lncRNA MIAT promotes cell invasion and migration in esophageal cancer.

Long non-coding RNAs (lncRNAs) serve crucial roles in carcinogenesis. Myocardial infarction-associated transcript (MIAT), originally isolated as a candidate gene for myocardial infarction, has been revealed to serve as an oncogene in chronic lymphocytic leukaemias and neuroendocrine prostate cancer. However, little is known about its expression pattern, biological function and underlying mechanism in esophageal cancer. Cell lines of esophageal cancer were used in the current study. The results of the present study revealed that MIAT knockdown decreased cell viability, migration, invasion and cell cycle arrest in the G1 phase. Mechanistic assessment revealed that MIAT interacts with histone methyltransferase mixed-lineage leukemia (MLL). The relative proteins expressions were measured by western blotting assay. MIAT knockdown suppressed cell invasion and migration by regulation MMP-2/9 protein expressions. The results of the current study indicated that MIAT expression was associated with esophageal cancer and may serve as a critical target in the progression and metastasis in esophageal cancer. Copyright: © Zhang et al.

Introduction

Esophageal cancer is one of the most common malignant tumors with 400,156 deaths worldwide in 2012 and it exhibits a high incidence in China (1). Of those patients with esophageal cancer, ~90% patients are diagnosed with esophageal squamous cell carcinoma (ESCC) (1). However, a lack of progress in chemotherapy and radiotherapy has resulted in little improvement of ESCC treatment, leading to a 5-year survival rate of 15-25% (2). Previous research has primarily focused on the role of protein encoded genes in the development of cancer and have not sufficiently assessed the effect of long non-coding RNA (lncRNA). A recent study has revealed that lncRNA may serve important biological roles in the formation, progression, invasion and metastasis of various tumors (3). lncRNAs also serve important roles in oncogenes and tumor suppressor genes by regulating their target genes or signaling pathways (4). H19 was the first tumor-associated lncRNA identified (5). It is abnormally expressed in many different types of cancer, including gastric, colon, liver and breast cancer and is involved in the regulation of tumor cell proliferation, apoptosis, invasion and migration (6,7). MEG3 is the first lncRNA that has been determined to inhibit tumor function (8). MEG3 inhibits proliferation and promotes cell apoptosis by regulating the expression of p53(9). A previous study reported that when the lncRNA myocardial infarction-associated transcript (MIAT) is knocked-out in mice, they do not exhibit any significant abnormality with cancer development, but are increasingly hyperactive (10). Furthermore a previous study demonstrated that MIAT is significantly increased in cancer lesions (11). In digestive tract cancer, previous studies have determined that MIAT is highly expressed in gastric cancer (12,13), colorectal cancer (14) and hepatocellular carcinoma (15,16).

However, the function and mechanism of the majority of lncRNAs are yet to be fully elucidated. The current study assessed the viability, apoptosis, invasion and migration of MIAT in esophageal cancer cells. The results of the current study may help identify novel therapeutic targets in esophageal cancer.

Materials and methods

Cell culture and transfection

HEEC, TE-1, Kyse 30, Kyse 180, Kyse 510, Kyse 150 and Eca 109 cell lines (American Type Culture Collection, Manassas, VA, USA) were respectively cultured in RPMI 1640 medium (Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.), 100 mg/ml streptomycin and 100 U/ml penicillin. Cells were then incubated at 37˚C with 5% CO2. Kyse 150 and Eca 109 cells were respectively transfected with 25 or 50 nM of small interfering (si)RNA using RNAiMAX Lipofectamine (Invitrogen; Thermo Fisher Scientific, Inc.) in accordance with the manufacturer's protocol. The lncRNA MIAT siRNA (siMIAT) sequence was 5'-ACUUCUUCGUAUGUUCGGCTT-3'. Kyse 150 and Eca 109 cells were divided into negative control (NC) transfected by siRNA-NC (5'-GCACCTTGAGTGAATGTCAGGGACTCCCTGATGATGTGA-3'), 25 and 50 nM groups, respectively. Samples were incubated for 24 h before subsequent experimentation.

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assay

Total RNA was extracted from HEEC, TE-1, Kyse 30, Kyse 180, Kyse 510, Kyse 150 and Eca 109 cells using a TRIzol kit (Thermo Fisher Scientific, Inc.) and cDNA was subsequently synthesized using the cDNA synthesis kit (Thermo Fisher Scientific, Inc.) and qPCR was performed using the qPCR kit (Takara Bio, Inc., Otsu, Japan) to measure mRNA expression by SYBR-Green method. The following primer sequences (synthesized by Shenzhen Huada Gene Biotechnology Co., Ltd., Shenzhen, China) were utilized as following: MIAT forward, 5'-GCTCACACCTCCTATTCCT-3' and reverse, 5'-CTTCACCAACTCTCCCACT-3'. U6 (nuclear reference) forward, 5'-CTCGCTTCGGCAGCACA-3' and reverse, 5'-AACGCTTCACGAATTTGCGT-3'; 18S (cytoplasmic reference forward, 5'-GTGGGCCGAAGATATGCTCA-3' and reverse, 5'-TTGGCTAGGACCTGGCTGTA-3'. The thermocycling conditions were as follows: 95˚C for 5 min; followed by 40 cycles of 95˚C for 30 sec, 60˚C for 30 sec and 72˚C for 30 sec.

