum-free defined medium (SFDM) in ultra-low adherent dishes during cell passaging. Under these conditions, cancer cells formed tumor spheres after 3–5 days (Fig. 1a). Eca109 formed bigger size of tumor spheres and more tumor spheres than Eca9706 (Fig. 1a). Tumor spheres were trypsinized and cultured in SFDM again for approximately five passages. Flow cytometry analysis demonstrated that enriched SFCs from Eca109 mainly expressed CD44 + CD24-, which was consistent with the results of previous studies [38]. The proportion of CD44 + CD24- of SFCs from Eca109 cells was 6.5 %. However, the CD44 + CD24- expression level from Eca9706 cells between parental and sphere cells was not significantly different (Fig. 1b).

To investigate the cancer stem cell characteristics of SFCs, stemness factor expression was analyzed. We first compared the expression levels of Nanog, Oct4, Sox2, and Bmi1 between parental cells and SFCs from Eca109 cells. qPCR analysis demonstrated that the expression levels of these stemness markers were clearly increased (Fig. 1c). However, an increase in stemness factor expression was not observed in both cells types from Eca9706 (Fig. 1d). Stem cell induction, metastasis, and dedifferentiation have been correlated with the epithelial-to-mesenchymal transition in different tumor cells [39]. To evaluate whether the transition to mesenchymal traits was coupled by increased stemness transcription factor expression, we quantified the expression of mesenchymal markers. Mesenchymal markers including Snail, Slug, N-cadherin, ZEB1, and ZEB2 were increased significantly (Fig. 1e). However, increased mesenchymal markers in SFCs from Eca9706 were not observed (Fig. 1f). Based on the above studies and to better understand the properties of esophageal cancer stem cell-like cells, we chose the Eca109 cell line to perform subsequent experiments.

To assess the clonogenic potential of SFCs, colony formation assays were performed in soft agar. The number of SFC colonies formed was greater than that of the parental cells, suggesting that the colony formation capacity of SFCs is stronger than that of the parental cells (Fig. 2a). To test the tumorigenic potential of SFCs in a xenograft mice model, we subcutaneously injected 5 × 10⁵ SFCs and parental cells into Balb/c mice. Both groups formed solid tumors. SFCs formed tumors in 6/6 mice. However, injection of parental cells resulted in tumor formation in 5/6 mice. Tumors of SFC xenografts were three-fold larger and more vascular than in the parental xenografts (Fig. 2b). The tumor weights formed by SFCs and parental cells were 0.15 g and 0.05 g, respectively (Fig. 2b). Next, we quantified and compared the mRNA expression level from the harvested tumors in both groups. qPCR analysis demonstrated that CD44 expression of SFC tumors were more significantly than that of the parental cells. However, CD24 expression in both groups was not significantly different (Fig. 2c). In addition, the mRNA expression of multi-drug resistance protein 1 (MRP1) and ABC transporter super-family ABCB1 and ABCG2 were increased by 2–3.5-fold in SFC xenograft tumors compared with that in parental cells forming tumors (Fig. 2d). These data suggest that SFCs have properties of cancer stem cells.

STAT3 is activated constitutively in many types of cancer cells and plays a critical role in proliferation, survival, metastasis, and angiogenesis, making it an attractive therapeutic target [40‐43]. Western blot analysis demonstrated that the p-STAT3 expression level in SFCs was higher than that in Eca109 parental cells (Fig. 3a). To investigate the role of STAT3 in SFCs, we performed colony formation experiments in soft agar. We first measured the efficiency of the small interfering RNA (siRNA) against STAT3 (Additional file 1: Figure. S1). The number of STAT3-depleted SFC colonies was significantly lower than by negative control (NC) cells (Fig. 3b and c). In contrast, STAT3-overexpressed in SFCs formed more colonies than did the NC (Fig. 3b and c). These results demonstrate that STAT3 plays a critical role in proliferation and colony formation.

miR-181b modulates drug resistance in gastric and lung cancer cells and promotes tumorigenicity in hepatocellular carcinoma [44, 45]. However, the relationship between STAT3 and miR-181b has not been investigated in SFCs. Our studies demonstrated STAT3 depletion reduced the expression level of miR-181b in SFCs (Fig. 3d). Consistently, the expression level of miR-181b was also decreased after the addition of JSI-124, which is a pharmacological inhibitor of STAT3. In contrast, overexpression of STAT3 strongly enhanced the expression level of miR-181b (Fig. 3d). Moreover, activated STAT3 by IL-6 treatment in SFCs resulted in upregulation of this miRNA (Fig. 3e). These results demonstrate that STAT3 trans-activates the transcription of miR-181b. After determining the molecular mechanism by which STAT3 promotes the expression of miR-181b, we performed a luciferase reporter assay.

