microRNA-194 suppresses osteosarcoma cell proliferation and metastasis in vitro and in vivo by targeting CDH2 and IGF1R

Studies have shown that miR-194 functions as a tumor suppressor and is associated with tumor growth and metastasis. We studied the effects of miR-194 in osteosarcoma and the possible mechanism by which miR-194 affected the survival, apoptosis and metastasis of osteosarcoma. Both human osteosarcoma cell lines SOSP-9607 and U2-OS were transfected with recombinant lentiviruses to regulate miR-194 expression. Overexpression of miR-194 partially inhibited the proliferation, migration, and invasion of osteosarcoma cells in vitro, as well as tumor growth and pulmonary metastasis of osteosarcoma cells in vivo. Potential miR-194 target genes were predicted using bioinformatics. Luciferase reporter assay, real-time quantitative PCR and western blotting confirmed that CDH2 (N-cadherin) and IGF1R were targets of miR-194. Using real-time quantitative PCR, we evaluated the expression of miR-194 and two miR-194 target genes, CDH2 and IGF1R in osteosarcoma samples from 107 patients and 99 formalin- or paraformalin-fixed paraffin-embedded tissues. The expressions of the target genes were also examined in osteosarcoma samples using immunohistochemistry. Overexpression of miR-194 inhibited tumor growth and metastasis of osteosarcoma probably by downregulating CDH2 and IGF1R. miR-194 may prove to be a promising therapeutic agent for osteosarcoma.


Introduction
Osteosarcoma (OS) is the most common primary malignant neoplasm in adolescents with an annual estimated worldwide incidence of 4 million, with a peak incidence at the age of 15-19 years (1). Osteosarcoma is associated with abnormal differentiation caused by genetic and epigenetic changes. Advances in osteosarcoma therapy have enhanced patient outcomes. The most effective regimens currently include neoadjuvant and adjuvant chemotherapy coupled with local control that usually consists of limb-sparing surgery (2). Unfortunately, the cure rate is still very poor due to pulmonary metastases (3). Therefore, the identification of effector molecules and signaling pathways underlying resistance to chemotherapy and malignancy is vital for osteosarcoma treatment. Studies have investigated the genes associated with metastasis of osteosarcoma, and microRNAs (miRNAs) have become a new research hotspot in gene therapy. microRNAs (miRNAs) are a class of 22-25 nucleotide RNA molecules that negatively regulate gene expression in animals and plants (4,5). Since the discovery of the role of miRNAs in Caenorhabditis elegans development (6), a frequent disregulation of miRNAs has been observed in diverse cancers, including synovial sarcoma, colon cancer (7), breast cancer (8), glioma (9), glioblastoma (10), hepatocellular carcinoma (11), lung (12) and gastric cancer (13). Some of these miRNA expression profiles showed downregulation in tumors compared with normal tissue (14), like miR-127 in human bladder cancers (15) and microRNA-34a in OS (16). However, other miRNAs are upregulated in tumors, such as miR-150 in gastric cancer (17) and miR-17-92 cluster in renal cell carcinoma (18). The alterations in miRNA expression may play a crucial role in the initiation and progression of the above cancers (19), functioning as a novel class of oncogenes and tumor suppressors (20,21). Thus, miRNAs play an essential role in basic physiologic processes, such as development, differentiation, proliferation and apoptosis (22). However, their biological function remains largely unknown. miR-194 is specifically expressed in the human gastrointestinal tract and is induced during intestinal epithelial cell differentiation (23). The regulatory role of miR-194 was first studied in normal and malignant cells of the gastrointestinal

microRNA-194 suppresses osteosarcoma cell proliferation and metastasis in vitro and in vivo by targeting CDH2 and IGF1R
tract (24). Overexpression of miR-194 in gastrointestinal cancer cells suppresses cell migration, invasion and metastasis (24). miR-194 functions as a tumor suppressor gene by downregulating targets such as SSH2, HBEGF, IGF1R, CDH2 (N-cadherin) and TLN2 (23)(24)(25)(26)(27). Hepatocyte nuclear factor (HNF) also induces miR-194 expression during intestinal epithelial cell differentiation (23). In colon cancer tissue, miR-194 was downregulated relative to normal mucosa (28). Low expression of miR-194 has been associated with large tumor size and advanced stage in gastric cancer (29). In endometrial cancer cells, miR-194 has been reported to inhibit self-renewal factor BMI-1, reduce cell invasion and inhibit epithelial-mesenchymal transition (EMT) (30). The mutations of p53 tumor suppressor gene, which directly regulates the expression of miR-194, were found in 20-60% of sporadic OS (31). The reports suggested that miR-194 may function as a tumor suppressor in OS. However, the effects of miR-194 in osteosarcoma have not been completely elucidated. Therefore, it is of great significance to further study the function and mechanism of miR-194 in osteosarcoma.
