Tumor suppressive microRNA-218 inhibits cancer cell migration and invasion by targeting focal adhesion pathways in cervical squamous cell carcinoma

Cervical cancer is one of the most common cancers in women. More than 275,100 women die from cervical cancer each year. Cervical squamous cell carcinoma (cervical SCC), one of the most frequent types of cervical cancers, is associated with high-risk human papilloma virus (HPV), although HPV infection alone may not be enough to induce malignant transformation. MicroRNAs (miRNAs), a class of small non-coding RNAs, regulate protein-coding gene expression by repressing translation or cleaving RNA transcripts in a sequence-specific manner. A growing body of evidence suggests that miRNAs contribute to cervical SCC progression, development and metastasis. miRNA expression signatures in SCC (hypopharyngeal SCC and esophageal SCC) revealed that miR-218 expression was significantly reduced in cancer tissues compared with adjacent non-cancerous epithelium, suggesting that miR-218 is a candidate tumor suppressor. The aim of this study was to investigate the functional significance of miR-218 in cervical SCC and to identify novel miR-218-mediated cancer pathways in cervical SCC. Restoration of miR-218 significantly inhibited cancer cell migration and invasion in both HPV-positive and HPV-negative cervical SCC cell lines. These data indicated that miR-218 acts as a tumor suppressor in cervical SCC. Our in silico analysis showed that miR-218 appeared to be an important modulator of tumor cell processes through suppression of many targets, particularly those involved in focal adhesion signaling pathways. Gene expression data indicated that LAMB3, a laminin protein known to influence cell differentiation, migration, adhesion, proliferation and survival, was upregulated in cervical SCC clinical specimens, and silencing studies demonstrated that LAMB3 functioned as an oncogene in cervical SCC. The identification of novel tumor-suppressive miR-218-mediated molecular pathways has provided new insights into cervical SCC oncogenesis and metastasis.


Introduction
Cervical cancer is one of the most common cancers in women. It has been estimated that more than 529,800 new cases will be diagnosed each year, and approximately 275,100 women worldwide will die of cervical cancer each year (1). Cervical squamous cell carcinoma (cervical SCC) is one of the most frequent types of cervical cancers, accounting for 80-90% of cervical cancers, and the most important risk factor for cervical SCC is persistent human papilloma virus (HPV) infection (2). Epidemiological studies have indicated that more than 99% of patients with cervical SCC are positive for high-risk HPV (HPV16, HPV18 and HPV31) (3,4). The high-risk HPVs contain oncoproteins, i.e., E6 and E7, which contribute to oncogenesis of cervical SCC by silencing the tumor-suppressive p53 and Rb proteins (5)(6)(7)(8). The molecular mechanisms of cervical SCC initiation, development and metastasis have not yet been fully elucidated. Therefore, an increased understanding of the molecular targets and pathways of cervical SCC progression and metastasis is necessary, preferably using latest approaches in genomic analysis, including non-coding RNA studies.
RNA can be divided into 2 categories: protein-coding RNA and non-coding RNA (ncRNA). It is important to examine the functions of ncRNAs and their association with human disease, including cancer. MicroRNAs (miRNAs) are endogenous small ncRNA molecules (19)(20)(21)(22) bases in length) that regulate protein-coding gene expression by repressing translation or cleaving RNA transcripts in a

