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FGFR3 silencing by siRNA inhibits invasion of A549 cells

The present study identified that fibroblast growth factor receptor 3 (FGFR3) was significantly upregulated in bone metastasis of lung adenocarcinoma. RNA interference (RNAi) is a powerful approach for treating a wide range of human diseases, including cancer, through downregulating the expression of selected genes. In the present study, the invasiveness of A549 cells cultured in vitro was altered by small interfering (si)RNA targeting FGFR3, and the regulatory effect of silencing FGFR3 on the expression levels of E-cadherin and matrix metalloproteinase (MMP)9 was investigated. Human lung adenocarcinoma A549 cells were transfected with synthetic specific siRNAs targeting a fragment of the FGFR3 gene (namely, siRNA-855, siRNA-1447 and siRNA-2076) or with negative control (NC) siRNA. Cells were divided into five groups (A, siRNA-855 group; B, siRNA-1447 group; C, siRNA-2076 group; D, NC-siRNA group; and E, blank control group). The effect of the above siRNAs targeting FGFR3 on the invasion capacity of A549 cells was detected by Transwell assay. siRNAs against FGFR3 were transfected into A549 cells with by Lipofectamine® 2000, and the expression levels of FGFR3, E-cadherin and MMP9 were measured by reverse transcription-quantitative polymerase chain reaction and western blot assay. The experimental findings indicated that the expression levels of FGFR3 and MMP9 were significantly reduced in the siRNA-FGFR3-transfected groups (A-C groups), compared with those in the D and E groups (P<0.01). In addition, the expression levels of E-cadherin were markedly elevated in the A-C groups, compared with those in the D and E groups (P<0.01). There was no significant difference in E-cadherin expression between the A-C groups, or between the D and E groups (P>0.05). These results indicated that siRNA-FGFR3 was able to decrease the invasiveness of A549 cells, inhibit the expression of MMP9 and increase the expression of E-cadherin by downregulating the expression of FGFR3. Taken together, the results of the present study indicated that the upregulation of E-cadherin expression and the downregulation of MMP9 expression are able to inhibit the migration of A549 cells, and siRNA silencing FGFR3 acts as a tumor suppressor in these cells.


Introduction
Non-small cell lung cancer (NSCLC) is the most common type of lung cancer and the leading cause of cancer-associated mortality worldwide, which mostly results from local invasion or distant metastases, rather than being due to primary tumors (1). The 5-year survival rate of advanced lung cancer has been reported to be <15% (2). It has been recognized that certain tumors present a predilection for metastasis to specific organs (3,4), being bone, brain, liver and lung the most frequent target organs of metastasis (4). The mechanism of tumor development in lung cancer is likely to be associated with genetic diseases that occur when certain genes are mutated or exhibit abnormalities such as deletions (3). Novel strategies for the treatment of lung cancer require the identification of novel molecules that are able to inhibit the invasiveness and metastasis of lung cancer cells (5).
Small interfering (si)RNA molecules consist of 21-23 nucleotides in length and display a characteristic and highly specific structure containing 2-3 nucleotides in the 3' overhangs and 5' phosphate and 3' hydroxyl groups to prevent erroneous gene silencing, in addition to a sense (passenger) strand and an antisense (guide) strand (6). The remarkable advances in biology and cancer therapy that had occurred during the past recent decades have led to the development of siRNA technology, which is an effective method to sequence-specific knockdown or reduce gene function through homology-dependent degradation of the corresponding target messenger (m)RNA, via a phenomenon known as RNA interference (RNAi), which is one of the most rapidly growing topics of research for the treatment of various diseases, including cancer (6,7). Despite the caveats and ongoing challenges such as off-target effects and false discovery rates, RNAi remains a powerful approach for reverse genetics in large-scale functional analysis conducted in cultured cells and in various in vivo systems (6). Furthermore, the development of siRNA therapeutics is rapidly gaining momentum at present (6).