We carried out in vitro and in vivo experiments to evaluate the effects of miR-194 and its possible direct targets, IGF1R and CDH2, in tumor growth and metastasis of SOSP-9607 and U2-OS cells. We also predicted its putative target genes, which are correlated with tumor growth and metastasis, using bioinformatics analysis. We report for the first time that overexpression of miR-194 inhibited growth and metastasis of osteosarcoma. In addition, miR-194 specifically downregulated the expression of IGF1R and CDH2. miR-194 gene therapy may prove to be a promising therapy for tumorsuppression in osteosarcoma.

Materials and methods
Ethics statement. All research involving human tissue samples and animals was approved by the Ethics Review Committee of Fourth Military Medical University, Xi'an, Shaanxi, China (approval ID:2013106) and written informed consent was obtained from all participating patients.
Human tissue samples. A total of 107 pairs of human osteosarcoma tissue samples were obtained from patients who underwent surgical resection at the Tangdu Hospital of Fourth Military Medical University between 2007 and 2010 and were diagnosed with osteosarcoma based on histopathological evaluation. The biopsies were immediately snap-frozen in liquid nitrogen after resection and stored at -80˚C. One section of each sample was stained with hematoxylin-eosin (H&E) for histopathological evaluation. The clinical stage of these osteosarcoma patients was classified according to the sixth edition of the tumor-node-metastases (TNM) classification of the International Union Against Cancer (UICC).
All 107 osteosarcoma patients were studied in a follow-up. The median follow-up was 42 months (range, 5-68 months). During the follow-up period, 62 patients (57.9%) died of disease. Distant metastases developed in 52 patients at a mean of 12.7 months (range, 3-41 months) after the original diagnosis. Of these patients, 13 had bone metastases and 43 had lung metastases (4 patients had both bone and lung metastases).
Cell culture. Human osteosarcoma cell lines SOSP-9607 were established and reserved in our laboratory as previously described (32). Human osteosarcoma cell lines U2-OS were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and used within 6 months of the purchase. SOSP-9607 cells were maintained in RPMI-1640 medium (HyClone, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS; HyClone), 2.0 mM L-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin, and incubated at 37˚C in a humidified incubator supplemented with 5% CO 2 and 95% air. U2-OS cells were maintained in the same conditions, except DMEM medium was used. Cell line authentication was performed by the Orthopedics Oncology Institute of Chinese PLA according to the UKCCCR guidelines every 2-3 months, including mycoplasma test by PCR and measurement of cell proliferation by counting.
Generation of stable cell lines. Recombinant lentiviruses containing overexpression of miRNA-194, for knocking down miRNA-194 and miRNA control were purchased from Shanghai Genechem Co., Ltd. (Shanghai, China). The precusor sequence of miR-194 was used for overexpression as follows: AUGGUGUUAUCAAGUGUAACAGCAACUCCAUGUGG ACUGUGUACCAAUUUCCAGUGGAGAUGCUGUUACU UUUGAUGGUUACCAA. The reverse complementary sequence of miR-194 was used for the knock-down as follows: TC CACATGGAGTTGCTGTTACA. Besides the multiple clone sites of lentivirus expression vectors, there also was a GFP reporter driven by an independent promoter (SV40 promoter) to indicate the infection rate of the virus timely.
To generate the stable cell line, 1x10 4 cells were transfected with 5x10 5 transducing units of lentiviruses. The supernatant was removed after 24 h and replaced with complete culture medium. Infection efficiency was confirmed by RT-PCR 96 h after infection and the cells were selected with 1 µg/ml puromycin for 2 weeks.
Reverse transcription and quantitative real-time PCR. Total RNA containing miRNA and mRNA was extracted from cells with TRIzol ® reagent (Invitrogen, Carlsbad, CA, USA), or from formalin-or paraformalin-fixed, paraffin-embedded (FFPE) tissues with RecoverAll™ Total Nucleic Acid Isolation kit (Ambion, Foster City, CA, USA; cat no. AM1975), according to the manufacturer's instructions. The RNA was quantified by absorbance at 260 nm and transcribed into cDNA using BioRT Two-Step RT-PCR kit (Bioer Technology, Inc., Hangzhou, China). To evaluate IGF-1R and N-cadherin expression levels, 1 µg of total RNA was used for reverse transcription with iScript cDNA Synthesis kit (Bio-Rad Laboratories, Hercules, CA, USA), according to the manufacturer's instructions. The sequences of the forward and reverse primers for IGF1R were 5'-CGACATTGAGGAGGTCAC AGA-3' and 5'-TGGGCACGAAGATGGAGTT-3'. The sequences of the forward and reverse primers for N-cadherin were 5'-GTCAGCAGAAGTTGAAGAAATAGTG-3' and 5'-GCAAGTTGATTGGAGGGATG-3'.
