ErbB4 is an important member of ErbB subfamily of tyrosine kinases receptor with overexpression in several tumors; however its biological role in esophageal cancer is poorly understood till date. The main objective of this study was to examine whether miRNA-140-5p could target and control ErbB4 expression at transcriptional level. The ErbB4 expressions in different cell lines were evaluated by western blotting and luciferase assay. Moreover, cell proliferation, apoptosis, and cell invasion studies were investigated using MTT, flow cytometry, and transwell assays. miRNA-140-5p remarkably downregulated the ErbB4 expression in EC9706 and TE-1A cell lines. Furthermore, miRNA-140-5p transfected cell significantly controlled the cell proliferation and enhanced the apoptosis of multiple cells. Additionally, miRNA-140-5p had marked effect on the DNA synthesis and caspase 3/7 activity in comparison to control cells. Specifically, miRNA-140-5p inhibited/repressed the cancer cell invasion and migration in a sign to have important biological role in esophageal carcinomas. Taken together, miRNA-140-5p could act as a potential molecular target in ErbB4 overexpressing ESCC cell lines paving the way for effective esophageal cancer treatment.
Esophageal cancer is one of the most fatal cancers worldwide with high prevalence in East Asian countries (China) [
Studies have shown that ErbB/HER receptors which govern the multitude functions including the cell proliferation, cell differentiation, and signaling pathway mediated cell death in cancer cells [
MicroRNAs (miRNAs) are small, endogenous, and noncoding RNAs of 20–22 nucleotides that suppress the translation/degradation and gene expression of messenger RNAs (mRNAs) at postblockade level [
Based on these observations, clinical potential of miR-140-5p as a cancer biomarker or therapeutic agent has been studied in the present work. Therefore, the main aim of this study was to examine the expression of miR-140-5p in ESCC cell lines and to draw a linear relationship between ErbB4 level and miR-140-5p in the given cell line. We have studied the effect of miR-140-5p and anti-miR-140-5p on the apoptosis (BrdU incorporation and caspase 3/7) level on ESCC cells. Furthermore, we have carried out experiments to prove ErbB4 as a potential molecular/pharmacological target in the treatment of esophageal cancers.
miR-140-5p mimics and miRNA negative control mimics (miR-NC), miR-140-5p specific antisense inhibitor (anti-miR-140-5p), and its negative control inhibitor (anti-miR-NC) were obtained from GenePharma (Shanghai, China). The esophageal cell lines, namely, EC9706 and TE-1, were procured from Cell Bank of Shanghai (China). The cells were immediately cultured in RPMI media supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin in a controlled incubator.
ES9706 and TE cells were seeded in a 6-well plate at a seeding density of 1 × 106 cells/well and allowed to attach overnight (15 h). The cells were then transfected with miR-140-5p and miR-NC (200 nm) until the cells reach 70–80% confluency. The cells were analyzed using Turbofect transfection reagent (Fermentas).
3′UTRs of ErbB-4 were amplified using PCR from genomic DNA and conjugated into pMIR-140-5p-report (Ambion). Followed by, miR-140-5p target sequence was induced from site-specific mutagenesis kit (Stratagene). Sequencing was done to confirm all the constructs. ESCC 9706 cells were transfected with miR-140-5p and miR-NC mimics and incubated for 48 h. The luciferase activities were evaluated through Dual Luciferase Reporter Assay System (Promega) which was normalized by dividing firefly luciferase activity with that of Renilla luciferase activity.
The cell proliferation/cell viability was performed by means of MTT assay. Briefly, cells were seeded and incubated in a 96-well plate at a density of 1 × 104 cells per well. The old media were removed and replaced with fresh media and the cells were incubated with pcDNA 6.2-GW/EmGFP-miR (mock) and anti-miR-inhibitors-negative control (control). And, miR-140-5p and anti-miR-140-5P were exposed to cancer cells and incubated for 24 h. The cells were washed and treated with MTT (5 mg/mL) solution, followed by absorbance that was measured by a microplate reader at a wavelength of 570 nm. The absorbance was measured using a sophisticated multidetection microplate reader (BMG LABTECH, Durham, NC, USA). All experiments were performed 8 times (8 wells).