MTT assay

A total of 24 h following treatment, Kyse 150 and Eca 109 cells in suspension (2x105 cells/ml) were inoculated into 96-well plates. Following culture for 48 h, 10 µl MTT was added to each well and cultured for 4 h. The supernatant was subsequently removed and 100 µl DMSO was added to wells to dissolve the purple formazan. Absorbance was measured at 570 nm and cell viability was determined.

Flow cytometry

Kyse 150 and Eca 109 cells of different groups (NC, 25 and 50 nM groups) were collected and adjusted to a concentration of 1x106 cell/ml. Cells then underwent centrifugation at 1,000 x g for 5 min, following which the supernatant was discarded. Samples were then washed twice with cold PBS and centrifuged for a further 5 min at 1,500 x g at 4˚C Cells were resuspended using cooled 70% EtOH and fixed with 70% EtOH overnight at 4˚C. The following day, samples were centrifuged (1,500 x g; 10 min; 4˚C), washed once with PBS, washed twice with normal saline and centrifuged a second time (1,500 x g; 5 min; 4˚C). Cells were then stained with Propidium iodide (50 mg/l; Triton X-100, 1.0%; RNase A, 10 mg/l; Thermo Fisher Scientific, Inc.) at 4˚C in the dark for 30 min. A flow cytometer was used to measure early and late stage cell apoptosis and the cell cycle by flow cytometry (Coulter Epics Altra flow cytometer; Beckman Coulter).

Transwell assay

Kyse 150 and Eca 109 cells of different groups (NC, 25 and 50 nM) were cultured in 20% culture medium, trypsinised then suspended in serum-free medium (Thermo Fisher Scientific, Inc.). Cells were plated at a density of 1x105 cells/well in the upper chamber with 20 µl Matrigel. Complete medium (600 µl; Thermo Fisher Scientific, Inc.) containing 20% FBS was added to the lower chamber. Samples were then routinely cultured for 24 h at 37˚C and washed twice with PBS. Following cell fixation with 4% polyoxymethylene at room temperature for 30 min and staining at 37˚C for 2 h with crystal violet, the number of cells in 5 random fields of view were counted using an optical light microscope (ECLIPSE Ts2; Nikon Corporation) at x200 magnification.

Wound healing assay

Kyse 150 and Eca 109 cells of different groups were cultured for 24 h at 37˚C. Cells were then suspended in culture medium, routinely digested by protein tryptase, (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) adjusted to a concentration of 5x105 cells/well and inoculated onto 6-well plates. When cells were completely confluent, a 200 µl pipette tip was used to create a scratch. A total of to 2 ml of serum free culture medium (RPMI 1640 medium) was added at room temperature for 30 min then incubated. The distance between cells following wound induction were observed and imaged using an inverted microscope (magnification, x100) at 0 and 48 h. Wound healing rate was then calculated using Image-Pro Plus software (Version X; Media Cybernetics, Silver Springs, MD, USA).

Western blotting

Total protein from Kyse 150 and Eca 109 cells in different groups were extracted using radioimmunoprecipitation lysis buffer (150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% NP-40; Sigma-Aldrich; Merck KGaA). Protein concentration was also measured using the bicinchoninic acid method. Equal quantities of total protein (50 µg) were separated on 10% SDS-PAGE gels and transferred to polyvinylidene difluroide membranes. Membranes were blocked using 1% bovine serum albumin (Beyotime Institute of Biotechnology) at room temperature for 2 h. The following primary antibodies (all 1:1,000; Abcam, Cambridge, UK) were then added to membranes and incubated overnight at 4˚C: histone methyltransferase mixed-lineage leukemia (MLL; cat. no. ab32400), cyclin-dependent kinase 2 (Cdk2; cat. no. ab32147), Cyclin D3 (cat. no. ab28283), matrix metalloproteinase-2 (MMP-2; cat. no. ab37150), MMP-9 (cat. no. ab73734) and GAPDH (cat. no. ab9485). Subsequently, membranes were incubated with horseradish-peroxidase conjugated goat anti-rabbit secondary antibodies (1:5,000; Santa Cruz Biotechnology, Inc.) for 1.5 h at room temperature. Electrochemiluminescence kit (EMD Millipore, Billerica, MA, USA) was used to visualize protein signals and bands were analyzed using ImageJ v1.42 software (National Institutes of Health, Bethesda, MD, USA). GAPDH was utilized as an internal control in this experiment.

