miR-33a-5p inhibits the progression of esophageal cancer through the DKK1-mediated Wnt/β-catenin pathway

Clinical value of miR-33a-5p and DKK1 in EC

We initially evaluated the clinical correlation of miR-33a-5p and DKK1 with EC. Comparison of clinical data between EC patients and concurrent healthy controls showed that there was no difference between the two groups in terms of gender, age, smoking history and drinking history (Table 1). After detection, qPT-PCR showed that miR-33a-5p expression was low expressed (Figure 1A and 1B) while DKK1 expression (Figure 1C and 1D) was highly expressed in EC tissues and serum samples. The expression of miR-33a-5p or DKK1 in EC tissues was positively correlated with the expression of miR-33a-5p or DKK1 in EC serum samples, respectively (Figure 1E and 1F). Meanwhile, the expression of miR-33a-5p was negatively correlated with DKK1 expression in both EC tissues and EC serum samples (Figure 1G and 1H). ROC analysis revealed that the AUCs of serum miR-33a-5p and DKK1 for EC occurrence were 0.914 and 0.900, respectively (Table 2). Further analysis of the relationship between miR-33a-5p, DKK1 and clinical characteristics showed that tumors in most cases were larger than 5cm in size, and were mostly moderately and highly differentiated (Table 3), indicating that miR-33a-5p and DKK1 were associated with higher TNM staging and differentiation degree. Then, the correlation of miR-33a-5p and DKK1 with 5-year OS of EC patients was determined, with the corresponding median value of serum miR-33a-5p and DKK1 expression as its boundary for defining their high and low expression (Figure 1I and 1J). Survival analysis found that patients with low miR-33a-5p expression or high DKK1 expression in serum had a worse 5-year OS (Figure 1I and 1J). Cox regression analysis further indicated that TNM staging, low miR-33a-5p expression and high DKK1 expression were independent prognostic indicators of 5-year OS in patients (Table 4).

Effects of miR-33a-5p on the biological functions of EC cells

Then, we further explored the effect of miR-33a-5p on EC progression in vitro. The qRT-PCR showed miR-33a-5p expression in EC cell lines KYSE150, TE-1, EC9706, Eca-109 was lower than that in human esophageal epithelial cell line HET-1A (Figure 2A). TE-1 and Eca-109 were selected for subsequent transfection. qRT-PCR results revealed that compared with miR-NC, miR-33a-5p expression in cells up-regulated significantly after transfection with miR-33a-5p-mimics, while decreased after transfection with miR-33a-5p-inhibitor (Figure 2B). CCK-8 assay indicated that miR-33a-5p upregulation inhibited cell proliferation, while miR-33a-5p significantly enhanced cell proliferation (Figure 2C). Transwell assay results exhibited that cell migration and invasion ability were inhibited after upregulation of miR-33a-5p, but enhanced after knockdown of miR-33a-5p (Figure 2D and 2E). AnnexinV-PI double staining test results validated that miR-33a-5p overexpression induced apoptosis, while miR-33a-5p knockdown decreased the apoptotic ability of cells (Figure 2F).

DKK1 is identified as the target of miR-33a-5p

Next, we explored the potential mechanism underlying miR-33a-5p-mediated EC progression. Given that miR-33a-5p could cause various biological functions of EC cells, we used TargetScan and miRDB database to predict its potential targets. Bioinformatics analysis in TargetScan and miRDB database indicated that DKK1 was the possible target of miR-33a-5p (Figure 3A). Subsequent DLR assay revealed that miR-33a-5p upregulation inhibited the activity of DKK1-WT, and conversely, miR-33a-5p downregulation significantly increased the activity of DKK1-WT; however, the luciferase activity was not affected after co-transfection with DKK1-MUT (Figure 3B). Then Western blot analysis exhibited that DKK1 protein expression was significantly reduced after transfection with miR-33a-5p-mimics, while was elevated after miR-33a-5p knockdown (Figure 3C). All these suggested that miR-33a-5p can directly regulate DKK1 expression in cells by binding to the 3′-UTR sequence.