Through bioinformatics analysis using rVista 2.0, we identified a conserved E-box motif (CANNTG) at 1650 bp relative to the transcription start site (+1) of the human miR-181b stem-loop (Fig. 3f). To determine whether STAT3 influences miR-181 expression, we constructed a 2-kb fragment upstream of the human miR-181b stem-loop and inserted the fragment into the luciferase reporter plasmid pGL4.11. This plasmid and the vector expressing STAT3 were cotransfected into SFCs. We observed a greater increase in luciferase activity by STAT3 compared with the empty vector (Fig. 3g). In addition, when the E-box sequence was mutated, luciferase activity was abrogated (Fig. 3g), indicating that the E-box sequence is necessary for STAT3 regulation of miR-181b and that STAT3 binds to the promoter of miR181b. Concordantly, forced expression of STAT3 increased the expression levels of primary-miR-181b and mature miR-181b (Fig. 3h). Collectively, these results suggest that STAT3 regulates SFCs proliferation and increases colony formation of SFCs by trans-activating miR-181b.

We found that the expression level of miR-181b in Eca109 SFCs increased significantly compared with in the parental cells (Fig. 4a). In addition, a miR-181b mimic led to a significant mRNA increase in CD44 (Fig. 4b). Interestingly, the CD24 expression level showed no obvious alterations after transfection with the miR-181b mimic (Fig. 4b). Moreover, SFCs transfected with the miR-181b mimic increased ABCB1, ABCG2, and MRP1 levels by more than two-fold (Fig. 4c). Next, we tested the function of miR-181b in colony formation in soft agar. The miR-181b mimic increased the number of SFC colonies formed (Fig. 4d). In contrast, the effect was reversed by miR-181b inhibitors (Fig. 4d). To further explore whether miR-181b regulates proliferation and colony formation, we performed in vitro gain-of-function analyses by overexpression of miR-181b with a lentiviral vector containing GFP in SFCs. We found that ectopic expression of miR-181b significantly increased colony size and the number of cells in the colonies (Fig. 4e). In contrast, colony formation was remarkably suppressed when miR-181b was silenced (Fig. 4e), suggesting that miR-181b promotes the proliferation and colony formation of SFCs.

To evaluate whether miR-181b regulates STAT3 expression, we conducted western blot analysis to detect STAT3. The expression of p-STAT3 protein was significantly increased after transfection with the miR-181b mimic, whereas its expression was suppressed by miR-181b inhibitors (Fig. 4f). Consistently, these results supported those observations obtained using the p-STAT3 inhibitor JSI-124 (Fig. 4f). Collectively, miR-181b positively regulates p-STAT3 in SFCs.

Based on the above observations, we investigated the biological significance of the reciprocal regulation mechanism between STAT3 and miR-181b. Consistent with the results of previous studies [40, 44], inhibition of STAT3 by JSI-124 or depletion of miR-181b with the inhibitor induced apoptosis compared with that measured in NC cells (Fig. 5a). miR-181b mimic transfection significantly protected SFCs from apoptosis induced by interruption of STAT3 activation with JSI-124 (Fig. 5b). Next, we analyzed the protein expression level of STAT3. Increased expression levels of p-STAT3 induced by the miR-181b mimic were reversed by the STAT3 inhibitor JSI-124 (Fig. 5c, lane 2 and lane 3). However, the miR-181b mimic increased the p-STAT3 expression level suppressed by JSI-124 (Fig. 5c, lane 3 and lane 4). In addition, forced STAT3 expression remarkably reversed apoptosis induced by the inhibitor of miR-181b in SFCs (Fig. 5d). To explore whether this effect was related to the miR-181b expression level, miR-181b expression was analyzed. qPCR analysis showed that forced STAT3 expression increased miR-181b expression (Fig. 5e). Thus, mutual regulation between STAT3 and miR-181b confers resistance to apoptosis in SFCs.

According to previous studies [36, 46], the 3′-untranslated region (UTR) of CYLD was identified as a target of miR-181b. However, it was unknown whether the CYLD 3′-UTR is a target of miR-181b in SFCs. Our western blot results demonstrated that forced STAT3 expression decreased CYLD expression in Eca109 SFCs, whereas siSTAT3 increaseded its expression (Fig. 6a). Consistently, the CYLD expression changes were observed in parental Eca109 cells (Fig. 6a). However, no appreciable alterations in CYLD mRNA expression were observed in SFCs regardless of whether STAT3 was present (Fig. 6b). These results suggest that the reduction in CYLD is regulated at the post-transcriptional level and that the CYLD 3′-UTR is a target of miR-181b in Eca109 SFCs.