Statistical analysis

Statistical data were analyzed using GraphPad Prism software (version 5.0; GraphPad Software, Inc., La Jolla, CA, USA). Values are expressed as the mean ± standard deviation from three independent experiments. The differences between two groups were analyzed using two-tailed Student's t-tests. The differences amongst more than two groups were analyzed using one-way analysis of variance followed by Tukey's post-hoc test. P<0.05 was considered to indicate a statistically significant difference.

Results

MIAT gene expression

The results of RT-qPCR revealed that the expression of MIAT in esophageal cancer cell lines were significantly increased compared with normal esophageal cells (HEEC; Fig. 1A). Furthermore, of the esophageal cancer cell lines used, the expression of MIAT was highest in Kyse 150 and Eca 109 cells. Following siMIAT transfection at 25 and 50 nM concentrations, the expression of MIAT was significantly decreased in Kyse 150 and Eca 109 cells compared with NC cells (Fig. 1B and C). MIAT expression was highest in Kyse 150 and Eca 109 cells therefore these cell lines were selected for further experimentation.

Figure 1

MIAT expression of siMIAT transfected Kyse 150 and Eca 109 cells. (A) The expression of MIAT in different cell lines, as determined via reverse-transcription-quantitative polymerase chain reaction. Cells were transfected with siMIAT (25 or 50 nM) and the expression of MIAT was subsequently determined in (B) Kyse 150 and (C) Eca 109 cells. *P<0.05, **P<0.01 and ***P<0.001 vs. the NC group. MIAT, myocardial infarction-associated transcript; si, small interfering RNA; NC, negative control.

si-MIAT affects cell viability

As determined by the MTT assay, the growth rate of Kyse 150 and Eca 109 cells were significantly and dose-dependently decreased following siMIAT transfection compared with NC cells (Fig. 2A and B). These results indicate that MIAT enhances the viability of certain ESCC cell lines.

Figure 2

Viability of siMIAT transfected Kye 150 and Eca 109 cells. The viability of (A) Kyse 150 and (B) Eca 190 cells was determined following (25 or 50 nM) siMIAT transfection via an MTT assay. **P<0.01 and ***P<0.001 vs. the NC group. NC, negative control.

MIAT knockdown improves cell apoptosis

The results of flow cytometry demonstrated that the rate of cell apoptosis in siMIAT-treated Kyse 150 and Eca 109 cells was dose-dependent and significantly upregulated compared with NC-treated cells (Fig. 3).

Figure 3

Apoptosis of siMIAT transfected Kyse 150 and Eca 109 cells. The apoptosis of (A) Kyse 150 and (B) Eca 109 cells transfected with (25 or 50 nM) siMIAT was determined via flow cytometry. **P<0.01 and ***P<0.001 vs. the NC group. siMIAT, small interfering RNA myocardial infarction-associated transcript; NC, negative control; FL4H:AV, wavelength 640-680 nm.

siMIAT affects the cell cycle

To determine whether MIAT causes cell cycle arrest, the cell cycle was analyzed via flow cytometry. The results revealed that 25 and 50 nM siMIAT transfection significantly and dose-dependently increased Kyse 150 and Eca 109 cell G1 phase compared with NC cells (Fig. 4). Transfection with 25 and 50 nM siMIAT significantly decreased Kyse 150 and Eca 109 cell G2 and S phase compared with NC cells in a dose-dependent manner.

Figure 4

Cell cycle analysis of siMIAT transfected Kyse 150 and Eca 109 cells. The cell cycles of (A) Kyse 150 and (B) Eca 109 cells transfected with 25 or 50 nM siMIAT were determined via flow cytometry. *P<0.05 and ***P<0.001 vs. the NC group. siMIAT, small interfering RNA myocardial infarction-associated transcript; NC, negative control; FL2-A, Pulse area.

MIAT knockdown affects cell invasion

To assess the efficiency of MIAT on the invasion of Kyse 150 and Eca 109 cells, a transwell assay was performed. The results revealed that transfection with siMIAT suppresses the invasion of Kyse 150 and Eca 109 cells in a dose-dependent manner when compared with NC treated cells (Fig. 5).

Figure 5

Invasion of siMIAT transfected Kyse 150 and Eca 109 cells. (A) The invasion of (A) Kyse 150 and (B) Eca 150 cells transfected with 25 or 50 nM siMIAT was determined via a Transwell assay. *P<0.05 and ***P<0.001 vs. the NC group. siMIAT, small interfering RNA myocardial infarction-associated transcript; NC, negative control.

