miR-200c inhibits the invasive activity of primary adamantinomatous craniopharyngioma cells CURRENT

Backgrounds Craniopharyngiomas are benign epithelial tumors and difficult to complete due to the digitate brain infiltration. miR-200c has been studied in terms of development, stemness, epithelial-mesenchymal transition (EMT), and therapy resistance in many cancers. However, the role of miR-200c remains to be elucidated in adamantinomatous craniopharyngioma. Methods Quantitative real-time polymerase chain reaction was used to evaluate the expression of miR-200c, ZEB1, ZEB2, and CTNNB1. Immunohistochemistry, Western blot, and immunofluorescence analyses were used to evaluate the expression of E-cadherin and β-catenin at the protein level. A Transwell assay was used to evaluate the invasiveness of ten primary craniopharyngioma cell. Results miR-200c was significantly downregulated in adamantinomatous craniopharyngioma compared with papillary craniopharyngioma. Conversely, ZEB1, ZEB2, and CTNNB1 were overexpressed in adamantinomatous craniopharyngioma. Inhibition of miR-200c significantly promoted the invasion of primary adamantinomatous craniopharyngioma cells. Moreover, E-cadherin was overexpressed and β-catenin was downregulated in miR-200c mimic primary adamantinomatous craniopharyngioma cell culture. Conclusion Our data demonstrated that miR-200c maybe reduce the invasive activity of adamantinomatous craniopharyngioma cells through E-cadherin/β-catenin. These findings suggest that the targets of miR-200c may regulate the EMT of adamantinomatous craniopharyngioma.

Craniopharyngiomas are benign epithelial tumors that are histopathologically divided into adamantinomatous craniopharyngioma (ACP) and papillary craniopharyngioma (PCP) [1]. However, increased morbidity and mortality was associated with the invasion of adjacent structures, including the sella turcica, hypothalamus, optic nerves and third ventricle [2]. Long-term morbidities of craniopharyngioma include hypopituitarism, increased cardiovascular mortality, reduced quality of life and impaired cognitive function [3]. A craniopharyngioma is a benign tumor, but it shows a clinically malignant tumor-like outcome.
MicroRNAs (miRNAs), which are short single-stranded noncoding RNAs (∼21 nucleotides in length), negatively regulate gene expression and induce mRNA degradation or translation inhibition [4]. miR-200c is a member of the miR-200 family, which consists of miR-200a, miR-200b, miR-200c, miR-141 and miR429. miR-200c has been demonstrated to be involved in many biological processes, including epithelial-mesenchymal transition (EMT), metastasis, cell invasion, proliferation, autophagy, apoptosis, and therapy resistance in several cancer types [5]. However, the role of miR-200c in craniopharyngioma remains to be elucidated.
In the present study, we hypothesized that miR-200c may reduce the invasive activity of adamantinomatous craniopharyngioma cells through EMT.

Patients and sample collection
This study was carried out after approval by the Ethics Committee of West China Hospital, Sichuan University (Sichuan China), and written informed consent was obtained from all participants. A total of ten tumor tissue samples were collected from patients with craniopharyngioma at West China Hospital, Sichuan University 4 (Sichuan China). The inclusion criteria were as follows: (1) Primary surgical removal in the neurosurgical department of West China Hospital, Sichuan University; (2) No preoperative or postoperative radiotherapy or chemotherapy; (3) Histological diagnosis of craniopharyngioma.

Cell culture
Primary cell culture was performed as described previously [6]. Briefly, the tumor

Transwell assay
Transwell assays were performed according to the manufacturer's instructions.
Briefly, the cells (∼5 × 104) were harvested and resuspended in serum-free medium and then added to the Transwell upper chambers, in which the upper surface of the 8-μm pore size membrane (Corning, Steuben, New York, USA) was coated without or with Matrigel (BD Biosciences, Waltham, Massachusetts, USA). After incubation for 24 h, the cells that had migrated through the membrane were fixed, stained, and counted with an inverted microscope.