Tumor suppressive microRNA-218 inhibits cancer cell migration and invasion by targeting focal adhesion pathways in cervical squamous cell carcinoma
sequence-specific manner (9). A growing body of evidence suggests that miRNAs are aberrantly expressed in many human cancers and that they play significant roles in the initiation, development and metastasis of these cancers (10). Some highly expressed miRNAs can function as oncogenes by repressing tumor suppressors, whereas low-level miRNAs can function as tumor suppressors by negatively regulating oncogenes (11). We previously performed miRNA expression signature analysis of hypopharyngeal, maxillary sinus, esophageal and lung SCCs, in addition to bladder cancer and renal cell carcinoma; these studies indicated that miR-218 was significantly reduced in cancer tissues compared with adjacent non-cancerous tissues, suggesting that miR-218 is a candidate tumor-suppressive miRNA in human cancers (12)(13)(14)(15)(16)(17)(18). The results of past functional studies of miR-218 in various cancers indicated that miR-218 inhibits cancer cell proliferation and invasion through targeting oncogenic genes (19)(20)(21)(22)(23). Interestingly, miR-218 was underexpressed in HPV-positive cell lines, cervical lesions, and cancer tissues containing HPV16 DNA, as compared to both C-33A cells and normal cervical tissues (24).
The aim of the study was to investigate the functional significance of miR-218 in both HPV-positive and -negative cell lines and to identify the molecular pathways mediating miR-218 in cervical SCC cells. Genome-wide gene expression data for miR-218 and in silico database analyses showed that the focal adhesion pathway was a promising miR-218 target pathway. The laminins LAMB3 and LAMC1 are an important and biologically active part of the basal lamina, influencing cell differentiation, migration, adhesion, proliferation and survival. In this study, we focused on LAMB3 and investigation of the functional significance of this gene in cervical SCC. The novel tumor-suppressive miR-218-mediated cancer pathways identified herein provide new insights into the potential mechanisms of cervical SCC oncogenesis and metastasis.

Materials and methods
Clinical specimens. A total of 18 primary cervical SCC specimens and 11 non-cancerous specimens were collected from patients who had undergone surgical treatment at Chiba University Hospital. The samples were processed and stored in RNAlater (Qiagen, Valencia, CA, USA) at -20˚C until RNA extraction. Patient information is summarized in Table I. Our study was approved by the Bioethics Committee of Chiba University; prior written informed consent and approval was given by each patient.
Cervical SCC cell lines. CaSki (HPV16-positive) and ME180 (HPV39-positive) cells were grown in RPMI-1640 medium supplemented with 10% fetal bovine serum. HeLa (HPV18-positive) cells were grown in E-MEM medium supplemented with 10% fetal calf serum, and Yumoto (HPV-negative) cells were grown in E-MEM medium supplemented with 10% fetal bovine serum. All cells were cultured in a humidified atmosphere containing 5% CO 2 at 37˚C. RNA isolation. Total RNA was isolated using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. RNA concentration was determined spectrophotometrically. RNA quality was confirmed using a NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA).
DNA isolation and HPV status. Genomic DNA was extracted by QIAamp DNA mini kit (Qiagen, Venlo, The Netherlands). Samples without HPV infection were determined by PCR amplification using the L1 consensus primers MY09 and MY11 as described previously (25). HPV-positive samples were analyzed to determine the presence of DNA for HPV16 and HPV18. We designed type-specific real-time PCR primers for the E6 and E7 regions of HPV16 and HPV18 (Table II),