Effects of DKK1 on the biological behavior of EC cells

Furthermore, we assessed whether DKK1 affected EC cell proliferation, migration and invasion, as well as apoptosis. The qRT-PCR showed a significant decrease in DKK1 expression after transfection with si-DKK1, while a significant increase in DKK1 expression after transfection with sh-DKK1 (Figure 4A). CCK-8 assay revealed that the proliferation of cells was inhibited after DKK1 knockdown (Figure 4B). Transwell assay exhibited that knocking down DKK1 lowered cell invasion and migration (Figure 4C and 4D). Flow cytometry analysis demonstrated that DKK1 knockdown significantly promoted cell apoptosis (Figure 4E). However, the opposite results were obtained by detecting these biological behaviors of cells after DKK1 overexpression (Figure 4E). Western blot analysis showed that knockdown of DKK1 could inhibit the expression of cyclin D1, c-jun, CD44, and c-Met proteins in cells, and correspondingly, overexpression of DKK1 can promote the expression of cyclin D1, c-jun, CD44, and c-Met proteins (Figure 4F).

miR-33a-5p targets DKK1-mediated Wnt/β-catenin pathway to influence biological behavior of EC

To further confirm whether miR-33a-5p affects EC by regulating DKK1, we transfect TE-1, Eca-109 cells with miR-33a-5p-mimics, miR-NC + si-NC, sh-DKK1, and miR-33a-5p-mimics + sh-DKK1. It was observed that DKK1 upregulation offset the effects of miR-33a-5p overexpression on the proliferation (Figure 5A), migration (Figure 5B), invasion (Figure 5C), and apoptosis (Figure 5D) of TE-1 and Eca-109 cells. Western blot detection demonstrated that cyclin D1, c-jun, CD44, and c-Met protein expression levels were not statistically difference between cells transfected with miR-33a-5p-mimics + sh-DKK1 and those transfected with miR-NC + si-NC (Figure 5E); however, compared with sh-DKK1 transfected cells, the expression levels of cyclin D1, c-jun, CD44, and c-Met protein expression were markedly decreased in cells transfected with miR-33a-5p-mimics + sh-DKK1 (Figure 5E).

miR-33a-5p inhibits the tumor growth of EC in vivo

Next, we tried to explore the impact o miR-33a-5p on the EC cell growth in vivo. To this end, the tumorigenicity analysis was carried out in nude mice injected with TE-1 cells treated with control mimic or miR-33a-5p-mimic. Significantly, the miR-33a-5p-mimic attenuated the tumor growth of TE-1 cells in vivo, as demonstrated by the decreased tumor size (Figure 6A), repressed tumor volume (Figure 6B), and reduced tumor weight (Figure 6C). As expected, the miR-33a-5p expression was up-regulated and DKK1 expression was down-regulated by miR-33a-5p-mimic in the tumor tissues of the mice (Figure 6D and 6E). Consistently, the levels of cyclin D1, c-jun, CD44, and c-Met protein were repressed by miR-33a-5p-mimic in the model (Figure 6F).

Clinical data

In total 68 patients with EC admitted and treated in Tumor Hospital of Liaocheng from March 2012 to March 2014 were selected. Inclusion criteria: Diagnosed as EC by pathological examination, all the patients met the TNM staging criteria issued by AJCC in 2017 [32] and had not received any prior treatment before surgery, with complete clinical data. Exclusion criteria: Patients with other malignant tumors, severe liver and kidney dysfunction, or infection were excluded, as well as those who lost to follow up; Patients with other diseases which might lead to the abnormal increase or decrease of miR-33a-5p or DKKl were excluded. Additionally, 68 healthy controls were selected. With the approval of the Medical Ethics Committee of Tumor Hospital of Liaocheng and the written informed consent obtained from all the participants, this study was conducted. EC patients were followed up for 5 years, and the total survival (OS) was from the time of surgery to the end of follow-up or the patient’s death [33].

Sample collection

Elbow vein blood (5 ml) was collected from EC patients (before surgery) and healthy controls and centrifuged at 4°C and 800g for 10 min. Healthy controls didn't have EC or any other type of cancer. The resulting supernatant was then transferred to the test tube for cryopreservation. After surgical resection, specimens of paired EC and normal tissues (5 cm from the edge of the tumor) were collected, frozen in liquid nitrogen tank, and then stored in a cryogenic refrigerator at -80°C for use.