Apoptosis, proliferation and cell cycle assays. Cultured cells were grown in 6-, 24-and 96-well plates. Apoptosis and cell cycle were measured using flow cytometry. The procedures were performed as previously described (33). Cell viability was examined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. MTT was performed at 24, 48, 72, 96, 120 and 144 h. The absorbance at 492 nm was measured after incubation with 20 µl of MTT for 4 h. The curve of cell proliferation was then drawn and the proliferation efficiency was examined. There were 6-wells in each group, the experiments were repeated three times independently and the results were given as means ± SD. The plate clone formation assay was performed as previously described (34). Clones with >50 cells were counted with an ordinary optical microscope and the clone formation rate was calculated with the following formula: Plate clone formation efficiency = (number of clones/ number of cells inoculated) x 100%.
Transwell cell migration and matrigel invasion assays. The invasive potential of cells was measured in 6.5 mm Transwell with 8.0-mm pore polycarbonate membrane insert (cat. 3422; Corning, NY), according to the manufacturer's instructions. The filter of the top chamber was coated with 50 µl of diluted Matrigel and incubated at 37˚C for 2 h. The lower chambers were filled with 600 µl of RPMI-1640 medium containing 5% fetal bovine serum (FBS) as chemoattractant. The suspension of 5,000 cells in 100 µl migration medium was added into each top chamber. After the cells were incubated for 16 h, the noninvading cells that remained on the upper surface were removed with a cotton swab. The invasive cells in the lower surface of the membrane insert were fixed with 4% paraformaldehyde for 30 min, permeabilized with 0.2% Triton X-100 at room temperature for 15 min and then stained with 0.1% crystal violet for 5 min. The number of cells in the lower surface, which had invaded through the membrane, was counted under a light microscope in five random fields at a magnification of x100. The experiments were repeated three times independently and results were expressed as means ± SD.
The procedure for Transwell migration assays was the same as the Transwell invasion assay except that the filter of top chamber was not coated with Matrigel.
Wound healing migration assay. When the transfected and untransfected SOSP-9607 and U2-OS cells were grown to confluence, a scratch in the cell monolayer was made using a micropipette tip. Following incubation of the cells under standard conditions for 24 h, the plates were washed twice with fresh medium and images were captured at different times. The migration potential was estimated by counting the cells that migrated from the wound edge. The cell migration rate was obtained by counting three fields per area and represented as the average of six independent experiments over multiple days.
Animal studies. Four-week-old female nude mice (BALB/c, nu/nu; Experimental Animal Centre of the Fourth Military Medical University in China), 17-22 g in weight, were maintained under specific pathogen-free conditions with 12-h light/12-h dark cycles at 26-28˚C and 50-65% humidity. Animal feed and underpad, which were purchased from the Experimental Animal Center, Fourth Military Medical University, were autoclaved and vacuum packed. The water was adjusted to a pH value of 2.8 and autoclaved before use.
Animal experiments were performed to evaluate orthotopic tumor growth and spontaneous pulmonary metastasis properties of osteosarcoma cells in vivo. Briefly, 4 groups SOSP-9607 cells (overexpression of miRNA-194, for knocking down miRNA-194, miRNA control and untransfected cells) suspension of 100,000 cells in 100 µl were injected into the proximal tibia of each anesthetized nude mouse (n=10 animals per group). Every 7 days post-inoculation, the length and width of individual orthotopic tumor were measured with calipers, and the volume (mm 3 ) was calculated according to the formula: 1/2 x length x width 2 (35). The curve of orthotopic tumor growth was depicted 42 days after inoculation mouse lungs and orthotopic tumors were harvested and weighed. miR-194 expression levels in the orthotopic tumors were tested using real-time RT-PCR, and the number of pulmonary metastatic tumor nodules was counted under a low-power dissecting stereomicroscope. Finally, mouse lungs were fixed with 10% neutral-buffered formalin, embedded in paraffin, sectioned at 6 µm and stained with H&E. The pulmonary metastases were imaged under a light microscope at magnifications of x40, x100, 2x00 and x400.