MIAT silencing depresses cell invasion in the wound healing assay

To further assess the effect of MIAT on Kyse 150 and Eca 109 cell invasion, a wound healing assay was performed. The results demonstrated that at 48 h following wound induction, siMIAT transfection significantly and dose-dependently decreased Kyse 150 and Eca 109 cell invasion compared with NC cells (Fig. 6).

Figure 6

Wound healing rate of siMIAT transfected Kyse 150 and Eca 109 cells. The wound healing rate of (A) Kyse 150 and (B) Eca 109 cells transfected with 25 or 50 nM siMIAT was determined via a wound healing assay. *P<0.05 and ***P<0.001 vs. the NC group. siMIAT, small interfering RNA myocardial infarction-associated transcript. NC, negative control.

MIAT knockdown affects relative protein expression

The results of western blotting revealed that the expression of MLL, Cdk2, Cyclin D3, MMP-2 and MMP-9 in Kyse 150 and Eca 109 cells transfected with siMIAT was significantly and dose-dependently decreased compared with NC treated cells (Fig. 7).

Figure 7

Relative protein expression in siMIAT transfected Kyse 150 and Eca 109 cells. The protein expression of MLL, Cdk2, cyclin D3, MMP-2 and MMP-9 in (A) Kyse 150 and (B) Eca 109 cells was determined via western blotting. *P<0.05 and ***P<0.001 vs. the NC group. siMIAT, small interfering RNA myocardial infarction-associated transcript; MLL, histone methyltransferase mixed-lineage leukemia; Cdk2, cyclin-dependent kinase 2; MMP, matrix metalloproteinase; NC, negative control.

Discussion

It is well known that lncRNA is abnormally expressed in many types of cancer and participates in the regulation of tumor development. It is therefore serves as a potential tumor marker and therapeutic target. For example, HOX Transcript Antisense RNA is highly expressed in breast, gastric and liver cancer, and increases the proliferation and invasion of tumor cells (17). Plasmacytoma variant translocation 1 is also highly expressed in colorectal cancer and functions to regulate the invasion and metastasis of tumor cells via the transforming growth factor-β signaling pathway (18). MIAT is a lncRNA that is located on the long arm of chromosome 22(19). Recent studies have revealed that MIAT serves an important role in the development of a variety of diseases (20,21). However, the effects and mechanism of MIAT in esophageal cancer are yet to be fully elucidated. The results of the current study revealed that the viability, migration and invasion of two ESCC cell lines (Kyse 150 and Eca 109 cells) were significantly and dose-dependently suppressed following treatment with a si-MIAT. Future work will investigate the underlying mechanism by measuring MIAT relative proteins expression.

A previous study has revealed that MIAT regulates the expression of the MLL protein in lung cancer (11). Previous studies have also demonstrated that the activation of MLL enhances cancer cell invasion and migration by regulating various MMPs (22,23). MMP overexpression closely correlates with cancer cell invasion and migration (24,25). MMP-2 and MMP-9 are two important members of the MMP family, which effectively breaks down the main components of the basement membrane (26,27). The overexpression of MMP-2 and MMP-9 may also promote cancer cell invasion and migration (28,29). Furthermore, it has been revealed that MMP-2 and MMP-9 were overexpressed in esophageal cancer (30,31). The results of the present study revealed that transfection with siMIAT suppressed the invasion and migration of Kyse 150 and Eca 109 cells dose-dependently. The underlying mechanism of MIAT attenuating esophageal cancer invasion and migration might be correlated with a reduction in MMP-2 and MMP-9 protein expression.

Cell proliferation is a process that is highly regulated and controlled by many factors including cyclin, cyclin dependent protein kinases (CDK), cyclin dependent suppressor protein (CKI). Different cells exhibit different proliferative phases, which primarily involve the G1 phase of the cell cycle. Once a cell surpasses the restriction point of the G1 phase, the cell cycle may continue such that mitosis is achieved. Therefore, the regulation of the G1/S phase checkpoint, which involves various proteins including cyclin Dl, cyclin D3, cyclin E, Cdk 2, Cdk 4 and Cdk 6 is important (32-35). The results of the current study revealed that the expression of cyclin D3 and Cdk 2 were significantly and dose-dependently decreased following siMIAT transfection. MIAT knockdown might suppress esophageal cancer cell proliferation by keeping the cell cycle in G1 phase. There were some limitations to the present study, for example the effects and mechanism of MIAT knockdown was only investigated in vitro therefore, future study will involve in vivo work.

In conclusion, MIAT knockdown suppresses esophageal cancer cell viability by enhancing the invasion, migration and G1 phase of the cell cycle in vitro and in future esophageal cancer treatment, MIAT might be used as a potential target gene.