Med Oncol (2015) 32:357 DOI 10.1007/s12032-014-0357-1

ORIGINAL PAPER

Effect and mechanism of RUNX3 gene on biological characteristics of human esophageal squamous cell carcinoma (ESCC) Huaxia Chen • Zhou Wang • Shuai Wang Zhiping Zhang • Shanshan Shi



Received: 23 October 2014 / Accepted: 12 November 2014 / Published online: 27 November 2014 Ó Springer Science+Business Media New York 2014

Abstract The aim of this study was to investigate the role of RUNX3 in esophageal squamous cell carcinoma (ESCC) cells biological behavior and the relationship between the expression of RUNX3 and MMP-9, TIMP-1, ICAM-1. RUNX3 levels in 90 esophageal squamous cell carcinoma specimens using immunohistochemical staining to examine the correlation between RUNX3 expression and clinical stage of ESCC. Furthermore, the role of RUNX3 in ESCC progression was evaluated in vitro by siRNA-mediated knockdown of RUNX3 or lentivirus-mediated overexpression of RUNX3 in ESCC cell lines. The expression and activities of MMP-9, TIMP-1, and ICAM-1 were analyzed. We found decreased expression of RUNX3 in ESCC tissue to be significantly related to T stage of tumor (p \ 0.01). In vitro, knockdown of RUNX3 in Eca9706 cells resulted in promoting cell growth, migration, and invasion. Additionally, MMP-9 and ICAM-1 were upregulated in RUNX3-knockdown cells. Notably, RUNX3 over-expression in Kyse150 cells could significantly decrease MMP-9 and ICAM-1. Tumorigenesis in vivo was significantly determined. The study indicates that low expression of RUNX3 in human ESCC tissue is significantly correlated with progression. Restoration of RUNX3 expression significantly inhibits ESCC cells migration, invasion, and tumorigenesis, which may be caused by RUNX3’s interaction with MMP-9 and ICAM-1; RUNX3 may be a potential therapeutic target for ESCC. Keywords ESCC  RUNX3  RNAi  Overexpression  ICAM-1  TIMP-1  MMP-9

H. Chen  Z. Wang (&)  S. Wang  Z. Zhang  S. Shi Jinan, China e-mail: [email protected]

Introduction Esophageal squamous cell carcinoma (ESCC) is a highly aggressive malignancy, with early lymphatic and hematogenous metastasis [1]. For the available prognostic factors for ESCC, the most important is the International Union Against Cancer (UICC) TNM stage as determined by the depth of invasion, involvement of lymph nodes, and presence of distant metastasis [2]. There are many types of treatments used or being tested in clinical trials for patients with ESCC currently; surgical resection, radiotherapy and chemotherapy are popularly used strategy in treatment with the ESCC [3]. However, for the patients with the advanced diseases, no treatment has a good therapeutic effect; the prognosis of ESCC is poor with a 5-year survival rate [4]. Many studies have shown that the occurrence and development of ESCC are the result of the multi-factors, multi-steps, and multi-genes participation [5]. Inactivation and mutation of tumor suppressor gene are one of mechanisms which involved in ESCC develops through the accumulation of genetic and epigenetic abnormalities, evolving from preinvasive lesions to invasive esophageal cancer, with the malignant cells commonly exhibiting abnormal important genes expression [6]. Consequently, it has a great clinical value to find new genes involved in ESCC tumorigenesis and progression, in order to explore the generation and development of ESCC. Runt-related transcription factor 3 (RUNX3) is regarded as a tumor suppressor gene in many human tumors, and its inactivation is believed to be related with the occurrence and development of tumor [7]. It is been well examined about its low expression in various solid tumors such as gastric cancer, breast cancer, colorectal cancer, renal cell carcinoma, and melanoma [8]. Therefore, it is hypothesized that the disruption of RUNX3 may also result in major