Protein extraction and western blot analysis. Protein extracts were prepared through a modified RIPA buffer with 0.5% sodium dodecyl sulfate (SDS) in the presence of a proteinase inhibitor cocktail (Complete Mini; Roche Diagnostics, Basel, Switzerland) and then were performed as previously described (36).
Luciferase reporter assay. To validate IGF1R and N-cadherin as target genes of miR-194 in osteosarcoma cells, luciferase assay was performed as previously described (33).
Immunohistochemistry. The dilution of CDH2 and IGF1R antibody used for immunohistochemistry was 1:100. Immunohistochemistry was carried out as previously described (37). The final scores of CDH2 and IGF1R expression were calculated as previously described (38) and classified as follows: 0-4, low; 5-9, high.
Statistical analysis. All values in the present study were expressed as the means ± SD, and all error bars represent the standard deviation of the mean. Student's t test, one-way analysis of variance and repeated measures data of ANOVA were used to determine significance. Patient survival and their differences were determined using the log-rank test. Cox regression (proportional hazards model) was used for multivariate analysis of prognostic factors. All statistical tests were two-sided. P<0.05 was considered statistically significant. Statistical analyses were performed using the SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA).

Results
Significant difference between F4 and control F5M2 cells. F4 and F5M2 were the sublines originated from SOSP-9607 using limited dilution method (32,39). F5M2 cells show stronger proliferation and invasion than F4 cells, which is useful in studies on metastatic mechanism of osteosarcoma (32). In the present study, we evaluated the expression of miR-194 using quantitative real-time PCR. The results showed that miR-194 expression was significantly lower in F5M2 cells compared with F4 cells (Fig. 1A; P<0.001). The results suggested that miR-194 might play a vital role in the metastatic processes.
Generation of stable cell lines. After transfection and selection of cells, the experiments with SOSP-9607 cells and U2-OS cells were divided into four groups including a blank group (untransfected cells), a control group (cells transfected with the control lentivirus), an OE group (overexpression of miRNA-194) and a KD group (knocked down miRNA-194). These GFP-labeled oligonucleotides were detected using fluorescence microscopy (Olympus, Tokyo, Japan) (Fig. 1B).
miR-194 expression levels in four groups were measured using microscopy and stem-loop real-time RT-PCR. The results ( Fig. 1C and D) showed that the level of miR-194 was significantly higher in the OE group and lower in KD group compared with control and blank groups, respectively. However, there were no significant differences between the control and blank groups. These results indicated that miR-194 recombinant lentiviruses could regulate miR-194 expression effectively in both SOSP-9607 and U2-OS cells. These strategies were then used as the basis of the remaining experiments.

miR-194 inhibits proliferation of osteosarcoma in vitro.
The results of MTT assay showed that SOSP-9607 cells in OE groups exhibited a significant decline in proliferation capacity compared with the other three groups (P<0.001), which were negatively correlated with the exogenous miR-194 level ( Fig. 2A). In contrast, cells in the KD group showed significantly enhanced proliferation (P<0.001). No statistical differ ence was found between the blank groups and control groups (P=0.541). We also tested U2-OS cells ( Fig. 2A). The results were similar to the stably transfected SOSP-9607 cells.
Cell cycle distribution was detected by flow cytometry. The results showed that more cells were in the G0/G1 phase in the OE group compared with the G0/G1 phase in KD group in SOSP-9607 and U2-OS cells (Fig. 2B). These data demonstrated that miR-194 could inhibit the proliferation in both SOSP-9607 and U2-OS cells.  Fig. 3B and D). These results showed that miR-194 could induce apoptosis in both SOSP-9607 and U2-OS cells.
The wound healing assay showed that cells in OE groups exhibited a significant decrease in migration rate compared to the other three groups. KD groups of SOSP-9607 cells (or U2-OS) nearly closed the wound 48 h after incubation, but not the other three groups (Fig. 5C and D).

miR-194 inhibits tumor growth and metastasis of osteosarcoma in vivo.