Immunohistochemistry
Immunohistochemical staining was performed as described previously [7]. Briefly, sections were blocked for endogenous peroxidase by incubation in 3% H2O2 for 20 minutes and then washed in PBS containing 0.05 M ethylenediamine tetraacetic acid

Western blotting
Western blotting was performed as described previously [7]. Briefly, 50 µg of protein lysate was loaded onto each well and resolved and then transferred to polyvinylidene fluoride (PVDF) membranes. The transferred membranes, blocked with 5% milk, were incubated overnight with primary antibodies against E-cadherin (Abcam) and β-catenin (CST) and washed with Tris-buffered saline with Tween-20 (TBST) (10 minutes, three times). The membranes were then probed with the appropriate secondary antibody (1:5000; Abcam). Immunoreactivity was determined and observed using enhanced chemiluminescence (Millipore, Billerica, MA, USA). β-Actin was used as a control. ImageJ was used to calculate the relative expression of E-cadherin and β-catenin at the protein level.

Immunofluorescence
Primary cells were washed with PBS and then fixed in 75% ethanol for 20 minutes at room temperature. After the cells were washed with PBS, they were blocked with 5% bovine serum albumin (BSA) for 1 h at 37°C. The cells were then incubated with a monoclonal anti-human E-cadherin (Abcam) and β-catenin antibody (CST) overnight at 4°C. The cells were counterstained with 4′,6-diamidino-2-phenylindole (DAPI, 10 µg/mL, 32670; Sigma-Aldrich) to mark the nuclei. The cells were examined with a confocal laser scanning microscope.

Statistical analysis
All data are presented as the mean ± SEM. All experiments were performed at least three independent times. Using one-way analysis of variance followed by the Duncan's multiple-comparison test using SPSS 19.0 (SPSS Inc., Chicago, IL), we calculated the statistical significance. P < 0.05, P < 0.01, or P < 0.001 was regarded as statistically significant.

8
The invasive activity between adamantinomatous and papillary craniopharyngioma Craniopharyngioma was defined as invasive if the tumor impaired the integrity of the brain parenchyma, hypothalamus, walls of the ventricles, cavernous sinus or optic chiasm or if it exhibited glio-arachnoidal adhesions [8]. To identify craniopharyngioma invasion, we examined the histological characteristics of the tumor tissues from a cohort of five ACPs and five PCPs. The results indicated that ACP had "whirl-like" or "island-like" cell clusters that penetrated into the surrounding brain tissue, showing obvious invasiveness (Fig 1a, as indicated by the black arrow); the PCP and the surrounding tissue boundary line were relatively flat, with no obvious signs of extension to the surrounding tissue (Fig 1b).

The invasive activity between primary adamantinomatouscraniopharyngioma cells and primary papillary craniopharyngioma cells.
To compare the invasive activity between the ACP and PCP, we performed Transwell assays of primary ACP tumor cells and PCP cells. We detected many more ACP cells that passed through the membrane than PCP cells (Fig 2a-c). Western blotting showed that the expression of E-cadherin was low and that β-catenin was high in primary ACP cells compared with primary PCP cells (Fig 2d-f). The expression levels of E-cadherin and β-catenin were significantly different between primary ACP and PCP cells. Immunofluorescence showed that β-catenin was distributed in the cytoplasm or nucleus of ACP but tended to be distributed in the cell membrane in PCP. The results indicated that E-cadherin was obviously downregulated in primary 9 ACP cell culture compared with primary PCP cell culture. In contrast, β-catenin was obviously upregulated in primary ACP cell culture compared with primary PCP cell culture (Fig 2g-h). These results suggest that ACP may be more aggressive than PCP.

miR-200c is downregulated in adamantinomatous craniopharyngioma
To confirm the involvement of miR-200c in craniopharyngiomas, we first tested the miR-200 family levels in five ACP and five PCP tissues. The results indicated that miR-200c expression was significantly lower in craniopharyngioma tissues than normal brain tissues, especially miR-200c expression, which was obviously lower in ACP than PCP (Fig 3a-b). Compared with the PCP tissues, the ACP tissues showed upregulation mRNA levels of ZEB1, ZEB2, and CTNNB1 (Fig 3c). These findings suggest that miR-200c may negatively regulate the expression of ZEB1, ZEB2, and CTNNB1 in ACPs.