qRT-PCR detection

miRNA was extracted from serum by miRNeasy kit (Invitrogen, Carlsbad, CA, USA, AM1561), while the RNA from cells and tissues was extracted using the TRIzol kit (Biolab Technology Co., Ltd., Beijing, China). The purity and the concentration of RNA were determined by SMA6000 microspectrophotometer (Merinton Instrument Co., Ltd., Beijing, China). For the determination of miR, the total RNA was reverse transcribed into cDNA using the TaqMan miRNA reverse transcription kit (Biolab Technology Co., Ltd., Beijing, China) according to the kit instructions, and PCR quantification was performed with the TaqMan Universal PCR Master Mix kit (Chundu Biotechnology, Co., Ltd., Wuhan, China). As to the determination of mRNA, reverse transcription of total RNA into cDNA was conducted by Prime Script RT kit (Future Biotechnology, Co., Ltd., Beijing, China), and PCR quantification was carried out using the SYBR Green qPCR superMix-UDG kit (Biolab Technology Co., Ltd., Beijing, China). On an ABI 7300 real-time PCR system (ThermoFisher Scientific, Co., Ltd., Shanghai, China), Then qPCR was performed on the ABI 7300 real-time PCR system (Thermo Fisher Scientific, Shanghai, China). The test was repeated 3 times. U6 and GAPDH were used as internal controls of miR-33a-5p and GAPDH respectively, and the expression levels of miR-33a-5p and GAPDH were calculated using 2^−ΔΔCt. The primers were synthesized and purchased (GenScript, China). Primer sequences: miR-33a-5p: Forward: 5′-GTGCATTGTAGTTGCATTGCA-3′; Reverse: 5′-GTGCAGGGTCCGAGGT-3′. DKK1: Forward: 5′-CCTTGAACTCGGTTCTCAATTCC-3′; Reverse: 5′-CAATGGTCTGGTACTTATTCCCG-3′. U6: Forward: 5′-GCTTCGGCAGCACATATACTAAAAT-3′; Reverse: 5′-CGCTTCACGAATTTGCGTGTCAT-3′. GAPDH: Forward: 5′-ATTCCATGGCACCGTCAAGGCTGA-3′, Reverse: 5′-TTCTCCATGGTGGTGAAGACGCCA-3′.

Cell culture and transfection

EC cell lines KYSE150, TE-1, EC9706 and Eca-109, as well as human esophageal epithelial cell line HET-1A (Otwo Biotechnology Co., Ltd., Shenzhen, China) were all cultured in Roswell Park Memorial Institute (RPMI1640) medium (Biolab Technology Co., Ltd., Beijing, China) containing 10% fetal bovine serum (FBS, Gibco, NY, USA) and penicillin/streptomycin double antibodies (SolarBio, China) in a 37°C and 5% CO₂ constant temperature cell incubator. Transfection was performed when the confluence reached 70%. miR-33a-5p-mimics, miR-335-3-inhibitor, miR-33a-5p negative control sequence (miR-NC), DKK1 RNA negative control (si-NC), targeted inhibition of DKK1 RNA (si-DKK1) and targeted overexpression of DKK1 RNA (sh- DKK1) were synthesized and obtained from ThermoFisher Scientific, Shanghai, China. The transfection process was carried out in strict accordance with the instructions of Lipofectamine 3000 transfection reagent (Biolab Technology Co., Ltd., Beijing, China).

Detection of cell proliferation

The cell proliferation test was carried out according to the instructions of the cell counting kit-8 (CCK-8; Yiyan Biotechnology Co., Ltd., Shanghai, China). Cells were seeded into 96-well plates. The absorbance (OD) values of the cells were measured at specific time points. CCK-8 solution (10 μL) was added to each well for another 2-hour culture at the time of detection, and then the OD value was measured at the 450nm wavelength on a AMR-100 automatic microplate reader (Allsheng Instrument Co., Ltd., Hangzhou, China).

AnnexinV-PI double staining for apoptosis detection

The collected cells were digested with trypsin and resuspended with 200 μL binding buffer. Then, 5 μL AnnexinV-FITC (Beyotime, China) was added to each well and incubated at 4°C in dark for 15 min before the addition of 10 μL PI solution. After incubation for 5 min, CytoFLEX flow cytometry (Beckman Coulter Commercial Enterprise (China), Co., Ltd.) was applied to detect cell apoptosis.