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alterations in gene expression patterns that may contribute to esophageal cell canceration [9]. In this study, we investigated the role of RUNX3 downregulation and up-regulation in ESCC progression. It is found that RUNX3 was low expressed in ESCC tissue, and its tumor levels were positively correlated with clinical stage. RUNX3 could cause the inhibition of ESCC cells in cell growth, migration, and invasion. Furthermore, we revealed that RUNX3 could upregulate the tissue inhibitor of metalloproteinases-1 (TIMP-1) expression while downregulate intercellular adhesion molecule-1 (ICAM-1), Matrix metalloproteinase-9 (MMP-9).

second antibody (1:100, Zhongshan Biotech, China) for 30 min at 37 °C, followed by streptavidin–peroxidase incubation at 37 °C for 30 min. Finally, sections were colored with diaminobenzidine tetrahydrochloride for 2 min. For negative controls, we replaced the primary antibody with phosphate-buffered saline. All sections were examined by two independent pathologists who were blinded to the clinical data. To evaluate RUNX3 expression, yellow cytoplasmic staining was considered as positive. Staining intensity was assigned a score as follows: 0, no staining; 1, weak intensity; 2, moderate intensity; 3, high intensity; and 4, very high intensity.

Materials and methods

RNA extraction and quantitative real-time PCR

Cell lines and transfection

Total RNA was extracted from cells by RNAiso Plus (Takara, JPN), according to the manufacturer’s protocol. All RNA samples were treated with RNase-free (Takara, JPN) and stored at -80 °C. Two micrograms of RNA was subjected to reverse transcription, using reverse transcriptase reagent Kit with gDNA Eraser (Takara, JPN) to obtain cDNA. b-actin was used as an internal control; the PCR primers (Takara, JPN) used were as follows:

Human ESCC cell Eca109, Eca9706, Kyse150, and TE-1 cell lines were first cultured and purchased from Chinese Academy of Sciences (Shanghai, China). All cells were cultured in Roswell Park Memorial Institute 1,640 enriched with 1 % penicillin/streptomycin and 10 % fetal bovine serum (FBS). Cell culture plates were maintained in humidified incubators at 37 °C in a 5 % CO2 incubator. Clinical samples Ninety esophageal squamous tissue samples were purchased from Department of Thoracic Surgery, Provincial Hospital Affiliated to Shandong University (Jinan, China) and were used for immunohistochemistry. Postoperative staging was based on the tumor-node-metastasis (TNM). TNM classification of the UICC in 2009 included 54 cases with early-stage (I–II) and 36 cases with advanced stage (III–IV). Sample collection was performed after receiving approval from the institutional ethics review committee of the Provincial Hospital Affiliated to Shandong University. No patient had undergone radiotherapy or chemotherapy before surgery. Surgical evaluation was used to determine the clinical stage and presence of metastases, whereas histopathologic analysis was performed by pathologists to assess cancer type and grade. Tissue immunohistochemistry Tissue sections (4 lm thick) were heat fixed, deparaffinized, and rehydrated by standard methods. After the formalin-fixed, paraffin-embedded tissues were deparaffinized and antigen retrieved, tissue sections were incubated overnight at 4 °C with the monoclonal mouse antihuman RUNX3 antibody (1:100, Santa Cruz, USA). After washing with PBS, the slides were incubated with biotinylated