Analysis using publicly available algorithms (TargetScan and microRNA) predicted CYLD as a target of miR-181b in Eca109 SFCs (Fig. 6c). Moreover, the 3′-UTR sequences of CYLD were found to be highly conserved among different species and showed three possible binding sites for miR-181b (Fig. 6c, D). To determine whether CYLD is direct target of miR-181b, we engineered the 3′-UTR fragments, in which wild-type and mutant binding sites were inserted into the region immediately downstream of the luciferase reporter gene (Fig. 6d). Luciferase reporter assays showed that miR-181b transfection significantly repressed the luciferase activity of the CYLD 3′-UTR, whereas mutations in the binding sites did not decreased the luciferase activity (Fig. 6e). Moreover, qPCR analysis showed that overexpression or inhibition of miR-181b had no effect on the CYLD mRNA expression level (Fig. 6f). However, western blot analysis showed that overexpression of miR-181b remarkably suppressed CYLD expression in SFCs and that inhibition of miR-181b increased CYLD expression in SFCs (Fig. 6g), indicating that miR-181b regulates CYLD in SFCs at the post-transcriptional level. Taken together, these results demonstrate that miR-181b regulates CYLD expression by directly targeting its 3′-UTR.

According to previous studies, CYLD is a deubiquitinating enzyme that negatively regulates NF-κB activity [36, 47, 48]. To further assess the effect miR-181b targeting CYLD, the activities of NF-κB and IL-6 were measured. miR-181b transfection increased NF-κB activity, while inhibition of miR-181b decreased this activity (Fig. 6h). This alteration was also observed for IL-6 activity, which is a target of NF-κB (Fig. 6i). NF-κB and IL-6 activity were increased, which in turn increased p-STAT3 level by western blot analysis. p-STAT3 expression level was decreased by IL-6 antibody treatment (Additional file 1: Fig. S2). These results are consistent with Fig. 4f. These results further suggest that CYLD is a target of miR-181b.

To further explore that miR-181b regulates the SFC proliferation through CYLD pathway, soft agar experiment was employed. The colonies efficiencies of SFCs was decreased by siCYLD, which was reversed by miR-181b (Fig. 7a).

To address whether the above observations in SFC are relevant to human cancer, we examine the relationship between STAT3 and miR-181b expression levels in human ESCC specimens. We collected clinical samples of ESCC (Additional file 1: Table S1) and enriched the SFCs using serum-free culture. There is a positive correlation between STAT3 and miR-181b expression levels in the cancer specimens (r = 0.683) (Fig. 7b). In addition, we found an inverse correlation between miR-181b and CYLD (r = -0.867) (Fig. 7c). These data further support the notion that there is a positive and mutual regulation between STAT3 and miR-181b and that CYLD is a target of miR-181b. Collectively, these studies demonstrate that miR-181b regulates the proliferation of SFCs through CYLD pathway.

Eca109 and Eca9706 cells were cultured in serum-free DMEM/F12 medium (SFDM) (Gibco, Grand Island, NY, USA) supplemented with B27 (Gibco), 20 ng/mL basic fibroblast growth factor (bFGF) (Pepro Tech, Inc., Rocky Hill, NJ, USA), 20 ng/mL epidermal growth factor (EGF) (Pepro Tech, Inc.), and 1 % penicillin and streptomycin using ultra-low attachment plates (Corning, Inc., Corning, NY, USA). Spheres were dissociated using trypsin every 5 days. Human esophageal cancer tissue were collected in Sun Yet-Sen University cancer center. Written informed consent was obtained from all esophageal cancer patients before the study. The use of the clinical specimens for research purposes was approved by the Jinan University Ethics Committee.

Total RNA was extracted using Trizol reagent (Tiangen, Beijing, China) and were reverse-transcribed into cDNA by using PrimerScript Master mix (TaKaRa Biotechnology, China) according to the manufacturer’s protocol. The PCR primers for miR-181b and U6 were purchased from RiBoBio (Guangzhou, China). The following PCR conditions were used on the Light Cycler: 95 °C for 5 s, 60 °C for 5 s, followed by 42 cycles of 95 °C for 15 s and 60 °C for 1 min in a 10 μL reaction volume. The expression of U6 or β-actin was used as an internal control. All experiments were conducted in triplicate. Real-time quantitative PCR was performed using the Bio-Rad system (Hercules, CA, USA) and TaqMan system (Applied Biosystems, Foster City, CA, USA).