Increased expression of miR-200c inhibits adamantinomatous craniopharyngioma cell invasion
To investigate the function of miR-200c in primary ACP cells, we performed Transwell assays and found that the invasion of primary ACP cells was higher than that of primary PCP cells (Fig 2c). The number of cells in miR-200c mimic group was significantly lower than that in miR-200c inhibitor group (Fig 4a-c). These findings indicated that miR-200c obviously decreased the invasion of ACP cells.

miR-200c affects the expression of E-cadherin and β-catenin
To explore the mechanism by which miR-200c inhibited invasion of primary ACP cells, we focused on the association between miR-200c and E-cadherin/β-catenin.
Immunofluorescence was used to determine the relative expression of E-cadherin and β-catenin at the protein level. The results indicated that E-cadherin was obviously downregulated by the miR-200c inhibitor and upregulated by the miR-200c mimic compared with the control. In contrast, β-catenin was obviously upregulated by the miR-200c inhibitor and downregulated by the miR-200c mimic compared with the control (Fig. 4 d-e). The expression levels of E-cadherin and βcatenin were significantly different in the miR-200c control, miR-200c mimic, and miR-200c inhibitor groups (Fig 5a). The expression of E-cadherin protein was the highest and β-catenin protein was the lowest in the miR-200c mimic group (Fig 5 bc). These results suggest that miR-200c suppresses ACP invasive activity through Ecadherin/β-catenin.

Discussion
Recently, an increasing number of studies have reported the dysregulation of miRNAs in many types of cancer, indicating that the expression of proto-oncogenes or tumor suppressor genes is regulated by interactions between miRNAs and their corresponding mRNAs, which determine the malignant characteristics of tumor cells [9]. In the present study, we found that the expression levels of miR-200c were significantly downregulated in ACP tissues compared with PCP tissues. Notably, these data indicated that a low expression of miR-200c was clearly associated with In early studies, miR-200c overexpression was shown to promote the invasive phenotype of A549 cells through inducing the upregulation of E-cadherin and downregulating the expression of ZEB1 [10], an important activator in EMT that inhibits the expression of basement membrane components and cell polarity factors [11]. All of the miR-200 family members were shown to suppress EMT by activating E-cadherin expression through direct targeting of ZEB1 and its homeobox ZEB2 [11]. Interestingly, ZEB1 is not only a target of the miR-200 family but could also strongly inhibit miR-200c and another miR-200 family member, miR-141, and promote invasion of cancer cells. In our study, miR-200c was shown to inhibit the expression of ZEB1, ZEB2, and CTNNB1. Therefore, miR-200c may play an important role in the invasive activity of ACP. E-cadherin, which is transcribed from CDH1 gene into a 135 kDa precursor polypeptide, plays cruial role in cell adhesion and cancer progression. It links to the polarized epithelial phenotype and tissue morphology [12]. Loss of the E-cadherin has been considered as the hallmark of the EMT. The previous research reported that E-cadherin and β-catenin can form a cadherin-catenin complex, which can stabilize the cell-cell contacts. Cadherin-catenin complex has been considered as the key regulator in the Wnt signaling pathway [13,14]. CTNNB1 and β-catenin overexpression are frequently present in adamantinomatous craniopharyngiomas, whereas papillary craniopharyngiomas do not show these variations [15,16].
Similarly, we found that miR-200c promoted the expression of E-cadherin and suppressed the expression of β-catenin at the protein level In ACPs. Therefore, these findings maybe demonstrate that miR-200c inhibit invasion through Ecadherin/β-catenin in ACP.

Availability of data and materials
All data generated or analysed during this study are included in this published article.