Cell invasion assay was performed using transwell chambers (Millipore, Billerica, USA) coated with matrigel. miR-140-5p or anti-miR-140-5p or mock transfected ESCC cells were trypsinized, washed, and resuspended in a serum-free RPMI 1640 medium. This suspension was added to the upper compartment of transwell chamber (with matrigel). Complete RPMI 1640 medium with 20% FBS was placed at the bottom part of matrigel chamber as a chemoattractant. The cells were allowed to incubate for 24 h after which noninvading cells were removed using a cotton swab. The number of cells invaded was fixed and stained with crystal violet (0.1%). The cells were counted using a light microscope.
Western blotting analysis was carried out by electrophoresing 25
EC9706 cells were seeded in a 6-well plate at a density of 3 × 105 cells/well and incubated for 24 h. Cells were exposed to different formulations at an equivalent concentration of 1
The cells were exposed to miR-140-5p and its corresponding mock (25 nm) mimics in a 96-well plate. The miRNA-transfected cells were trypsinized/harvested and incubated with 10
The cells were exposed to miR-140-5p and its corresponding mock (25 nm) mimics in a 96-well plate. The miRNA-transfected cells were trypsinized/harvested and used to measure caspase 3/7 activity using Caspase-Glo 3/7 assay (Promega) as per the manufacturer’s instruction.
To validate our postulation, miRNA-140-5P expression in ESCC surgical samples was investigated by qRT-PCR technique. As can be seen (Figure
miR-140-5p expression in esophageal squamous cell carcinoma and matched adjacent normal cells. The expression of miR-140-5p in ESCC and normal cells was analyzed using immunoblot analysis.
ErbB4 expression has been reported in multiple cancers including colon, ovarian, lung, breast, and esophageal cancers [
Earlier, literature surveys have suggested that miRNAs play a central role in gene expression regulation and tumorigenesis. Studies especially suggested that miRNA expression is dysregulated in multiple cancers and miRNA may be a new targeting agent in the treatment of pathological cancers in the clinics [
miR-140-5p controls the ErbB4 expression. ErbB4 expression in esophageal cancer cell lines (ECA 109, EC9706, and TE-1) and normal cell line (HET-1A) via western blot analysis (a). miR-140-5p expression in these cell lines (b). Western blot analysis of effect of miR-140-5p on ErbB4 protein expression (c and d). RT-PCR analysis of effect of miR-140-5p on ErbB4 mRNA expression (e). Analysis of luciferase reporter activity in EC9706 cancer cell line (f). The luciferase activity was examined by Renilla luciferase method.
Next, we investigated whether ErbB4 is the pharmacological target of miR-140-5P or not. For this, luciferase reporter was constructed with 3′UTR of ErbB4 which was cloned with firefly luciferase gene. Now, EC-9706 cells were transfected with either miR-140-5P or miR-NC to examine the effect of miRNAs on the ErbB4 3′UTR with regard to relative luciferase activity. As can be seen (Figure
Having confirmed ErbB4 as the functional target for miR-140-5p, we next examined whether the miRNA can be effective to control or limit the cell proliferation activity in esophageal cancer cell lines such as EC-9706 and TE-1A cells. For this, EC-9706 and TE-1A cells were transfected with miR-140-5p and anti-miR-140-5p and cell viability was analyzed using the colorimetric MTT assay at 570 nm. As can be seen, cancer cells treated with anti-miR-140-5p remarkably increased the cell viability in comparison to the cells transfected with anti-miR-140-5p (Figures
Effect of miR-140-5p on cell proliferation of esophageal cancer cells. Cell proliferation of EC9706 (a and b) and TE-1 (c and d) was analyzed after transfection with miR-140-5p and anti-miR-140-5p. The cells were exposed to respective mRNA sequence and incubated for 24, 48, and 72 h and growth inhibition was evaluated using the MTT assay.