Lung cancer is the leading cause of cancer-associated mortality and morbidity worldwide (8), and NSCLC is responsible for almost 80% of all lung cancer-associated mortalities, mostly resulting from tumor invasion and metastasis (8,9). Fibroblast growth factor receptor 3 (FGFR3) is a member of the transmembrane receptor tyrosine kinase (RTK) family, which interacts with FGF ligands and participates in the regulation of cell proliferation, differentiation and tumorigenesis (10,11). Previous studies demonstrated that the expression levels of FGFR3 were significantly upregulated in metastasis of NSCLC, compared with normal lung tissues, according to the results of quantitative analysis of FGFR3 expression in humans (3,10,12). In addition, FGFR3 has been demonstrated to be involved in the RAS/RAF/mitogen-activated protein kinase (MAPK) kinase (MEK)/MAPK signaling pathway through the activation of p90 ribosomal S6 kinase (13).
Migration and invasion are critical steps in the initial progression of cancer that facilitate metastasis (14,15). In particular, the critical steps required for the initiation and progression of tumor invasion are epithelial-mesenchymal transition (EMT) and extracellular matrix (ECM) degradation (14). E-cadherin is an important marker of EMT (15), while matrix metalloproteinase (MMP)9 is a member of the MMP family of enzymes, which are involved in the degradation of the ECM (16,17). Increased levels of MMP9 have been correlated with tumor aggressiveness in numerous types of cancer (18).
RNAi is a popular method for exploring gene function, which suppresses the expression of a specific gene of interest in transfected mammalian cell cultures (19). In the present study, siRNA-FGFR3 significantly inhibited the expression of FGFR3 by directly targeting FGFR3 in A549 cells. The present authors hypothesized that siRNA-FGFR3 was able to silence FGFR3 and inhibit the migration of A549 cells by decreasing the expression levels of E-caherin and MMP9 in these cells. To test the above hypothesis, cell invasion ability was assessed by Transwell invasion assay, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis in A549 cells transfected with siRNA-FGFR3.
Transwell invasion assay. Cell invasion was assessed by Transwell invasion assay using 8-µm Matrigel-coated invasion chambers (Costar; Corning Life Sciences), according to the manufacturer's protocol. Brief ly, A549 cells (0.5x10 5 cells/well) were suspended in 200 µl serum-free medium and seeded on the upper chamber of a 24-well Transwell migration chamber (Costar; Corning Life Sciences). The lower compartment was filled with 750 µl complete medium. Following 16-h culture at 37˚C in a humidified incubator with 5% CO 2 , those cells that were retained on top of the membrane were removed with a cotton swab, while those cells that had migrated to the bottom side (invaded cells) were fixed with 10% formalin (Costar; Corning Life Sciences), stained with 0.1% crystal violet (Costar; Corning Life Sciences) and observed under a light microscope (Olympus BX41; Olympus Corporation, Tokyo, Japan) at 200x magnification. The number of penetrating cells present in 9 random fields was counted, and the experiment was repeated ≥3 times.
RT-qPCR. Total RNA was isolated from A549 cells using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) at 36 h post-transfection, according to the manufacturer's protocol. The concentration and purity of the extracted RNA was quantified by its absorbance at 260 nm using a BioPhotometer (D30; Eppendorf, Hamburg, Germany).