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RUNX3 forward: 50 -ACCTGTCACAACGGCCAGAA C-30 ; RUNX3 reverse: 50 -TTCCAGTGAGGACAGGCCA AG-3; TIMP1 forward: 50 -CTGTTGTTGCTGTGGCTGAT-30 ; TIMP1 reverse: 50 -TGGTATAAGGTGGTCTGGT TGA-3; MMP-9 forward: 50 -GCACCACCACAACATCACCT A-30 ; MMP-9 reverse: 50 -GGACCACAACTCGTCATCGT-3; ICAM1 forward: 50 -GCAAGAAGATAGCCAACCA A-30 ; ICAM1 reverse: 50 -CAGTGCGGCACGAGAAA-3; b-actin forward: 50 -GGCGGCACCACCATGTACCC T-30 ; b-actin reverse: 50 -AGGGGCCGGACTCGTCATAC T-30 . RUNX3 mRNA expression was evaluated by LightCycler480 sequence detection system (Roche Diagnostics GmbH, USA) according to the supplied protocol. ACTB (b-actin) was used as an internal control. Amplification conditions were as follows: reverse-transcription reaction: 42 °C, 30 min per cycle. PCR cycling conditions were as follows: enzyme activation 95 °C 15 min per cycle, denaturation 95 °C at 15 s per 40 cycles, and annealing/ extension at 60 °C for 60 s. Measurements between samples were compared by the threshold cycle of amplification (CT).

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RUNX3 knockdown with siRNA

Cell proliferative analysis

RUNX3 siRNA oligonucleotides were transfected using lentivirus vector according to the manufacturer’s instructions. The sequence of RUNX3 siRNA was 50 -GCCGCTT CCACCATACCTA-30 .

Cell Counting kit (CCK)-8 (Zhongshan Biotech, China) is the routine checks of the cell growth and was performed to assess the proliferation of the transduced cells. Cells were seeded at an initial density of 5 9 104 cells/ ml in 96-well plates (three wells per group, total five plates) for 12, 24, 48, 72, 96 h post-transfection, respectively. CCK-8 was added to the wells (10 lL per well) at the experimental period. After 2 h of incubation at 37 °C in 5 % CO2, optical density was determined by a Tecan Infinite M200 Multimode microplate reader at the absorbance of each well at 450 nm wavelengths. Triplicate wells were used for each data point. All experiments were performed in triplicate, and the average of the results was calculated. Three independent experiments were performed.

Lentivirus production and transduction of target cells The RUNX3 sequences were amplified by PCR, confirmed by sequencing, and then inserted into a pWPT vector by replacing GFP to generate pWPT-RUNX3. To produce virus particles, 20 mg of pWPT-RUNX3 was transfected with 15 mg of packaging plasmid psPAX2 and 5 mg of G protein of the vesicular stomatitis virus (VSV-G) envelope plasmid pMD2.G (generous gifts from Dr. T. Didier) into 293T cells using a calcium phosphate transfection system. HEY cells were transduced with recombinant lentiviral particles to produce polyclonal cells with stable expression of RUNX3 and confirmed by western blotting. Protein extraction and western blot assay Cells were collected by trypsinization. Total protein was extracted using the RIPA (Reusable Industrial Packaging Association, ZSGB-BIO) plus PMSF (phenylmethylsulfonyl fluoride, ZSGB-BIO) according to the manufacturer’s instructions. The total protein extracted from cells was diluted in 49 loading buffer, separated on a 10 % SDS–polyacrylamide gel., underwent electrophoresis, using PageRuler Prestained Protein Ladder (Fermentas) as size markers. The proteins were transferred to Hybond-P polyvinylidene difluoride membranes (Amersham) in transbuffer containing 25 mM Tris and 185 mM glycine, pH8.3 together with 20 % methanol. After transfer, the membranes were blocked for 1 h in blocking buffer (TBS containing 0.1 % Tween-20, TBST, supplement with 5 % nonfat dry milk) and then the membranes were incubated overnight at 4 °C with mouse anti-human RUNX3, MMP9, ICAM-1, and GAPDH monoclonal antibodies in blocking buffer. After being washed three times with TBST, the membranes were incubated with HRP-conjugated goat anti-mouse secondary antibodies (1:10,000, Zhongshan Biotech, China) for 30 min, the immunoblots were exposed to X-ray film. Western blotting densitometric analysis was performed using Image Reader LAS-4000 analysis software. The band densities were normalized to the densities of GAPDH. The electrophoretic results were scanned into images. Data were collected by Multi Gauge software, and the ratio of strip density to GAPDH density served as index for statistical analysis. All experiments were repeated at least three times.

