Histological and Pathological Assessment of miR-204 and SOX4 Levels in Gastric Cancer Patients

Gastric cancer is one of the most common cancers and the efficient therapeutic methods are limited. Further study of the exact molecular mechanism of gastric cancer to develop novel targeted therapies is necessary and urgent. We herein systematically examined that miR-204 suppressed both proliferation and metastasis of gastric cancer AGS cells. miR-204 directly targeted SOX4. In clinical tissue research, we determined that miR-204 was expressed much lower and SOX4 expressed much higher in gastric cancer tissues compared with normal gastric tissues. Associated analysis with clinicopathological parameters in gastric cancer patients showed miR-204 was associated with no lymph node metastasis and early tumor stages whereas SOX4 was associated with lymph node metastasis and advanced tumor stages. In addition, miR-204 and SOX4 were negatively correlated in tissues from gastric cancer patients. Our findings examined the important role of miR-204 and SOX4 played in gastric cancer, and they could be used as candidate therapeutic targets for gastric cancer therapy.


Introduction
Gastric cancer remains one of the most common cancers and the second leading cause of cancer-related death in the world [1][2][3]. Most gastric cancers are diagnosed at advanced stages, so efficient therapeutic methods are limited [4]. High recurrence and metastasis rate of gastric cancer is the biggest obstacle [5,6]. Many studies tried to uncover the mechanism of gastric cancer, but the exact molecular pathways remain unclear. Further study of gastric cancer to develop novel targeted therapies is an urgent issue.
SOX4 belongs to sex-determining region Y (SRY) box family and was recently found to be an oncogene in prostate cancer, colon cancer, lung cancer, bladder cancer, gastric cancer, and so forth [24][25][26][27][28][29]. SOX4 was also reported to be one target of miR-204 in renal cancer, T-cell acute lymphoblastic leukemia, and H. pylori-associated gastric cancer [24,30,31]. But correlated expression of miR-204 and SOX4 in gastric cancer tissues from patients remains unknown, and it is necessary for better understanding the functions and mechanisms of miR-204 and SOX4 in gastric cancer patients.

BioMed Research International
In this study, we systematically examined that miR-204 suppressed both proliferation and metastasis of gastric cancer AGS cells. miR-204 directly targeted SOX4. We collected 54 normal gastric tissues and 54 gastric cancer tissues from patients and evaluated the expression of miR-204 and SOX4, respectively. We found miR-204 was expressed much lower and SOX4 expressed much higher in gastric cancer tissues compared with normal gastric tissues, and miR-204 was associated with no lymph node metastasis and early tumor stages whereas SOX4 was associated with lymph node metastasis and advanced tumor stages. Moreover, miR-204 and SOX4 showed a negative correlation in tissues from gastric cancer patients, which indicated that the pathway miR-204 targeting SOX4 played an important role to suppress tumorigenesis and progression of gastric cancer. Therefore, we provided new evidence that miR-204 and SOX4 can be used as new therapeutic targets for gastric cancer therapy.

Cell Lines and Cell
Culture. Human gastric cancer cell AGS was obtained from the American Type Culture Collection (Rockville, MD) and cultured in a humidified incubator at 37 ∘ C and 5% CO 2 as recommended.

Luciferase Reporter
Assay. Cells were seeded in a 24well plate and cotransfected with 0.2 g of psiCHECK2-SOX4 3 UTR or psiCHECK2 control vector and 30 nM miR-204 mimics or its negative control using Lipofectamine 2000. 48 h after transfection, cells were harvested, and reporter assays were performed using a dual luciferase assay system (Promega).

Patients and Tissue
Samples. 54 normal gastric tissues (nontumorous tissues) and 54 gastric cancer tissues were collected from archive of the Department of Pathology, Anhui Medical University, and the patients underwent surgery at the Second Affiliated Hospital of Anhui Medical University (Hefei, China) between 2013 and 2015. Tumors were graded according to Edmondson-Steiner grading system and staged according to American Joint Committee on cancer staging system [35,36]. Informed consent documents were got from all of the patients. Research related to patients' tissues was approved by the Institutional Review Board of the Anhui Medical University.

In Situ Hybridization.
We performed in situ hybridization in formalin-fixed paraffin-embedded tissues to evaluate the expression of miR-204. Experiments were carried out essentially as previously described [37]. The intensity and the percentage of stained cells were used to score the expression of miR-204 in each section under an Olympus microscope (Olympus America Inc., Melville, NY). 10% or more tumor cells stained were considered as positive expression of miR-204, whereas less than 10% tumor cells stained with any intensity were considered as negative expression.
2.6. Immunohistochemistry. Immunohistochemistry analysis in formalin-fixed paraffin-embedded tissues was carried out as described earlier [38]. Rabbit polyclonal antibody against SOX4 (1 : 200, Abcam, Cambridge, UK) was used in this study. The stained sections were scored by two experienced pathologists to calculate the expression of SOX4 under an Olympus microscope. 10% or more tumor cells stained were considered as positive expression of SOX4, whereas less than 10% tumor cells stained with any intensity were considered as negative expression.

Statistical Analysis.
In this study, we used SPSS software for Windows (version 13.0; SPSS, Chicago, IL, USA) to perform statistical analyses. The relationship of miR-204 level and SOX4 expression in gastric tissues was analyzed using Pearson's chi-square test. In the statistical analyses, < 0.05 was considered as statistically significant.

miR-204 Suppressed Proliferation of Gastric Cancer Cells.
Recently, several studies demonstrated the important role of miR-204 to suppress tumor initiation and development of several human cancers including gastric cancer [11,20,21]. Herein, we systematically examined the role of miR-204 in gastric cancer cells and made an exhaustive analysis of miR-204 in human gastric cancer tissues. In our study, we selected human gastric cancer cell AGS for research. After transfected with miR-204 mimics, total number of AGS cells significantly decreased over a period of 5 days (Figure 1(a)). Concordantly, in MTT assay, cell viability decreased significantly after transfected with miR-204 mimics (Figure 1(b)). In addition, AGS-miR-204 cells showed a decreased cell colony formation compared with AGS-NC cells (Figure 1(c)). Moreover, we carried out soft agar colony formation assay to evaluate the anchorage-independent cell growth ability of AGS cells after transfected with miR-204. Figure 1(d) showed miR-204 dramatically suppressed the soft agar colony formation. As a whole, miR-204 suppressed proliferation of gastric cancer cells.

miR-204 Was Expressed Lower and SOX4 Expressed Higher in Gastric Cancer Tissues Than Normal Gastric Tissues.
Since miR-204 suppressed both proliferation and metastasis in gastric cancer cells, we want to know the expression of miR-204 in gastric cancer tissues compared with normal gastric tissues from patients. Zhou [24,30]. We performed luciferase reporter assay to confirm that miR-204 directly targeted the SOX4 3 UTR (Figure 3(a)). In Zhou et al. 's article, they lacked convincible clinical evidence about miR-204 and SOX4 in gastric cancer study. Herein, we collected 54 normal gastric tissues and 54 gastric cancer tissues to analyze the association of miR-204 and SOX4 expression. The expression of miR-204 was measured using in situ hybridization and the expression of SOX4 was measured using immunohistochemistry. Figure 3 gastric tissues, whereas SOX4 was expressed much higher in gastric cancer tissues compared with normal gastric tissues. Moreover, Table 1  Expression of miR-204 in normal gastric and gastric cancer tissues was measured using in situ hybridization. Expression of SOX4 in normal gastric and gastric cancer tissues was measured using immunohistochemistry. Representative pictures showed that miR-204 was expressed much lower in gastric cancer tissues compared with normal gastric tissues, whereas SOX4 was expressed much higher in gastric cancer tissues compared with normal gastric tissues * * < 0.01. further study, we analyzed the relationship between miR-204 and SOX4 levels and clinicopathological parameters in gastric cancer patients. Clinicopathological parameters in our study involved patients' age, gender, tumor size, lymph node metastasis, tumor grade, and tumor stage. miR-204 level was significantly associated with no lymph node metastasis ( = 0.003) and early tumor stages (stages I-II; = 0.048). SOX4 protein level was significantly associated with lymph node metastasis ( = 0.001) and advanced tumor stages (stages III-IV; = 0.003). But the associations between expression level of miR-204/SOX4 and patients' age, gender, tumor size, or tumor grade were not significant ( Table 2).

Correlation of miR-204 and SOX4 Expression in Gastric
Cancer Patients. For further study, we analyzed the correlation of miR-204 and SOX4 expression in gastric tissues from patients. Concordant with former data, miR-204 and SOX4 were statistically negatively correlated in the 54 normal gastric tissues and 54 gastric cancer tissues ( < 0.001).
Pearson's correlation coefficients were −0.801 and −0.582, respectively (Table 3). Therefore, the pathway miR-204 targeting SOX4 may play a combined role in suppressing gastric cancer development and progression.

Discussion
In this study, we systematically examined that miR-204 suppressed cell proliferation including cell total number, cell viability, cell colony formation, and soft agar colony formation in gastric cancer cells ( Figure 1); miR-204 also suppressed cell metastasis including wound healing, migration, and invasion ( Figure 2). miR-204 directly targeted SOX4. We found that miR-204 was expressed much lower in gastric cancer tissues compared with normal gastric tissues by in situ hybridization method, and SOX4 (one target gene of miR-204) was expressed much higher in gastric cancer tissues compared with normal gastric tissues by immunohistochemistry method (Figure 3 and Table 1). Moreover, we documented that miR-204 and SOX4 were negatively correlated in tissues from gastric cancer patients (Table 3). We for the first time made comprehensive analysis of miR-204 and SOX4 with clinicopathological parameters in patients and determined that miR-204 and SOX4 were dramatically associated with lymph node metastasis and tumor stages ( Table 2). The important roles of miRNAs played in tumorigenesis and metastasis become much clearer recent years. miRNAs contribute to nearly all kinds of human cancers including breast cancer, liver cancer, lung cancer, and gastric cancer [7][8][9][10]. In this study, we focused on miR-204 in gastric cancer.  [20]. In this study, we examined that miR-204 not only suppressed proliferation but also suppressed migration and invasion of gastric cancer cells. In other studies, Zhang et al., Zhang et al., and Liu et al. also documented that miR-204 was a suppressor in gastric cancer, which supported our findings [11,22,23]. Therefore, miR-204 played a comprehensive suppressing role in many kinds of human cancers. For further study of this miRNA, we made a systematical histological and pathological analysis of miR-204 in tissues from gastric cancer patients including comparing miR-204 level in normal tissues and gastric cancer tissues and analyzing the relationship between miR-204 level and clinicopathological parameters in gastric cancer patients. We for the first time determined that miR-204 was dramatically associated with lymph node metastasis and tumor stages. This is of great significance for clinical diagnosis and treatment. SOX4 is one of the sex-determining region Y-box 4 (SOX4) genes and is a developmental transcription factor [24,31,41]. Numerous studies have found SOX4 plays an oncogenic role in many kinds of human cancers [24][25][26][27][28][29]. Bilir et al. demonstrated that SOX4 promoted tumor initiation and development in prostate cancer [25]. Song et al. documented that SOX4 was associated with malignant status of breast cancer [42]. Lin et al. showed that high expression of SOX4 was associated with poor outcome of colon cancer patients [26]. SOX4 was also demonstrated to promote gastric cancer in several studies [24,29]. We also determined that SOX4 was overexpressed in human gastric cancer tissues compared with normal gastric tissues. And we for the first time document that SOX4 was associated with lymph node metastasis and tumor stages of gastric cancer.
In a word, we documented miR-204 suppressed proliferation and metastasis of gastric cancer cells and found miR-204 and SOX4 were negatively correlated and associated with clinicopathological parameters in gastric cancer patients. These findings were helpful for better understanding the exact molecular pathways of miR-204 and SOX4 in gastric cancer and provided potential therapeutic targets for gastric cancer therapy.