Abstract
Background/Aim: Basaloid squamous cell carcinoma of the oesophagus (BSCCE) is a variant of oesophageal squamous cell carcinoma (ESCC), that has a more aggressive biological behaviour than that of typical ESCC. miR-3687 has been previously identified to be highly expressed in BSCCE. This study aimed to evaluate the prognostic impact of miR-3687 in ESCC and determine the role of miR-3687 in ESCC motility. Materials and Methods: miR-3687 expression in human ESCC cell lines and in primary tumour samples obtained from patients with ESCC who underwent esophagectomy were analyzed via real-time polymerase chain reaction (PCR). Knockdown and over-expression experiments were conducted with miR-3687 siRNA and miRNA mimic, and the effect on cell proliferation, migration, and invasion was analyzed. Results: A total of 92 samples were analyzed. High miR-3687 expression was correlated with poor prognosis. miR-3687 upregulation promoted cell migration and invasion. Conclusion: miR-3687 expression negatively impacts the prognosis of patients with ESCC.
Oesophageal cancer is one of the primary malignant tumours with poor prognosis, and its morbidity and mortality rank eighth and sixth worldwide, respectively, among malignant tumours (1). Unlike other organs, the oesophagus has no serous membrane and thus the tumour easily invades other organs. Moreover, the location of the oesophagus makes it adjacent to large blood vessels and lymph nodes, the lungs, heart, and stomach. As such, the tumour can not only migrate to nearby organs, but also to distant organs such as the brain and adrenal gland. Given that oesophageal carcinoma is often asymptomatic or presents with non-specific symptoms, patients are usually diagnosed at the late stage of the disease. Therefore, accurate diagnostic modalities and effective treatment targets need to be identified to improve the prognosis of oesophageal carcinoma.
Basaloid squamous cell carcinoma of the oesophagus (BSCCE) was first identified 20 years ago as a variant of squamous cell carcinoma with a distinct basaloid morphology (2, 3). It commonly develops in the mid to distal oesophagus and among elderly men. The incidence of BSCCE accounts for approximately 1% of all cases of oesophageal squamous cell carcinoma (ESCC). Histologically, it is characterised by a solid growth pattern with a nesting, lobular, or trabecular arrangement of basaloid cells; small cystic spaces; and foci of comedo-type necrosis in central areas of the tumour (4). Several studies have shown that BSCCE has relatively undifferentiated features and undeveloped cell organelles and its biological behaviour is more aggressive than that of typical ESCC (2, 5, 6). Furthermore, it is reported that BSCCE and ESCC retain different cellular phenotypes with distinct genetic and epigenetic alterations (7). Therefore, analysing the characteristics of BSCCE is important in improving the prognosis of oesophageal cancer.
MicroRNA (miRNA) is a single-stranded RNA molecule measuring 21-25 bases in length that is involved in the regulation of post-transcriptional expression of genes in eukaryotes. miRNA binds to multiple, partially complementary sites of the 3 ‘UTR of target genes, destabilizing the target mRNA, and suppressing translation and protein production (8). miRNA mediated transcriptional repression plays an important role in various biological processes such as development, cell proliferation and differentiation, apoptosis, and metabolism (9-12). MiRNAs also play an important role in cancer. Previous studies have reported that miRNA expression is altered in various cancers, linking miRNA expression to either initiation or progression of cancer (13-17). Variation of miRNA expression affects the characteristics of carcinoma by regulating the expression of oncogenes or tumour suppressors (12). Therefore, miRNAs may serve as biomarkers for cancer diagnosis, prognosis, and pathogenesis (18).
In this study, we aimed to investigate the impact of miR-3687 on the prognosis of ESCC.
Materials and Methods
Patients and ethical concerns. ESCC tumour samples were obtained from patients with histologically-proven primary ESCC who underwent esophagectomy at the Yamanashi University Hospital (Yamanashi, Japan) between 2004 and 2012. The eligibility criteria were as follows: no synchronous or metachronous cancers (in addition to ESCC) and no preoperative chemotherapy or radiation therapy. Patients with non-curative resected tumours were excluded. Relevant clinicopathological and survival data were obtained from the hospital database. Staging was according to the International Union Against Cancer (UICC)/TNM Classification of Malignant Tumors (7th edition) (19).
