Esophageal cancer (EC) is the eighth most common cancer worldwide and the sixth most common cause of cancer death. There are two main types of EC—squamous cell carcinoma (ESCC) and adenocarcinoma (EAC). Although some advances in the exploration of its possible etiological mechanism were made recently including behaviors and environmental risk factors as well as gene alterations, the molecular mechanism underlying ESCC carcinogenesis and progression remains poorly understood. It has been reported that miR-21 was upregulated in most malignant cancers, the proposed mechanism of which was through suppressing expression of programmed cell death 4 (PDCD4). In present study, it is firstly reported that miR-21 was upregulated in Kazakh’s ESCC and that miR-21 played a negative role in regulating PDCD4 using in situ hybridization (ISH) and luciferase reporter approach. Morever, in model of ESCC xenografted nude mice, miR-21 maybe used as an effective target in the treatment. The present results demonstrated that miR-21 may be a potential therapeutic target in management of ESCC.
EC is the eighth most common malignancy and the sixth leading cause of cancer death in the world [
MicroRNAs (miRs) are short noncoding RNAs which control gene expression by targeting specific genes in a posttranscriptional way [
Pairs of primary ESCC and adjacent normal tissues were obtained from 50 patients, who were hospitalized from 2007 to 2008 at the First Affiliated Hospital, Xinjiang Medical University, China. The present study was approved by the local Medical Ethics Committee and signed informed consent was obtained. None of the recruited patients received treatment before surgery. All tissues were formalin-fixed and paraffin-embedded (FFPE) for pathological diagnosis. Eca109 cell line was purchased from WuHan University (Hubei; WuHan).
Eca109 cells were maintained in DMEM (Gibco) supplemented with 10% FBS (Gibco) in a 5% CO2 humidified incubator at 37°C and transfected with miR-21 mimics, miR-21 inhibitor, and scramble sequence by Lipofectamine 2000 in Eca109 cells as described previously [
Eca109 cells were transfected with two luciferase reporter vectors using Lipofectamine 2000, each of which contains full length of pre-miR-21 sequence and 3′-UTR of PDCD4, respectively (Origene). Luciferase reporter vector with PDCD4 mutant target was transfected in parallel as control. Luciferase activities in the cells were assayed using a luciferase assay kit (Promega).
As Eca109 cells were grown to 85% confluence in 6-well plates, a wound was incised with a sterile 10 uL pipette tip, in the center of the dishes, to create extended and definite scratches with a bright and clear field. Phosphate-buffered saline (PBS) was used to remove the detached cells by washing the cells once. After transfection with miR-21 mimics, inhibitor, and scramble sequence for 24 h, 48 h, and 72 h, respectively, images of cellular morphology from different groups and migratory cells images from the scratched boundary were observed and acquired under the light microscope. The distance between the two sides of the wound was measured with a graduated ruler and relative scratch breadth was determined by a ratio of average breadth in treatment cells versus the average breadth in control cells.
Five-micrometer-thin sections were cut from the FFPE blocks. Expression of miR-21 was detected by ISH with probes for miR-21 according to manufacturer’s protocol of microRNA ISH Optimization Kit (Exiqon). The slides of IHC were dewaxed and rehydrated in a gradient of ethanol and xylene. The antigen retrieval step consisted of microwaving sections in 0.01 M citrate buffer at pH 6.0 for 20 min in 800 W microwave oven operated at full power. The sections were then allowed to cool at room temperature (RT), and then it was treated with 0.3% H2O2 in methanol for 15 min at RT. After washing three times with PBS, they were incubated with mouse monoclonal antibody against PDCD4 (Cell Signaling Technology) from human source.
To evaluate
All statistical analysis was carried out using SPSS for Windows version 13.0 (SPSS). Student’s
To determine whether PDCD4 protein was one of the downstream targets of miR-21, Eca109 cells were transfected simultaneously with two different luciferase reporter vectors with full length of 3′-UTR of PDCD4 and pre-miR-21 sequence, respectively, as described previously [
miR-21 directly regulated PDCD4-3′-UTR. (a) Prediction result of targetscan (
Wound-healing assay showed that miR-21 could dramatically promote the mobility of Eca109 cells after exogenous upregulation (Figure
miR-21 promoted tumor cell migration after transfection. (a) Cell migration ability was assessed using wound-healing assay. Eca109 cells were transfected with miR-21 mimics, inhibitor, and scrambled sequence and were assessed for migration by wound-healing assay at 0 h, 24 h, 48 h, and 72 h after transfection. Images of migratory cells from the scratched boundary were observed and acquired with light microscope (10 × 10). Similar results were obtained in three independent experiments, and shown were representative figures. (b) Statistical analysis of wound-healing. There was significant difference between miR-21 mimics and control group (
In statistical analysis of wound-healing (Figure
To determine and visualize the expression status of miR-21 in Kazakh’s ESCC and paired control, ISH was employed for its advantages in localization and qualitative expression in FFPE blocks in comparison with qRT-PCR techniques, which was adopted as classical approach in the detection of miRNAs. There were 50 paired Kazakh’s ESCC tissues and matched adjacent normal controls were enrolled. MiR-21 was extremely significantly overexpressed in Kazakh’s ESCC tissues in comparison with paired adjacent normal control (
The expression of miR-21 and PDCD4 in paraffin-embeded ESCC and matched tissues by ISH and IHC.