Western blot analysis. To analyze the protein expression levels of FGFR3, MMP9 and E-cadherin in A549 cells, transfected and untransfected cells were rinsed twice with precooled phosphate-buffered saline (Costar; Corning Life Sciences), prior to being homogenized in radioimmunoprecipitation assay (RIPA) buffer (Costar; Corning Life Sciences) supplemented with a protease and phosphatase inhibitor cocktail (Roche Diagnostics GmbH, Mannheim, Germany) at 36 h post-transfection. Cell lysates were centrifuged (GTR 21-1; Shanghai Medical Instruments Co., Ltd., Shanghai, China) at 14,000 x g for 10 min at 4˚C, and the supernatants were then mixed with 5X sodium dodecyl sulfate (SDS; Costar; Corning Life Sciences) loading sample buffer, boiled for 5 min and separated using 10% SDS-polyacrylamide gel electrophoresis at 60 V for 5 h. Total cellular protein levels were quantified by Bradford assay (21) using standard protein solution and Coomassie Briliant Blue (R-250) (Generay Biotech Co., Ltd., Shanghai, China). Following electrophoresis, the proteins were transferred  were mixed at a 1:1 ratio, prior to be added to the blots, which were subsequently exposed and imaged. The relative intensity of the bands was analyzed using ImageJ software (National Institutes of Health, Bethesda, MA, USA). All experiments were performed in triplicate.
Statistical analysis. Results were analyzed with SPSS statistical software version 18.0 (SPSS, Inc., Chicago, IL, USA). Data are represented as the mean ± standard deviation of 5 replicates. Differences between multiple sets of data were compared by analysis of variance, while differences between 2 groups were compared by homogeneity of variance test. The significance level was considered to be α= 0.05, and P<0.05 was considered to indicate a statistically significant difference.  (Table I). Thus, the rate of inhibition of invasion for groups A, B and C was determined to be 63.9, 61.4 and 61.8% (P=0.000004, P=0.000006 and P=0.00005, respectively; Table I). No significant differences were observed for the NC-siRNA group, where the number of invasive cells was determined to be 0.97 times that of the blank control group (P= 0.745000; Fig. 1 and Table I Tables II-IV). These results indicated that siRNA transfection with Lipo2000 markedly inhibited the mRNA expression of FGFR3 in a specific manner, which led to a marked reduction in the mRNA expression levels of MMP9 and a marked increase in the mRNA expression levels of E-cadherin in siRNA-FGFR3-transfected A549 cells.

Protein expression of FGFR3, MMP9 and E-cadherin was altered in siRNA-transfected A549 cells.
Western blot analysis demonstrated that, compared with groups D and E, the protein levels of FGFR3 and MMP9 were downregulated in A-C groups, while the protein levels of E-cadherin were upregulated in these groups, according to the lower and higher gray, respectively, of the protein bands in the A-C groups (Fig. 1H). ImageJ software was used to analyze the gray values of the different protein bands. Compared with the E group, the FGFR3/GAPDH and MMP9/GAPDH ratios in the A-C groups were significantly decreased, while the E-cadeherin/GAPDH ratio in these groups was significantly increased, compared with the E group (P<0.01; Tables V-VII). No significant differences   in FGFR3, MMP-9 and E-cadherin expression were observed between the A-C groups or between groups D and E (P>0.01; Tables V-VII). These results suggest that the protein expression of FGFR3, encoded by FGFR3 mRNA, was inhibited, possibly due to the specific degradation of FGFR3 mRNA induced by siRNA-FGFR3, which led to a decrease in the protein expression levels of MMP9 and an increase in the protein expression levels of E-cadherin.

Discussion
The process of carcinoma metastasis consists of a series of sequential steps (4). Initially, tumor cells are detached from the ECM, and the surrounding tissue is then invaded (4).
Losing cell-cell adhesion and gaining motility are important for carcinoma cells to separate from neighboring cells and invade other tissues (4). EMT is a multistage process involving major alterations in cell morphology, cell-cell and cell-matrix adhesions. ETM is important for the motile and invasive capabilities of cells, which are essential in malignant tumor progression and metastasis (14). A hallmark of EMT is the loss of expression of E-cadherin, which is a key cell-cell adhesion molecule (22). It has been demonstrated that the ability of invasion and metastasis of certain invasive carcinoma cells may be altered by forcing the expression of E-cadherin (22-24). ECM dysregulation and remodeling are essential for the progression of neoplastic processes, and MMP-mediated ECM degradation has been demonstrated to lead to cancer cell invasion and metastasis (25). Previous studies on stromal cells undergoing EMT have revealed that EMT may be the most critical and abnormal signaling pathway that occurs during development and metastasis of lung cancer (22). MMP9 has been identified as a critical component of the multifunctional MMP family of enzymes, which act as ECM-degrading proteases and may promote metastasis (26,27). The hypothetical role of MMP9 as a metastasis promoter is supported by a previous study (28).