A
Migration and Matrigel invasion assays demonstrated that the number of invading cells significantly decreased in miR-218 transfectants in comparison with mock and miR-control transfectants in all cell lines tested. In fact, migration in miR-218 transfectants was reduced to only 11  Identification of miR-218-mediated molecular pathways and putative miR-218 target genes. We first obtained putative miR-218 target genes by searching the TargetScan database. According to the database, 4,946 conserved targets, with a total of 1,865 conserved sites and 4,372 poorly conserved sites, were deposited in this database. These genes were analyzed and characterized in KEGG pathway categories using the GeneCodis program. This analysis revealed 105 signaling pathways (Table III). In these pathways, we focused on the focal adhesion pathway and the 48 genes contained within this pathway (Table IV). To search for genes regulated by tumor-suppressive miR-218 in cervical SCC, we applied gene expression profiles in the GEO database (accession no. GSE6791). Among 48 genes, 24 genes were upregulated in cervical SCC compared to adjacent non-cancerous tissues. The expression levels of up-or downregulated genes in clinical specimens are shown in Table IV. As a result of our expression data, we identified LAMB3 as one of the most highly upregulated genes in clinical specimens; this gene has 1 putative miR-218 binding site. LAMB3 is a laminin that is an important and biologically active part of the basal lamina, functioning in a variety of pathways, such as cell differentiation, migration, adhesion, proliferation and survival. Thus, we focused on LAMB3 as a promising target gene of miR-218 in cervical SCC.   Fig. 3A). Moreover, LAMB3 expression was significantly inversely correlated with miR-218 expression (r=-0.377; P=0.0461; Fig. 3B).
LAMB3 is a direct target of miR-218. We performed quantitative real-time RT-PCR and western blot analysis to investigate whether LAMB3 mRNA and protein were downregulated by restoration of miR-218. Importantly, both LAMB3 mRNA and protein levels were significantly repressed in miR-218-transfectants in comparison with mock transfectants (Fig. 4A and B).  GAPDH was used as a loading control. The expression ratio of LAMB3 was evaluated using ImageJ software. (C) A putative miR-218 binding site in the 3'UTR of LAMB3 mRNA was identified using the TargetScan database (Upper). Luciferase reporter assays were performed using a vector encoding the partial sequences of the 3'UTR containing the putative miR-218 target site. The vector (10 ng) and miR-218 or miR-control (10 nM) were cotransfected into CaSki cells. Renila luciferase activity was measured 24 h after transfection. The results normalized by firefly luciferase values are shown. * P<0.0001.
To determine whether the 3'UTR of LAMB3 had an actual target site for miR-218, we performed a luciferase reporter assay by using a vector encoding the 3'UTR of LAMB3 mRNA. We found that the luminescence intensity was significantly reduced in miR-218 transfectants as compared to mock and miRNA-control transfectants (P<0.0001; Fig. 4C).
Silencing of LAMB3 mRNA and protein in a cervical SCC cell line. Next, we examined the impact of si-LAMB3 transfection in CaSki cells. The expression of LAMB3 mRNA was reduced in 2 si-LAMB3 transfectants in comparison with mock and si-control transfectants (P<0.0001; Fig. 5A). Additionally, the expression of LAMB3 protein was reduced in si-LAMB3-1 and si-LAMB3-2 transfectants in comparison with mock and si-control transfectants (P<0.0001 and P=0.0002, respectively; Fig. 5B). These results showed that the 2 siRNAs were useful for loss-of-function assays in this study.

Effects of LAMB3 silencing on cell proliferation, migration and invasion in cervical SCC cell lines.
To investigate the functional role of LAMB3, we performed loss-of-function studies using si-LAMB3 transfectants. The XTT assay revealed that cell proliferation was not inhibited in the 2 si-LAMB3 transfectants as compared with mock and si-control transfectants in CaSki cells (82.4±10.7%, 100.2±6.1%, 100.0±14.6% and 95.6±11.2%; P>0.0083; Fig. 6A).