In our previous analysis, 4 miRNAs were identified to be highly expressed in BSCCE tissues, namely, miR-200c-3p, miR-205-5p, miR-3687, and miR-4732-5p. Patients with ESCC were divided into two groups, those with low miR-3687 expression [<7.9 (relative expression to normal tissue)] and those with high expression (≥7.9) by the top 10 percentile.
Written informed consent for use of samples and data were obtained from all patients. This study was approved by the Ethics Committee of Yamanashi University (approval number: 1888).
Sample collection
Formalin-fixed paraffin-embedded tissue specimens. The formalin-fixed paraffin-embedded (FFPE) samples of ESCC were collected from Yamanashi University Hospital. Total RNA was extracted from 5-μm thick FFPE using the RNeasy FFPE Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions.
Cell lines. Human ESCC cell lines TE13 and TE14 were obtained from the Cell Resource Center for Biomedical Research Institute of Development, Aging, and Cancer (Tohoku University, Sendai, Japan) (20). The human ESCC cell lines KYSE30 and KYSE150 were obtained from the Japanese Collection of Research Bioresources Cell Bank (21). These cells were grown in RPMI-1640 medium (NacalaiTesque, Kyoto, Japan) supplemented with 100 U/ml of penicillin, 100 μg/ml of streptomycin, and 10% FBS. The cells were cultured in dishes in a humidified incubator at 37°C and 5% CO2 in air.
RNA extraction, reverse transcription, and RT-qPCR. Total RNA was extracted using TRIzol Reagent (Invitrogen: Thermo Fisher Scientific, Inc.), and refined using the RNeasy Mini Kit (Qiagen, Hilden, Germany). RNA concentration was measured using a NanoDrop 2000/2000c (Thermo Fisher Scientific, Inc.). cDNAs were synthesized using the TaqMan miRNA Reverse Transcription Kit (Applied Biosystems, Tokyo, Japan) based on the specific stem-loop RT primer design. Reverse transcriptase reaction contained 10 ng RNA samples. We used 3 μl of stem-loop primers (hsa-miR-3687, Assay ID: 464645_mat), 1.5 μl of 10×RT buffer, 0.15 μl of 100 mM dNTPs, 1 μl of MultiScribe reverse transcriptase, 0.19 μl of RNase inhibitor and 4.16 μl of nuclease free water in a final volume of 15 μl. The reaction mixtures were incubated for 30 min at 16°C, 30 min at 42°C, 5 min at 85°C, and then held at 4°C. Next, 1.33 μl of cDNA was amplified using 10 μl of 2×TaqMan Universal PCR master mix, 1 μl of primers and probes, and 7.67 μl of nuclease free water in a final volume of 20 μl. The initial PCR step was a 10 min hold at 95°C followed by 1 min of annealing/extension at 63°C. The PCR reactions were run on a 7500 Real-Time PCR machine (Applied Biosystems) and analysed using the 7500 System SDS software. miRNA expression was normalised against the expression of U6 small nuclear RNA (RNU6B) and analysed via the 2-ΔΔCq method.
Cell transfection. Cells were plated (2×105/well) in a 6-well plate 24 h prior to transfection. For downregulating miR-3687, the miR-3687 inhibitor (mirVana miRNA Inhibitor, Anti-miR ID: MH20273) or negative control inhibitor miRNA (mirVana miRNA Inhibitor Negative Control #1) was used to transfect the ESCC cells. For miR-3687 overexpression, the miR-3687 mimic (mirVana miRNA Mimic, Pre-miR ID: MC20273) or negative control mimic miRNA (mirVana miRNA Mimic Negative Control #1) was used.
For the preparation of transfection reagents, 90 pmol of mimic or inhibitor were diluted in Opti-MEM media (Gibco; Thermo Fisher Scientific, Inc.) and mixed with 7 μl of Lipofectamine RNAiMAX Reagent (Invitrogen Life Technologies) diluted in Opti-MEM. After incubating the cells for 24 h, several assays were performed.