The FGFR3 gene is located in the 4p16.3 region of the chromosome, which consists of 19 exons and 18 introns expanding across 16.5 kb and encodes a 4.4-kb mRNA molecule (29)(30)(31). FGFR3 is a member of the FGFR family of tyrosine kinase receptors, and consists of an extracellular domain that includes a    (3). Subsequently, the trans-autophosphorylation of tyrosine residues in the cytoplasmic domain of FGFR3 stimulates the intrinsic catalytic activity of the receptor, and leads to the activation of downstream signaling pathways (13,33). FGFR3 has been demonstrated to be involved in the RAS/RAF/MEK/MAPK signaling pathway through the activation of p90 ribosomal S6 kinase (13,33). Previous studies have reported that FGFR3 is overexpressed in lung cancer and bone metastasis of lung adenocarcinoma; thus, FGFR3 may be regarded as a potential target for cancer therapy (3,10,11). However, the effect of silencing FGFR3 on the inhibition of invasion of lung carcinoma remains unknown. Therefore, the present study aimed to inhibit the expression of FGFR3 in lung cancer cells in order to evaluate its effects on the invasion ability of NSCLC. For that purpose, the association between FGFR3, E-cadherin and MMP9 expression in A549 cells was investigated by siRNA targeting FGFR3. In addition, the mRNA and protein expression levels of FGFR3, E-cadherin and MMP9 were analyzed in siRNA-FGFR3-transfected and control groups.
Previous in vivo and in vitro studies have demonstrated that the upregulation of FGFR3 increased cancer cell invasion and metastases in breast (12) and lung cancer (10). A hallmark of cancer is its self-sufficiency in terms of growth signals, which is frequently driven by RTK-dependent growth factor signaling pathways (34). These pathways are important during the development and adult life of multicellular organisms (2,3). FGFR3 belongs to the RTK family, and FGF/FGFR3 signaling has been frequently observed to occur in certain NSCLC cell lines via the Ras/RAF/MEK/MAPK signaling pathway, which regulates key cellular processes such as cell proliferation, migration and survival (25,35). A previous study has reported that FGFR signaling is active and important in cells undergoing EMT, which is induced by transforming growth factor-β (14). EMT is involved in the progression and metastasis of cancer (14). In the present study, marked upregulation of E-cadherin and downregulation of MMP9 via siRNA targeting FGFR3 was observed in lung cancer A549 cells. Furthermore, the results of Transwell assay demonstrated that the invasion ability of siRNA-FGFR3-transfected cells significantly decreased, compared with control cells. Taken together, these results indicate that FGFR3 is associated with tumor invasion and may promote the expression of E-cadherin and MMP9, which are associated with EMT and ECM remodeling, respectively, in lung cancer. However, the direct mechanism between FGFR3 and MMPs remains to be elucidated. Therefore, siRNA targeting FGFR3 may act as a tumor suppressor through the regulation of FGFR3. Thus, RNAi technology may serve as a potential target of NSCLC therapy.
In conclusion, the present study suggests that downregulation of FGFR3 via siRNA-FGFR3 in NSCLC may lead to a decrease in tumor cell invasion. In addition, the expression of FGFR3 was observed to be significantly correlated with the expression of MMP9 and E-cadherin in the present study.
According to these results, FGFR3 may be an important marker of NSCLC metastasis and a potential therapeutic target. Further studies are required to clarify the mechanisms of FGFR3 in EMT and ECM remodeling in NSCLC.