Cell migration and invasion assay Cell migration and invasion assay were determined by using a modified two-chamber migration assay with a pore size of 8 lm (Merck Millipore Bioscience, Germany). For migration assay, each upper well was loaded with 2.5 9 105 cells in a total volume of 200 ll of serumfree medium. The lower wells of the chamber were loaded with 600 ll of 1,640 medium with 10 % FBS. At the end of the experimental time period, any cells remaining on top of the insert were removed by scraping. Cells that migrated to the bottom surface of the insert were fixed with ethanol-based crystal violet solution for 10 min. Cells were counted based on 12 random high-power fields (2009) under a light microscope (LEICA DM4000B, Leica, Wetzlar, Germany). BALB/c nude mice transplantation Six-week-old BALB/c nude mice were purchased from Vital river co. [Certificate of Quality No.: SCXK (Beijing) 2012-0001] and maintained in specific pathogen-free facilities at the Experimental Animal Center of Provincial Hospital Affiliated to Shandong University. Mice were provided with free access to food, water, and bedding at all time and were housed with a 12 h light/dark cycle in filter top cages containing a maximum of five mice per age. The mice were randomly separated into two groups of ten each. Under aseptic conditions, the cells were digested and, respectively, injected subcutaneously in a total volume of 100 ll (1 9 106) cells resuspended by PBS without FBS. Mice were sacrificed by cervical dislocation, and growth of the tumor was evaluated up to day 28 after cell inoculation.

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Statistical analysis SPSS 19.0 software was used for all statistical analysis. The relationship between negative expression of RUNX3 and clinicopathological characteristics was analyzed by Chi-square test or Fisher’s exact test. Significance was assessed by comparing mean values (mean ± SD) using the Student’s t test for independent groups. p \ 0.05 was considered to indicate a statistically significant difference.

Med Oncol (2015) 32:357 Table 1 Clinical data of 90 patients with ESCC Clinical characteristic

Total patients (n = 90)

Negative RUNX3 expression (n and %)

Male

69

38 (55.1 %)

Female Age

21

10 (47.6 %)

C50 years

65

33 (50.8 %)

\50 years

25

15 (60.0 %)

Yes

31

16 (51.6 %)

No

59

32 (54.2 %)

\3 cm

46

26 (56.5 %)

3–5 cm

35

17 (48.6 %)

[5 cm

9

5 (55.6 %)

Gender

0.549

0.432

Weight loss

Results Loss or drastic decrease of RUNX3 expression in advanced stage of esophageal cancer tissues In view of that pTNM-status is an important prognostic marker for patients with ESCC, to further confirm the relationship between RUNX3 expression and pTNM-status. We analyzed RUNX3 expression in different stage ESCC tissue by immunohistochemistry. By immunohistochemical staining, the positive expression of RUNX3 protein showed as yellow or brownish yellow stain in the cytoplasm and/or nucleus of tumor cells. The average staining score for ESCC in early-stage (I–II) was significantly higher than that in advanced stage (III–IV) (p \ 0.01), suggesting that RUNX3 expression was reduced in metastatic esophageal squamous cancer; RUNX3 protein expression varied significantly with pTNM-status (Fig. 1; Table 1). Decreasing RUNX3 promoted ESCC cells proliferation, migration, and invasion in vitro To elucidate the role of RUNX3 in ESCC progression, RUNX3 siRNA was used to reduce RUNX3 expression in the human Eca9706 cell line, which has a high level of

Fig. 1 Immunohistochemical staining picture and staining score of RUNX3 in different stage ESCC tissue (original magnification = 100)

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0.813

Length

0.769

pTNM-status

0.000*

Stage I–II

54

21 (38.9 %)

Stage III–IV

36

28 (77.8 %)

62 28

33 (53.2 %) 10 (35.7 %)