Cell proliferation assay. To measure cell growth rate, the number of viable cells at various time points after transfection was assessed via the colorimetric water-soluble tetrazolium salt assay (Cell Counting Kit 8; Dojindo Laboratories, Kumamoto, Japan). According to the manufacturer's protocol, cells transfected with miRNA mimics or inhibitor were plated in 96-well plates. Ten μl cell counting assay kit-8 solution were added to each well and the absorbance was measured at 450 nm using a microplate reader.
Analysis of cell migration and invasion. The migration assay was conducted using a Cell Culture Insert with a pore size of 8 μm (BD Biosciences). Biocoat Matrigel (BD Biosciences) was used to evaluate cell invasion potential. Briefly, cells (1.0×105 cells per well) were seeded in the upper chamber in serum-free medium 24 h after transfection. 10% FBS and medium were added to the lower chamber. The chambers were incubated at 37°C for 24 h in 5% CO2, and non-migrated or non-invaded cells were then removed from the upper side of the membrane by scrubbing with cotton swabs. Meanwhile, migrated or invaded cells were fixed on the membrane and stained with Diff-Quick staining reagents (Sysmex, Kobe, Japan). The migrated or invaded cells on the lower side of the membrane were counted in 10 independent fields of view at 100× magnification for each insert. Each assay was performed in triplicate.
Statistical analysis. The Mann–Whitney U-test and the t-test for unpaired data were performed for comparing sample data. The Chi-square test or Fisher's exact probability test was used to evaluate correlations between the miRNA levels and clinicopathological factors. For the survival rate analysis, Kaplan–Meier survival curves were constructed for groups based on univariate predictors, and differences between the groups were analysed with the log-rank test or the Wilcoxon test. Univariate and multivariate survival analyses were performed using the likelihood ratio test of the stratified Cox proportional hazards model. For all analyses, a p-value of <0.05 was considered statistically significant using GraphPad Prism® version 7 (GraphPad Software, San Diego, CA, USA).
Results
miR-3687 expression in ESCC cell lines. ESCC tumour samples were obtained from 92 patients. We first evaluated the expression of miR-3687 in ESCC cell lines (Table I). Although all the cell lines used were ESCC, different values were observed. miR-3687 was highly expressed in KYSE30 and TE13 cells, while it had low expression in KYSE150 and TE14 cells (Figure 1A). Analysis of the association between miR-3687 expression and the characteristics of each cell line showed no significant difference regarding age, sex, and differentiation (Figure 1B, C, and D). This indicates that the histological features of ESCC were not related to miR-3687 expression in these cell lines.
Relationships between miR-3687 expression and clinicopathological features of ESCC. miR-3687 expression in the 92 ESCC tissues was then correlated with clinicopathological features, such as T and N classification and stage. High miR-3687 expression was associated with T and N classifications and stage (Figure 2A, B, C). Meanwhile, similar to the results from cell lines, miR-3687 expression was also not associated with histological differentiation.
The miR-3687 low-expression group and the miR-3687 high-expression group comprised 83 and 9 patients, respectively. miR-3687 expression was associated with lymphatic invasion, T classification, N classification, and stage (Table II). This result indicates that high miR-3687 expression in BSCCE is related to the clinicopathological features of the malignancy, including tumour development and lymph node metastasis.
Effect of miR-3687 expression on the survival of patients with ESCC. We compared the 5-year overall survival (OS) of patients with ESCC with various clinicopathological features and miR-3687 expression (Table III). Similar with lymphatic invasion, venous invasion, T classification, and N classification being related to 5-year OS, miR-3687 expression was also associated with 5-year OS. The 5-year survival rate of patients with high miR-3687 expression was significantly poorer than that of patients with low expression (40.5% vs. 58.1%, p=0.0487; Figure 3). Disease-free survival (DFS) was also significantly different between the high and low miR-3687 expression groups at 1-year after surgery (51.3% vs. 72.7%, p=0.0302; Figure 4). This shows that high miR-3687 expression was related to poor prognosis and early recurrence.