Sample |
miR-21 expression |
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PDCD4 expression |
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Negative (−) | Positive (+) | Negative (−) | Positive (+) | |||
ESCC | 13 (26.4%) | 37 (73.6%) | 0.004 | 35 (70.0%) | 15 (30.0%) | 0.000 |
Normal | 27 (51.0%) | 23 (49.0%) | 12 (24.0%) | 38 (76.0%) |
miR-21 negatively correlated with PDCD4 expression in ESCC tissues. ((a), (b)) miR-21 was detected as a weak positive staining in normal epithelium, strongly positive staining of the tumor. ((c), (d)) PDCD4 was detected as a strongly positive staining in normal epithelium, weak positive staining of the tumor.
To further confirm the upregulation and the localization of PDCD4 in Kazakh’s ESCC and paired adjacent normal tissues, the rabbit anti-PDCD4 antibody was used for IHC in enlarged 50 pairs of tissues. Significantly low expression of PDCD4 protein was displayed in ESCC compared with matched tissues. Positive immunoreactivity of PDCD4 was located in cell nucleus. Most of normal esophagus epithelium showed high expression of PDCD4 whereas low expression was detected in ESCC (Figures
PDCD4 protein, a putative downstream target of miR-21, has been proposed several times in previous studies. The expression of PDCD4 was determined using IHC with necessary controls in the same panel of FFPE blocks as detected for miR-21. It was shown that PDCD4 was significantly lower expressed in ESCC compared with paired adjacent normal controls, the trend of which was quite opposite with that of miR-21. Clinicopathological analysis showed that PDCD4 protein correlates inversely with miR-21 expression (
Correlation between expression level of miR-21 and PDCD4 in 50 pairs of ESCC.
PDCD4 expression |
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| ||
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Negative (−) | Positive (+) | ||||
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miR-21 expression | |||||
Negative (−) | 3 (6.0%) | 10 (20.0%) | 7.144 | 0.010 | −0.371 |
Positive (+) | 32 (64.0%) | 4 (8.0%) | |||
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miR-21 expression | |||||
Negative (−) | 3 (6.0%) | 24 (48.0%) | 5.346 | 0.020 | −0.327 |
Positive (+) | 9 (18.0%) | 14 (28.0%) |
To investigate the prognostic significance of miR-21 and PDCD4 expression in Kazakh’s ESCC, Kaplan-Meier analysis was performed illustrating that there is no significant correlation between poorer overall survival and disease-free survival in the ESCC case. But the tendency of survival status tended to be significantly associated with miR-21 and PDCD4 expression (Figure
Prognostic analysis of miR-21 and PDCD4 expression. 50 ESCC patients with their following information available were classified into negative expression group and positive expression group. Shown are Kaplan-Meier overall survival curves of two group patients; log-rank test was used. (a) Expression of PDCD4 protein is associated with overall survival of ESCC patients. (b) Expression of miR-21 is associated with overall survival of ESCC patients.
As miR-21 inhibitor can prevent ESCC cell growth
miR-21 promotes tumorigenesis of ESCC in xenografted nude mice. Twice per week intratumor injection of miR-21 mimics, inhibitor, and scramble reduces tumor volume followed for 28 d after implantation. (a) Evaluation of tumor size at different therapeutic times; (b) gross morphology of tumors resected from different groups.
In present study, miR-21 was significantly upregulated in Kazakh’s ESCC tissues compared with paired adjacent normal controls, and that miR-21 directly negative-regulated PDCD4 protein and that miR-21 could be used as potential therapeutic target in the treatment of ESCC.
With regard to the interplay between miR-21 and PDCD4, which was definitely verified by a series of similar studies, that miR-21 was generally overexpressed in cancerous tissues versus paired normal controls, regardless of its tumor types or origins [
As regards the inverse relationship between miR-21 and PDCD4 expression, our results were consistent with Asangani et al.’s [
Taken together, our results in ESCC xenografts imply that target miR-21 may hold great promise for designing novel therapeutic strategies against ESCC carcinogenesis and development. MiRNA AMO or mimics could potentially be used as single therapeutic agents or in combination with other conventional chemotherapies or radiotherapies to achieve optimal therapeutic effect.
Finally, the present study demonstrated that miR-21 promoted proliferation, migration of ESCC both
The authors have declared no conflict of interests.
Tao Liu and Qing Liu contributed equally to this work.
The project was supported by Natural Science Foundation of Xinjiang Uygur Autonomous Region (no. 201211B30).