Differentiation Well or moderate Poor

p value

0.124

* p \ 0.01, Chi-square test or Fisher’s exact test, pTNM-status pathological diagnosed TNM stage according to 2009 NCCN

RUNX3 expression in four esophageal squamous cell lines (Eca109, Kyse150, TE-1, and Eca9706) (Fig. 2). Eca9706 cells were randomly divided into three groups: Eca9706 group, Eca9706 EV group (plasmid-empty-vector group), and Eca9706 RUNX3i group (plasmid -RUNX3ivector group). Seventy-two hours after transfection, RUNX3 siRNA significantly reduced RUNX3 expression in Eca9706 group cells (Fig. 3). Three cell groups were determined by CCK-8 assay. It was found that the cell growth rate for Eca9706 RUN3i group was significantly

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higher compared to those of control cells (p \ 0.01) (Fig. 4). The rate of cell growth inhibition was similar in the two control groups. Then, the transfected cells were subjected to cell migration assay and invasion assay. In cell migration assay, we found that silence of RUNX3 in Eca9706 cells promoted the proliferation, migration, and invasion of Eca9706 cells (p \ 0.01). However, restoration of RUNX3 had no effect on two control cells (p [ 0.05) (Fig. 5). Restoration of RUNX3 expression inhibited ESCC cells proliferation, migration, and invasion in vitro

Fig. 2 RUNX3 expression levels in esophageal cancer cell lines. Eca9706 had significantly highest RUNX3 expression, and Kyse150 had the lowest expression among the esophageal cancer cell lines studied

Fig. 3 In 72 h after transfection, relative mRNA and protein of RUNX3 expression level in three Eca9706 groups

To further evaluate whether RUNX3 upregulation could promote tumor proliferation, migration, and invasion, Kyse150 cells were also divided into three groups under the same conditions; lentivirus-mediated delivery of RUNX3 cDNA was used to increase RUNX3 expression in the human Kyse150 cell line, which has low RUNX3 expression (Fig. 2). Seventy-two hours after transfection, real-time PCR and Western blot analysis showed that the mRNA expression levels of RUNX3 in the Kyse150 RUNX3 group were significantly higher than that in control groups (p \ 0.05) (Fig. 6). The cell growth curves showed that over-expression of RUNX3 could suppress the growth of Kyse150 cells more than that of in the other two groups (p \ 0.01) (Fig. 7). Transfected cells were subjected to cell migration assay and invasion assay. Compared with that in Kyse150 group and Kyse150 EV group (plasmid-emptyvector group), over-expression of RUNX3 in Kyse150 RUNX3 group cells significantly suppressed their migration through the transwell (p \ 0.01). Furthermore, the invasive potential, which is determined by the cells’ ability to invade a Matrigel barrier, was also considerably suppressed in Kyse150 RUNX3 group cells (p \ 0.01) (Fig. 8). Restoration of RUNX3 expression repressed the tumorigenicity of ESCC cells in vivo

Fig. 4 Cell growth rate of each Eca9706 cell group. Cell proliferation was determined at 12, 24, 48, 72, and 96 h, respectively. Absorbance from the plates was read at 490 nm

Next, in vivo subcutaneous tumor formative assay was adopted to examine the tumorigenesis of ESCC cells in nude mice. All the nude mice survived after injections with cells. In vivo experiment showed that the tumor mass could be touched in the mice 7 days after inoculation, the tumor formation rate being 100 %. The cells were injected 2 weeks later; the tumor’s volume of two groups began to appear differently. After 4 weeks after inoculation, compared to control cells, the Eca9706 RUNX3i group (2.410 ± 0.588 cm3, 2.83 ± 0.47 g) led to a significantly increase in tumor volume and weight than Eca9706 group (1.340 ± 0.351 cm3, 1.45 ± 0.26 g) (p \ 0.05) (Fig. 9). Compared with the Kyse150 group (1.712 ± 0.233 cm3, 1.87 ± 0.31 g),