Analysis of effect of miR-140-5p on EC9706 cells via flow cytometer after staining with Annexin V (a). Percentage cell apoptosis was examined using flow cytometer after transfecting EC9706 and TE-1 cells with miR-140-5p (b and c). The percentage of cells in early and late apoptosis was quantified by interpretation of quadrant (UR and LR) with regard to control. The apoptosis assay was performed by staining Annexin V/PI on cells and analyzed by flow cytometry (d). The expression of apoptotic-related proteins, cleaved caspase 3, and cleaved PARP-1 was detected by western blot analysis, and
Consistent with the profound antiproliferative effect and apoptosis effect of miR-140-5p on esophageal cancer cells, its effect on the DNA synthesis to inhibit cell proliferation (EC-9706) was analyzed. To analyze DNA synthesis level, BrdU incorporation was examined. As can be clearly seen in Figure
Antiproliferative effects of miR-140-5p on DNA synthesis (a) and caspase 3/7 activity (b). The miR-140-5p transfected cells were incubated with BrdU, followed by BrdU incorporation that was measured. Caspase 3/7 activity was measured as per the manufacturer’s instruction. Values are expressed as mean ± SD from three independent experiments. Light microscopic photomicrographs of EC9706 cells after exposure to different concentration of miR-140-5p (c).
Flow cytometry analysis confirmed the role of miR-140-5p in apoptosis pathways. In order to further investigate its effect on the apoptotic pathways, caspase 3/7 activity was measured (Figure
Apoptosis was further visualized by light microscopy wherein viable cells remained connected with specific morphology; however, for the addition of miR-140-5p, a slight change in morphology was observed (Figure
Transwell assay was carried out to investigate the correlation between miR-140-5p and ESCC cell invasion. It can be clearly seen that overexpression of miR-140-5p significantly controlled the cell invasion in EC9706 cell lines (Figure
Effect of miR-140-5p on cell invasion of esophageal cancer cells. miR-140-5p and anti- miR-140-5p were transfected on EC9706 (a and b) and TE-1 (c and d) cells and cell invasion was analyzed using the transwell invasion assay.
Consistent with cell invasion, migration of cells was examined in matrigel-coated polystyrene membrane. As expected, ectopic expression of miR-140-5p remarkably inhibited the migration of EC9706 cells by around 30–40% in comparison to control cells. Similar trend was observed for TE-1A cells which also showed remarkable inhibition of cell migration (data not shown).
Therefore, miR-140-5p has been shown to have a pronounced role in the tumorigenesis and progression of tumors. In the present study, we have showed that miR-140-5p remarkably downregulates the ErbB4 protein expression in both the esophageal carcinoma cell lines. Furthermore, our results suggest that miR-140-5p could directly target the ErbB4 receptor and could effectively prohibit the cell proliferation and repress cell invasion and cell migration of EC9706 and TE-1A cell lines. Therefore, overexpression of single strain of RNA can easily coordinate/regulate the gene expression on the cell related functions that will further encourage the therapeutic use of miRNA as a targeting agent. However, challenges in using therapeutic RNA are to precisely predict the molecular pathway or signaling pathway as a result of the binding interactions. In this regard, transcriptome analysis by microarray can be utilized to find out the miRNA target identification. The results therefore indicate that onco-miRNAs and suppressor-miRNAs could control the same gene with two different roles, either as oncogenes or as tumor suppressing genes depending on the targets.
Summing up, we have successfully showed that miRNA-140-5p could directly target ErbB4 protein in EC9706 and TE-1A esophageal cancer cell lines to control the cell proliferation and repress the cell invasion and migration. To the best of our knowledge, we for the first time demonstrated the correlation between ErbB4 and miRNA-140-5p in ESCC cell lines. For this first we examined the ErbB4 expression in normal and cancerous cell lines, followed by miRNA expression in these cell lines. Result suggested a significant downregulation of ErbB4 receptor in cancerous cell lines in comparison to normal cell lines. We have also demonstrated the cell proliferation regression in the miRNA-140-5p transfected cell in comparison to anti-miRNA-140-5p transfected cells. Finally we showed a remarkable apoptosis level, augmented DNA synthesis inhibition, and elevated caspase 3/7 activity with miRNA-140-5p transfected cell lines. The RNA transfected cells were observed to repress the cell invasive ability with parallel inhibition of migrations. Overall, our results suggest the potential application of miRNA-140-5p as a potential therapeutic moiety in the treatment of ErbB4 mediated/overexpressed esophageal cancers.
The authors report no conflict of interests.
The authors thank all the participants in the study. They thank Dr. Chen and Dr. Zhang for their technical assistance in the study.