Discussion
The discovery of non-coding RNA during the human genome sequencing project had a significant impact in cancer research (26). The reconstructing of genome-wide studies to include non-coding RNA is therefore necessary for cancer research. miRNAs are a class of small non-coding RNAs, and a growing body of evidence has suggested that miRNAs also contribute to cancer initiation, development and metastasis in many types of cancers, including cervical cancer (10).
It is believed that normal regulatory mechanisms can be disrupted by aberrant expression of tumor-suppressive or oncogenic miRNAs in cancer cells. Therefore, identification of aberrantly expressed miRNAs is the first step toward elucidating miRNA-mediated oncogenic pathways. Based on this, we identified the miRNA expression signatures in several human squamous cell carcinomas, including esophageal SCC, hypopharyngeal SCC, maxillary sinus SCC and lung SCC, allowing the elucidation of multiple tumor-suppressive miRNAs (12)(13)(14)(15). Our previous studies showed that miR-218 is a frequently downregulated miRNA and that restoration of this miRNA inhibited cancer cell migration and invasion in head and neck SCC (HNSCC) cells (19). We also searched for downregulated miRNAs in cervical SCC by examining expression signatures published in public databases (27)(28)(29)(30)(31). These data indicate that miR-218 is frequently downregulated miRNA in cervical SCC. Thus, we focused on miR-218 and investigated the functional significance of this miRNA in mediating cancer pathways.
In the human genome, 2 miR-218 precursor genes, miR-218-1 and miR-218-2, have identical sequences in the mature miRNA and map to human chromosomes 4p15.31 and 6q35.1, respectively. Interestingly, the genomic regions of miR-218-1 and miR-218-2 are located in the introns of the SLIT2 and SLIT3 genes, respectively. Downregulation of miR-218 in cancer cells has been shown to be caused by promoter hypermethylation of SLIT2 and SLIT3 genes (20). Silencing of miR-218 by DNA hypermethylation has also been reported in oral SCC using a function-based screening approach (21). On the other hand, several reports have indicated that silencing of miR-218 in cervical SCC was caused by HPV infection (24,32). Our expression data showed that miR-218 expression was significantly reduced in both HPV-positive cells (CaSki, HeLa and ME180) and HPV-negative cells (Yumoto), in addition to clinical specimens. Since the molecular mechanisms of miR-218 silencing in cervical SCC are still unclear, further study is necessary to solve this problem.
In the current study, we found significant inhibition of cancer cell migration and invasion in cervical SCC cell lines (CaSki, HeLa, ME180 and Yumoto) by miR-218 restoration, suggesting that miR-218 is a tumor-suppressive miRNA in cervical SCC. Our previous reports in HNSCC also showed that miR-218 contributes to cancer cell migration and invasion (19). The tumor-suppressive function of miR-218 has also been reported in several types of cancers, and miR-218 has been shown to target several oncogenic genes, such as Rictor (oral cancer), survivin and ROBO1 receptor (nasopharyngeal cancer and gastric cancer) (20)(21)(22). Our recent report also indicated that restoration of miR-218 inhibited cancer cell proliferation, migration, and invasion in bladder cancer (23,33). These data suggested that miR-218 is an important tumor-suppressive miRNA that is deeply involved in human cancers.
miRNAs are unique in their ability to regulate many protein-coding genes. Bioinformatic predictions have indicated that miRNAs regulate more than 30% of proteincoding genes (34). A single miRNA is capable of targeting a number of genes to globally regulate biological processes. The identification of novel cancer pathways and responsible genes regulated by tumor-suppressive miR-218 in cervical SCC is the next step for our understanding of cervical SCC oncogenesis. Thus, we pursued GeneCodis analysis to reveal the functional significance of these genes potentially regulated by miR-218 in cervical SCC. The GeneCodis analysis applies many genes to known pathways in the KEGG Pathway Database, and these data facilitate our understanding of tumorsuppressive miRNA-mediated molecular pathways in human cancer. This method of analysis has previously been used to efficiently identify tumor-suppressive miRNA-mediated cancer pathways in our laboratory (19). In the current study, the GeneCodis analysis revealed 105 signaling pathways, as highlighted in Table II. In these pathways, we focused on the focal adhesion pathway and the 48 genes contained within this pathway.
To search for genes regulated by tumor-suppressive miR-218 in cervical SCC, we used gene expression profiling in this study (deposited in the GEO database as accession no. GSE6791). Among the 48 genes in the focal adhesion pathway, 10 were upregulated in cervical SCC clinical specimens, indicating that these genes were candidates for regulation by tumor-suppressive miR-218 in cervical SCC. From these genes, we focused on LAMB3, a component of laminin-332, and investigated the functional significance of this gene.
Laminin-332, a heterotrimer composed of 3 chains (LAMA3, LAMB3 and LAMC2), is an adhesion substrate for epithelial cells and regulates epithelial cell migration during epithelial regeneration and repair processes (35,36). Several immunohistochemical studies have shown that laminin-332 or its subunit LAMC2 is expressed in tumor cells at the invasion front or in budding tumor cells in many types of human cancers, such as adenocarcinomas of the colon, breast, pancreas and lung, SCC of the esophagus and melanoma (35). Our data demonstrated that LAMB3 was directly regulated by miR-218 and functioned as an oncogene, contributing to cancer cell migration and invasion in cervical SCC. Many studies have indicated that laminin-332 binds to several cell-surface receptors, such as integrins, epidermal growth factor receptor and syndecan-1 (37)(38)(39). Among these binding partners, integrins are cell surface transmembrane proteins that mediate extracellular signals and intracellular pathways, leading to control of the cell cycle, cell migration, and invasion in cancer cells (40). It will be necessary to analyze the signal pathways associated with the interactions of integrins and laminin-332 in cervical SCC in the future.