Association between miR-3687 upregulation and cell migration and invasion. miR-3687 was upregulated in the ESCC cell lines with low miR-3687 expression, namely, KYSE150 and TE14. High miR-3687expression in KYSE150 and TE14 promoted cell migration and invasion, but did not alter proliferation compared to the negative control (Figure 5). Next, miR-3687 was downregulated in ESCC cell lines with high miR-3687 expression, namely, KYSE30 and TE13. miR-3687 down-regulation inhibited migration and invasion in KYSE30 and TE13, but did not alter proliferation (Figure 6).
Discussion
In this study, miR-3687 was related to prognosis and early recurrence of ESCC. Several studies have shown miRNAs to be related to esophageal cancer (22-24). miR-9, miR-25, miR-92a, miR-96, miR-1179, and miR-1290 have been reported as onco miRs related to invasive capability and metastatic potential (25). Target genes of these onco miRs include E-cadherin, reversion-inducing-cysteine-rich protein with kazal motifs, slit homolog 2 (SLIT 2), and suppressors of cancer cell invasion. miR-9, miR-25, and miR 92a targeting the E-cadherin region promotes epithelial-to-mesenchymal transition by inhibiting e-cadherin expression and increasing the expression of c-myc, and CD44 in the nucleus. Further, miR-9 and miR-92a are associated with poor prognosis (26-28). Other miRNAs related to prognosis have also been reported (29-31). According to Target scan (32), miR-3687 is associated with DNA binding proteins and ubiquitin related proteins. In addition, miR-3687 target genes which are expected to affect esophageal cancer cells are FGFRL1, MTA2, PGRMC2 and SIRT2. In this study, high miR-3687 expression was related to poor prognosis and a metastatic and invasive ability. miR-3687 was highly expressed in BSCCE tissue. Therefore, cases with high expression of miR-3687 resemble BSCCE with aggressive biological features, resulting in poor prognosis.
It was reported that miR-3687 was highly expressed in conjunctival melanoma; moreover, miR-3687 upregulation was also related to high risk of local recurrence (33). However, no study to date has assessed the impact of miR-3687 in oesophageal cancer. To the best of our knowledge, this is the first report on miR-3687 in oesophageal cancer.
In general, BSCCE is reported to be more aggressive and have a poorer prognosis than that of typical ESCC (34). Imamhasan et al. reported EGFR and TP53 gene mutations in BSCCE that are associated with poor prognosis (6). Zhang et al. reported that BSCCE had high local recurrence and risk of distant metastasis, and the median relapse-free survival was 10 months (4). Moreover, they reported that BSCCE should be considered an equivalent to ESCC with moderate to poor differentiation.
Our study showed that miR-3687 is highly expressed in BSCCE, and this was related to poor prognosis. Regarding DFS, high miR-3687 expression was related to poor survival rate at 1 year postoperative. Therefore, miR-3687 expression is a potential prognostic marker of early recurrence.
In conclusion, miR-3687 is among the onco-miRs that are related to poor prognosis in ESCC. Thus, miR-3687 is a potential biomarker or treatment target in ESCC. Our findings may have valuable implications for improving the prognosis of BSCCE, but further studies on the specific mechanisms by which miR-3687 affects the metastatic potential and invasive ability in BSCCE are needed.
Acknowledgements
The Authors are grateful to Motoko Inui and Makiko Mishina for their expert technical assistance.
Footnotes
Authors' Contributions
Kotaro Hagio performed the majority of experiments and wrote the manuscript. Shinji Furuya and Daisuke Ichikawa designed the research and helped to draft the manuscript. All other Authors have contributed to data collection and interpretation, and critically reviewed the manuscript.
This article is freely accessible online.
Conflicts of Interest
The Authors have no conflicts of interest to disclose regarding this study.
- Received December 28, 2018.
- Revision received January 16, 2019.
- Accepted January 17, 2019.
- Copyright© 2019, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved