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Expression of combined interference of slug and FoxC2 in endometrial carcinoma and its clinicopathological relationship

Background Endometrial carcinoma is one of the three major malignant tumors in female reproductive system. The current research aimed to investigate the relationship between the Slug and FoxC2 expression and the proliferation, invasion and metastasis of endometrial carcinoma. Methods The expression of Slug and FoxC2 genes between 124 endometrial carcinoma tissues and 35 normal endometrial tissues was analyzed through immunohistochemistry. The endometrial carcinoma cell lines Ishikawa and RL-952 were cultured, the Slug-shRNA and FoxC2-shRNA expression vectors were constructed, and the endometrial carcinoma cells interfering with the expression of Slug and FoxC2 genes were also established. Western blotting and RT-PCR were employed to verify whether shRNA could down regulate the expression of Slug and FoxC2 genes. Additionally, the proliferation, migration and invasion capacities in both cell lines after interfering with Slug and FoxC2 was detected through CCK-8 and Transwell assay respectively. Furthermore, MMP2 and MMP9 were detected by ELISA and epithelial-mesenchymal transition (EMT) related proteins including E-cadherin, N-cadherin and Vimentin were assessed by Western blotting analysis. Results Compared with normal endometrial tissues, the Slug and FoxC2 expression levels in endometrial carcinoma tissues were remarkably increased. shRNAs transfection significantly down-regulated expressions in both endometrial carcinoma cell lines. The proliferation, invasion and migration ability were significantly inhibited by Slug-shRNA and FoxC2-shRNA compared with the control group. The expression of E-cadherin was increased while the expression of N-cadherin, Vimentin, MMP-2 and MMP-9 was suppressed by the Slug-shRNA and FoxC2-shRNA. Conclusions Slug and FoxC2 could be used as a prognostic factor of endometrial carcinoma. Interfering with the expression of Slug and FoxC2 in endometrial carcinoma cell lines could effectively inhibit the proliferation, invasion and migration, and its mechanism is related to the inhibition of EMT. Slug and FoxC2 are potential targets for the treatment of endometrial carcinoma.


Introduction
Endometrial carcinoma is one of the most malignant tumors in female reproductive system, which has severely damaged the life health in women. However, there is still lacking effective prognostic indicators and treatment options for the clinical treatments. Slug is a transcription factor of SNAIL family that can encode the zinc finger protein, which is reported to be up-regulated in multiple malignant tumors. FoxC2 is a member of the Fox protein family, which can regulate the expression of a series of downstream genes, and participate in the epithelial-mesenchymal transition (EMT) (1)(2)(3). Transcription factors Slug and FoxC2 have been reported to play important roles in tumor invasion and metastasis, which are the important regulatory molecules during tumor cell migration and metastasis (4).
Short hairpin RNA (shRNA) is a kind of RNA sequence that can form a tight hairpin turn, which can silence gene expression. The double-strand small molecule RNA or shRNA has been utilized in clinical test to treat several diseases, and certain achievements have been attained in terms of tumor treatment. In the current study, shRNAs were used to down-regulate the Slug and FoxC2 expression in Ishikawa and RL-952 endometrial carcinoma cell lines, and cell proliferation, migration and invasion were further evaluated. Then we tested the relevant indicators of EMT after transfection to gain a deeper understanding of the role of Slug and FoxC2 in endometrial carcinoma.

Patients
The endometrial carcinoma tissues were retrospectively collected from 124 patients in the Pathology Department of the First Affiliated Hospital of Bengbu Medical College from January 2014 to December 2015. Before surgery, all endometrial carcinoma patients did not receive any anticancer treatments, including chemotherapy and radiotherapy. Patients with endometrial carcinoma were confirmed by pathologic diagnosis. The normal endometrial tissues were collected into the control group, which were derived from 35 patients receiving endometrial tissue biopsy and hysterectomy due to other benign disease in Gynecology.
The study was conducted in accordance with the

CCK-8 colorimetry assay
Ten μL CCK-8 was added into each well at 24, 48, 72 and 96 h, respectively, and further cultured for 4 h, and the optical density value in each well was measured using a microplate reader. Subsequently, the growth curve was plotted, and the average of 4 wells in each group was used to calculate the cell proliferation capacity. Each experiment was repeated for three times.

Transwell chamber migration assay
Cells in each group were digested with trypsin and washed with the serum-free culture medium. Cell suspension was prepared, and 200 μL was evenly added into the upper Transwell chamber (Beyotime Biotechnology, Haimen, China), while 600 μL complete medium was added to the lower Transwell chamber for 48 h. Then, the chambers were taken out, washed with PBS, fixed with 4% paraformaldehyde for 20 min, and stained with 1% crystal violet staining solution for 10 min. Subsequently, cells on the basilar membrane surface were wiped using the cotton swab, and the number of cells penetrating the membrane was counted under microscope. Each experiment was repeated for three times.

Transwell invasion assay
Before Transwell invasion assay, cells were deprived of serum for 24 h, resuspended (5×10 4 ) with 200 μL serumfree medium after trypsin digestion, and added onto the Matrigel surface in the upper Transwell chamber, while 600 μL complete medium was added to the lower Transwell chamber as the chemotaxin. The number of cells penetrating the Matrigel was counted under microscope. Each experiment was repeated for three times.

ELISA assay
The concentrations of MMP-2 and MMP-9 were further evaluated in both cell lines. Cell culture supernatants were collected at 48 h after transfection. MMP-2 and MMP-9 concentrations were measured using human MMP-2 ELISA Kit and human MMP-9 ELISA Kit (Invitrogen Corporation, Carlsbad, United States) according to the manufacturer's instructions. The optical density (OD) value at 450 nm was record using a microplate reader.

Statistical analysis
Statistical analyses were performed using SPSS 18 for Windows (SPSS Inc., Chicago, IL, USA). Data are given as means ± SD and One-way analysis of variance followed by post-hoc Bonferroni analysis of variance. The two groups were compared with Student's t-test. Each experiment was repeated for three times. P<0.05 was taken as a significant difference between groups.

Slug and FoxC2 were highly expressed in endometrial carcinoma tissues
As shown in Figure 1, immunohistochemical results indicated positive expression in the nuclei of endometrial carcinoma tissues. Compared with normal endometrial tissues, the Slug and FoxC2 expression levels in endometrial carcinoma tissues were remarkably increased. The positive rate of Slug and FoxC2 in endometrial carcinoma was 61.3% (76/124) and 71.0% (88/124), and the difference was statistically significant compared with that in normal endometrial tissues ( Table 1).

Relationships of Slug and FoxC2 expression with the clinicopathological factors of endometrial carcinoma
There was no relationship between the Slug and FoxC2 expression and age in each group (P>0.05). Slug expression in endometrial carcinoma was notably correlated with the histological grade, muscular layer infiltration and lymph node metastasis (P<0.05), but it was not markedly correlated with The International Federation of Gynecology and Obstetrics (FIGO) stage. FoxC2 expression was distinctly related to the FIGO stage and lymph node metastasis (P<0.05), but it was not correlated with histological grade  and muscular layer infiltration ( Table 2).

Slug and FoxC2 protein expression were detected by Western blotting and RT-PCR
Ishikawa and RL-952 cells were first observed at 48h after shRNAs transfection ( Figure 2). The fluorescence images indicated that the cells were successfully transfected with the shRNAs. Western blotting and RT-PCR results also indicated that, compared with control group, Slug and FoxC2 protein and mRNA levels in Ishikawa ( Figure 3) and RL-952 ( Figure 4) cells were remarkably reduced after Slug-shRNA and FoxC2-shRNA interference (P<0.01). Nonspecific shRNA group showed no significant difference compared with those in blank control group (P>0.05).

Slug-shRNA and FoxC2-shRNA inhibited endometrial carcinoma cell proliferation
As shown in Figure 3D and Figure 4D, the proliferation rates of both cell lines in the control group were markedly higher than those in shRNAs treatment groups. Independent sample t-test results suggested that, the differences in the proliferation capacity of cells in expression vector treatment  groups were statistically significant compared with that in the other two groups at days 2, 3, 4, 5, 6 and 7 of growth (P<0.05). CCK-8 assay suggested that, the proliferation capacity of cells in shRNAs treatment groups was apparently lower than that in the control group.

Slug-shRNA and FoxC2-shRNA inhibited endometrial carcinoma cell migration and invasion
The

Slug-shRNA and FoxC2-shRNA reduced MMP-2 and MMP-9 in endometrial carcinoma cells
Subsequently, we compared the MMP-2 and MMP-9 of the cell culture supernatant in each group of both cell lines by ELISA assay. As shown in Figure 7, the expressions of MMP-2 and MMP-9 after shRNA-Slug and shRNA-FoxC2 transfection were significantly reduced. This is consistent with the results of our previous migration and invasion results.

shRNA-Slug and shRNA-FoxC2 inhibited EMT in endometrial carcinoma
To gain insights into the mechanism underlying the Slug and FoxC2 inhibiting endometrial carcinoma migration and invasion, we evaluated the protein level of EMT related protein including E-cadherin, N-cadherin and Vimentin by Western blotting. As shown in Figure 8, after transfection of shRNA-Slug and shRNA-FoxC2 in Ishiyama and RL-952 endometrial carcinoma cells, the expression of E-cadherin increased while the expression of N-cadherin and Vimentin decreased. These results indicated that EMT capability is suppressed by shRNA-Slug and shRNA-FoxC2.

Discussion
In the current study, we found that Slug and FoxC2 expressions in endometrial carcinoma tissues were remarkably increased. In addition, Slug and FoxC2 silences by shRNA significantly inhibited endometrial carcinoma proliferation and metastasis, which may be related to the downregulation of MMPs protein expression and inhibition of EMT. As a member of the Zinc finger transcription factor family members, Slug is mainly involved in neoplasm malignant phenotype regulation, and could also promote EMT, which is closely related to tumor cell migration and invasion (2,5). Recent researches have indicated that, Slug antisense could prevent EMT, indicating that, the Slug gene could act a treatment target for tumor invasion and metastasis (6)(7)(8).
FoxC2 is also referred to as mesenchyme forkhead 1 (MFH1), which belongs to the forkhead transcription factor family member and is encoded by genes located at human chromosome 16q24.1 (9,10). It has been highlighted that FoxC2 plays a central role in multiple signaling pathways, which are closely related to EMT and could further promote angiogenesis in tumor development (11,12).
In our current study, we found that the positive expression rates of Slug and FoxC2 proteins in endometrial carcinoma of 68.75% and 59.34%, respectively, which were remarkably elevated compared with that in normal endometrial tissue group (25.00%). Besides, the Slug and FoxC2 expressions were evidently correlated with the endometrial carcinoma development and poor prognosis In order to verify the Slug and FoxC2 roles in endometrial carcinoma, Ishikawa and RL-962 endometrial carcinoma cells were transfected with shRNA-Slug and shRNA-FoxC2 to silence the expression of Slug and FoxC2 respectively, which were confirmed by the fluorescence microscopy, Western blotting, and RT-PCR. Cell proliferation, invasion and migration were significantly inhibited by shRNA-Slug and shRNA-FoxC2 in both cell lines. The matrix metalloproteinases (MMP) family is a class of Zn 2+ and Ca 2+ -dependent endopeptidases that play an important role in multiple pathological processes such as inflammation, tumors, and cardiovascular diseases. It has found that MMP-2 and MMP-9 can promote tumor neovascularization in vivo, and upregulation of MMP-2 and MMP-9 in stromal cells could significantly increase the tumor cell metastasis (13). In the current study, our results also highlighted that the expressions of MMP-2 and MMP-9 in both cell lines after shRNA-Slug and shRNA-FoxC2 transfection was significantly decreased compared with the control group. Infiltration and metastasis are also the main causes of treatment failure and poor clinical prognosis of endometrial carcinoma. It has been reported that metastasizes of endometrial carcinoma related to by EMT was induced by Slug and FoxC2 (14)(15)(16)(17)(18)(19). EMT was an originally physiological process that occurred during the development of mammalian embryos, and recent research showed that EMT played an important role in the metastasis of tumors. Epithelial-derived tumor cells can be transformed into tumor cells with stronger migration ability and the enhanced invasions could also be promoted by interstitial cells. EMT could be activated by both intracellular signaling pathways and integrin signaling pathways, such as Slug, Snail, Twist and FoxC2 (20)(21)(22)(23)(24)(25). Then the expressions EMT markers (such as Slug, Snail, and FoxC2) in tumor cells will be significantly increased, which will enhance cell invasiveness and lead to transformation into tumor cells (11,26,27), and the migration and resistance to apoptosis of the tumor epithelial cells will be enhanced (28)(29)(30). As EMT markers, E-cadherin, N-cadherin and Vimentin were analyzed in each group in both cell lines. We found that, after transfection of shRNA-Slug and shRNA-FoxC2, the expression of E-cadherin was markedly increased while the N-cadherin and Vimentin expression was significantly decreased, which was in consistence with previous studies (5,31), indicating the suppression of EMT by shRNA-Slug and shRNA-FoxC2.

Conclusions
In summary, our current study found that, compared with normal endometrial tissues, the Slug and FoxC2 expression levels in endometrial carcinoma tissues were remarkably increased. Interfering with Slug and FoxC2 through shRNAs can effectively suppress the in vitro proliferation, invasion, and migration of Ishikawa and RL-952cells, which may be related to the downregulation of MMPs protein and inhibition of EMT. Further studies will more focus on the detailed mechanism of the regulation of EMT in endometrial carcinoma by Slug and FoxC2.