Proteins were harvested in cold RIPA buffer. Samples were collected and measured for protein concentration (BCA protein A assay, Byotime, Haimen, China). Proteins were separated by SDS–PAGE and transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). Membranes were blocked in TBST-0.1 % (0.1 % Tween-20 in Tris-base buffer) skim milk and blotted with primary antibodies including STAT3, p-STAT3, CYLD, and β-actin (#9139, #9145, #8462, #4970,respectively, all from Cell Signaling Technology, Danvers, MA, USA) at 4 °C overnight. IL-6 neutralising antibody (#ab6672) was purchased from abcam (USA). Next day the membranes were washed three times TBST-0.1 % buffer. Then the membranes were then incubated with secondary antibodies (Millipore). The signals on the membranes were revealed with ECL reagent (Thermo Scientific, Waltham, MA, USA).

For flow cytometry analysis, 1 × 10⁶ cells were incubated with anti-CD44 (eBioscience, San Diego, CA, USA) PE-conjugated antibody, and anti-CD24 (BD Biosciences, Franklin Lakes, NJ, USA) FITC-conjugated antibody for 30 min, washed three times, and resuspended in PBS. Data were analyzed using Flow Jo software (Tree Star, Ashland, OR, USA). CD44 and CD24 double-negative and single-positive staining controls were used for compensation. Cell staining was visualized using a Nikon inverted microscope (Tokyo, Japan).

According to previous studies [20, 63], sphere formation assays were performed using 96-well ultra-low attachment cell culture plates. Eca109 cells were mixed with serum-free DMEM/F12 sphere culture media containing B27, EGF, and bFGF and then seeded into each well. Plates were incubated for 2 weeks until spheres formed; wells containing spheroid cells were counted.

According to previous studies [20, 63], colony formation assays were performed using 6-well cell culture plates coated with 0.5 mL bottom soft agar mixture (DMEM/F12, 20 % FBS, 0.6 % soft agar). After the bottom layer had solidified, the cells were mixed with top agar (DMEM/F12, 20 % FBS, 0.3 % soft agar), and seeded into each well (3 wells for each concentration). Plates were incubated for 2 weeks until colonies were large enough to be visualized. Colonies were stained with 0.5 % crystal violet for 30 min and counted.

The miR-181b mimic, miR-181b inhibitor, and NC cells were purchased from RIBOBIO (Guangzhou, China). The sequence containing the pre-miR-181b was cloned into the pGCSIL-GFP lentiviral. The miR-181b mimic and siRNA were used with Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. The siRNA against CYLD was refered to Yang’s study [64] and was purchased from Genechem (Shanghai, China). Targeted cells were infected using 1 × 10⁸ lentivirus transducing unit with 8 μg/mL polybreneaccording to the manufacturer's instructions.

The plasmid pcDNA3.1-STAT3-wt UTR was constructed by inserting the STAT3 cDNA into the pcDNA3.1 vector (Invitrogen) at the Xhol and Xbal sites. To construct a luciferase reporter vector, the wild-type 3′-UTR of CYLD, containing three putative binding sites for miR-181b, was PCR-amplified using genomic cDNA from SFCs as templates. The corresponding mutant constructs were created by mutating the seed regions of the miR-181b-binding sites. Both wild-type and mutant 3′-UTRs were cloned downstream of the luciferase gene in the luciferase vector. For luciferase reporter assays, SFCs were transiently transfected with the reporter plasmid and miRNA using Lipofectamine 2000. After 48 h, the cells were harvested and lysed, and luciferase activity was measured using the Luciferase Reporter Assay System (Promega, Madison, WI, USA). Renilla luciferase was used for normalization. For each plasmid construct, the transfection experiments were performed in triplicate.

The handling of mice and experimental protocol were approved by the Experiment Animal Care Committee of Jinan University (Guangzhou, China). 5-week-old male Balb/c mice were purchased from the Animal center of Guangdong Province (Guangzhou, China). Cells from Eca109 cancer parental cell lines or from the SFCs were trypsinized, washed twice, and counted. Next, 5 × 10⁵ cells were resuspended in 100 μL PBS, mixed an equal volume of Matrigel (BD Biosciences) and injected subcutaneously into the neck area of each mouse. Mice were sacrificed after 4 weeks and the tumors were harvested and measured. Tumor weight was measured for statistical analysis. Xenografts were divided into two parts and one was snap-frozen in liquid nitrogen for RNA isolation while the other was used for western blot analysis. Animal studies were approved by the Jinan University Ethics Committee.

The SPSS19.0 program (SPSS Inc., Chicago, IL, USA) was used for statistical analysis. Experimental data are presented as the means ± SD for at least three independent experiments. Student’s t-tests were used for two-group comparisons. Differences between groups were assessed by one-way analysis of variance when more than two groups were compared. Spearman analysis were employed to analyzed the relationship between STAT3 and miR-181b, miR-181b and CYLD. Differences were considered statistically significant at *P < 0.05 and **P < 0.01.