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Fig. 5 Transwell assays were performed to investigate changes in cell migration and invasiveness

the tumor in Kyse150 RUNX3 group was significantly lighter and smaller (0.818 ± 0.362 cm3, 0.66 ± 0.48 g) (p \ 0.05). The results indicating that the over-expression of RUNX3 could suppress the growth of tumors in Kyse150 RUNX3 group cells, which is reverse to low expression in Eca9706 RUNX3i group cells.

expression. Consistently, RUNX3 over-expression in Kyse150 cells upregulated TIMP-1, while downregulated MMP-9 and ICAM-1 expression compared to control cells (p \ 0.05) (Fig. 10). Our results further confirmed that the inhibition of MMP-9 protein level is due to the increased expression of TIMP-1 after RUNX3 over-expression in ESCC cell lines.

RUNX3 suppresses MMP-9 and ICAM-1 expression Migration and invasive ability of cancer cells can be regulated by MMPs and ICAM-1. To investigate the mechanisms of RUNX3 regulating cell invasion, we performed Western blot, RT-PCR to detect the MMPs and ICAM-1 levels in ESCC cells. Our results showed that siRNAmediated RUNX3 low expression in Eca9706 cells decreased TIMP-1, increased MMP-9 and ICAM-1

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Discussion Surgical resection is the most popularly used strategy in treatment with the ESCC [10]. Although local tumor was completely excised, more than 50 % of patients develop lymphatic metastatic recurrence in the 2 or 3 years after surgery. Thus, it is essential to predict the risk of

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Fig. 6 In 72 h after transfection, relative mRNA and protein of RUNX3 expression level in three Kyse150 groups

Fig. 7 Cell growth rate of each Kyse150 cell group. Cell proliferation was determined at 12, 24, 48, 72, and 96 h, respectively. Absorbance from the plates was read at 490 nm

recurrence to minimize the adverse effects and maximize the therapeutic effect of treatment of ESCC. Metastasis is the leading cause for tumor death, and tumor cell migration and invasion are essential steps in the process of metastasis [11]. RUNX3 is a target gene of TGF-b-mediated tumor suppressor pathway. Given the potential role of RUNX3 in TGF-b signaling, it is possible that the tumor suppressor activity of RUNX3 is realized by regulating cell migration and invasion [12]. Better to understand the exact role of RUNX3 in ESCC development, we used clinical specimens immunohistochemical technology, vitro cell model, and vivo animal model to investigate the role of RUNX3 in ESCC. Our clinical evidence clearly supported that compared with different clinical stage of ESCC, RUNX3 was lower expressed in later stage ESCC tissue, the observation suggests that RUNX3 in tumor participates in ESCC progression. The vitro studies revealed that over-expression of RUNX3 in Kyse150 cells significantly inhibited ESCC cell