Transwell chamber assay for cell migration and invasion

Migration detection: 24 hours after transfection, cells were seeded into the Transwell upper chamber (Corning, NY, USA) at 5 × 10⁵ cells/well and supplemented with serum-free Dulbecco's modified eagle medium (DMEM) to 200 μL, while 700 μL DMEM containing 10% FBS was added to the lower chamber for culture in a 37°C and 5% CO₂ thermostatic cell incubator.

Twenty-four hours later, the chamber was taken out, and the cells remaining in the upper chamber were cleared away. Then the cells were immobilized with formaldehyde for 10 min and dyed with crystal violet for 30 min at room temperature. Finally, cell counting was performed under a microscope, with 5 fields being randomly observed in each hole, and the number of cell transmembrane was compared. Invasion detection: serum-free DMEM and Matrigel glue were used to prepare Matrigel diluent at 8:1. Then the diluent was added to the bottom of the upper chamber at 50 μL per well and placed overnight at 4°C. The follow-up experiment steps were the same as the migration assay.

Western blot detection

After cell lysis, bicinchoninic acid (BCA) (Abbkine, CA, USA) method was used to detect the protein concentration, and 30 μL protein was separated using 12% sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE). Then the protein was transferred to a Polyvinylidene Fluoride (PVDF) membrane (Millipore, MA, USA), sealed with 5% skim milk powder for 2 h, and added with cyclin D1, c-jun, CD44, c-Met and β-actin primary antibodies purchased from Shanghai Beyotime Biotechnology Co., Ltd. (Dilution ratio: 1: 1000, AF1183, AF1612, AF0105, AF1432, AF0003), for overnight incubation at 4°C. Then TBST buffer was applied for three rinses of the membrane, 10min each, followed by a 3-minute incubation with the corresponding horseradish peroxidase-labeled goat anti-rabbit (HRP)-labeled secondary antibody (Beyotime Biotechnology Co., Ltd., Shanghai, China, dilution ratio: 1:1000, A0208). The membrane was finally developed by ECL (Millipore, Germany), and the grayscale values were analyzed using Quantity One 1-D software.

Dual luciferase reporter (DLR) assay

TargetScan and miRDB database [34] predicted that DKK1 was a possible target of miR-33a-5p. The wild type sequences of the 3′UTR region of DKK1 were cloned into the pmirGLO vectors and purchased (GeneScript, China). The site-specific mutated sequences (shown in Figure 3A) of the 3′UTR of DKK1 were inserted into pmirGLO vectors and purchased (GenScript, China). Lipofectamine 3000 was used to co-transfect DKK1-WT or DKK1-MUT with miR-33a-5p-mimic, miR-33a-5p-inhibitor or miR-NC into cells. Forty-eight hours after transfection, DLR gene assay kit (Shanghai Beinuo Biotechnology Co., Ltd., China, SLDL-100) was utilized to measure luciferase activity.

Xenograft tumor model

All animal experiments in this study were approved by the Tumor Hospital of Liaocheng. BALB/c nude mice aged 5 weeks were purchased from Shanghai Laboratory Animal Center of China. TE-1 cells were transfected with miR-33a-5p mimic or negative control, and suspended in saline at a density of 1×10⁷ cells/ml. A total of 200 μL TE-1 cell suspension was subcutaneously injected to left back of mice. The mice were euthanized at day 30, and the tumors were isolated, weighted and processed for subsequent analysis. Tumor size was measured every 5 days and calculated using the following formula: width² × length/2.

Statistical analysis

Data in line with normal distribution were expressed as mean ± SD, and the inter-group comparison was conducted by the independent sample t-test. Receiver operating characteristic curve (ROC) was employed to evaluate the correlation of miR-33a-5p and DKK1 with EC occurrence, and the area under the curve (AUC) was calculated. Pearson test was utilized for correlation analysis between miR-33a-5p and DKK1. Multiple time point data were analyzed by repeated measures analysis of variance (ANOVA), and Bonferroni method was used for post hoc test. Data comparison among multiple groups was performed by one-way ANOVA, and the post-hoc test was performed by LSD-t. The survival curve of patients was visualized using Kaplan-Meier method, and Log-rank was used to evaluate the difference of survival time in patients with high or low expression of miR-33a-5p and DKK1. Univariate and multivariate Cox regression analysis was used to analyze the prognostic factors of patients. A statistically significant difference was assumed at P < 0.05. SPSS 20.0 and GraphPad Prism 8.0 (IBM Corp, Armonk, NY, USA) were used for statistical analysis.