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proliferation as well as migration and invasion, whereas siRNA-mediated RUNX3 low expression promoted biological activities in Eca9706 cells, which were consistent with the function of RUNX3 in vivo. MMP-9 is a key member of MMPs gene which are a family of zinc-dependent endopeptidase that are capable of degrading components of the basement membrane and extracellular matrix, allowing cancer cells to invade and migrate [13]. In addition, previous in vitro studies also show that the expression of MMP-9 promote invasion and lymph node metastasis of cancer cells [14]. ICAM-1 is one of transmembrane glycoprotein belongs to the immunoglobulin superfamily of adhesion molecules [15]. It was identified as an important protein involved in the process of cancer metastases, facilitating the process of metastases, whereby invasive cells dissociate from a tumor proper, enter circulation, increases the adhesion of metastatic tumor cells, and escape from immunological recognition and cytotoxicity by effector cells [16–19]. On the other hand, ICAM-1 is released from the cell membrane by proteolytic cleavage of its extracellular domain, with a parallel increase in expression and proteolytic activity of certain MMPs, including MMP-1 and MMP-9 [20]. It was demonstrated the participation of MMP-9 in the activity of ICAM-1 [21]. Therefore, it contributes to tumor resistance, against NK cell cytotoxic activity. Given its association with many cancer types, ICAM-1 and MMP-9 may serve as important diagnostic tool for assessing the progression and prognosis of tumors. In this research, we focused on elucidating the relationship between RUNX3 and MMP-9 and ICAM-1 which has been reported to participate closely in tumor progression, in order to determine how RUNX3 inhibits ESCC cell migration and invasion [22]. Compared with the control groups, the Kyse150 RUNX3 group can suppress ICAM-1, MMP-9 protein expression level, whereas low expression of RUNX3 in Eca9706 cells upregulated ICAM-1 and MMP-9 expression. ICAM-1 and MMP-9 were highly expressed in ESCC metastasis, so the RUNX3 likely inhibited the metastasis of ESCC by regulating the expression of ICAM-1 and MMP-9. However, it remains to be elucidated how RUNX3 regulates MMP-9 and ICAM-1 expression and its signal pathway to regulate ESCC cell progression. Chen et al. [23] showed that MMP-9-inhibiting activity of RUNX3 due to the direct interaction of RUNX3 with the TIMP-1 promoter in human gastric carcinoma cell. They recognized that RUNX3 suppresses tumor metastasis due to the imbalance between MMP-9 and TIMP-1. Our results further confirmed that the inhibition of MMP-9 protein level is due to the increased expression of TIMP-1 after RUNX3 over-expression in ESCC cell lines. Previous studies have demonstrated that RUNX3 factors regulate the expression of integrins like LFA-1, CD11c,

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Fig. 8 Transwell assays were performed to investigate changes in cell migration and invasiveness

CD49d, implying that RUNX factors simultaneously control the expression of the integrin LFA-1 [24]. ICAM-1 is originally identified as a molecule involved in lymphoblastoid cell adhesion to purified LFA-1 [25]. Fiore et al. [26] found ICAM-1 which could specifically bind LAF-1 could induce the expression of MMP-9 gene in HL-60 cell line, induce MMP-9 dock and activation on membranes. It was also confirmed in MCF10A and SW620 cell line [27]. Thus, we infer that RUNX3 may regulate MMP-9 gene expression effectively by the other pathways, one factor of tumor immunological escape in ESCC is that loss or low expression of RUNX3 might increase ICAM-1 expression,

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further induces MMP-9 over expressed and persistently activated in ESCC cells. The enzymatic hydrolysis of MMP-9 produced ICAM-1 shed from the plasma membrane into sICAM-1. It makes immune cells lost contact to ESCC cells, and inactivate cytotoxic effect by binding with sICAM-1, and thus tumors can escape from body’s immunization mechanism. In conclusion, our clinical and mechanistic data provide evidence that decreased RUNX3 expression is significantly correlated with pTNM-status of patients with ESCC. The expressions of MMP-9 and ICAM-1 after RNA-dependent transcriptional RUNX3 silencing were increased greatly.

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Fig. 9 Comparison of the size of the harvested implanted tumors in nude BALBC/c mice

Fig. 10 Relative mRNA expression of MMP-9 and ICAM-1 levels in esophageal cancer cell lines

Our findings suggest that RUNX3 is an important player in the malignancy of ESCC cells by promoting cell proliferation and invasion. Further experiments are needed to confirm the link between RUNX3 modulation of the

(TGF-b) signaling pathway, ICAM-1, and MMP-9. RUNX3 constituted a potential treatment modality for human ESCC cancer;these findings identify RUNX3 as a potential therapeutic target for ESCC.

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357 Page 10 of 10 Conflict of interest We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, this manuscript.

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Effect and mechanism of RUNX3 gene on biological characteristics of human esophageal squamous cell carcinoma (ESCC).

The aim of this study was to investigate the role of RUNX3 in esophageal squamous cell carcinoma (ESCC) cells biological behavior and the relationship...
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