Four groups of stable cells (OE, control, blank and KD SOSP-9607) were injected into proximal tibia of young nude mice. To evaluate tumor growth, the length (L) and width (W) of orthotopic tumor were measured every 7 days post-inoculation. The volume of tumor was calculated according to the formula: Volume = 1/2 x L x W 2 , and the growth curve of orthotopic tumor obtained. Progressive solid tumors were seen in all mice. By contrast, cells in OE groups produced much smaller tumors, while KD group generated the biggest size ( Fig. 6A and D; P<0.05). The mice were sacrificed 42 days post-inoculation. The mean tumor weight ± SD of orthotopic tumors were as follows: OE group 1.04±0.159 g, control group 1.598±0.198 g, blank group 1.622±0.240 g and KD group 2.082±0.134 g (Fig. 6A and E). No significant difference was observed between the blank and control cells (P=0.842). The number of metastatic nodes was dramatically reduced in the nude mice in OE group compared with the other groups ( Fig. 6A and F). Tumor and metastases were confirmed based on histopathological evaluation ( Fig. 6B and C). Orthotopic tumors in the OE group expressed higher miR-194 levels compared with other groups (Fig. 6G) indicating that exogenous miR-194 significantly inhibited the tumor growth and metastasis in vivo. Expression of miR-194 in osteosarcoma and corresponding non-cancerous tissues. miR-194 expression was decreased in 59 of 107 (55.14%) tumor samples compared with their non-malignant counterparts by real-time PCR (Fig. 7A). U6 was used as a control. However, no statistically significant difference was observed between the cancer tissues (mean ± SD, 3.6467±7.44944) and matched non-tumor adjacent tissues (NATs) (mean ± SD, 5.3679±15.09357) (P= 0.291) (Fig. 7B).
Downregulation of miR-194 is associated with advanced clinicopathological features of osteosarcoma. The median miR-194 expression level in 107 patients with osteosarcoma was 3.647. Patients were divided into two groups according to their expression levels of miR-194, using its median as a cut-off: high miR-194 expression group (n=41) and low miR-194 expression group (n=66). As shown in Table I, we found statistically significant relationships between miR-194 expression and age (P= 0.0015), clinical stage (P=0.019), distant metastasis (P= 0.0251) and patient mortality (P= 0.0065). No significant difference was observed between the expression of miR-194 and the patient gender (p=0.4038) and tumor size (P= 0.6264).
The median of miR-194 expression levels in all 99 paraformalin-fixed, paraffin-embedded (FFPE) tissues with osteosarcoma was 5.74. The FFPE tissues were also divided: high miR-194 expression group (n=21) and low miR-194 expression group (n=78). As shown in Table II,  To identify whether miR-194 was an independent prognostic covariate for osteosarcoma, we performed a multivariate Cox proportional hazards analysis. In the final multivariate Cox regression model, low levels of miR-194 expression in osteosarcoma (P=0.001, relative risk = 0.390) and distant metastasis (P=0.001, relative risk =2.386) were associated with a poor prognosis in terms of overall survival, independent of other clinical covariates (Table III). Similar results were obtained in FFPE tissues (Table III; P=0.023, relative risk = 0.371).
CDH2 and IGF1R are potential targets of miR-194. We examined the potential targets of miR-194 by searching the PicTar miRanda and TargetScan databases. We identified a conserved domain within the 3'-UTR of CDH2 (N-cadherin) and IGF1R with a potential miR-194 binding site (Fig. 8A). We examined the expression of CDH2 and IGF1R on mRNA and protein levels in OE, blank, control and KD SOSP-9607 cells using real-time PCR and western blot analysis. The results showed that miR-194 had no effect on CDH2 and IGF1R mRNA levels ( Fig. 8C and D). However, the level of endogenous N-cadherin protein in OE SOSP-9607 cells was reduced compared with the other three cells normalized to an endogenous reference β-actin protein (Fig. 8B). Overexpression of N-cadherin protein was also found in KD SOSP-9607 cells compared with the other three groups of cells (Fig. 8B). Western blot analysis demonstrated a significant decrease in endogenous IGF1R levels in OE group compared with the other three groups (Fig. 8B). The results indicate that miR-194 may target CDH2 and IGF1R.
We assessed the interaction of miR-194 with luciferase reporter assay in SOSP-9607 cells using a pMIR-REPORT™ Luciferase vector containing the 3 '-UTR of CDH2 or a control pMIR-REPORT™ Luciferase vector containing the same 3'-UTR with mutated miR-194 seed nucleotides. Renilla luciferase vector was used for normalization. The miR-194-up cells significantly repressed the luciferase activity of the vector with the wild-type CDH2 3'-UTR, whereas mutation of the seed sequence abolished this repression (Fig. 8E and F). Similar results of the IGF1R were also obtained ( Fig. 8G and H).  Table I. Relationship between expression of miR-194, N-cadherin and IGF1R and clinicopathological factors in 107 osteosarcoma patients.
We then examined IGF1R and N-cadherin protein expression in 99 paraffin specimens of osteosarcoma using immunohistochemistry analysis. Representative images of IGF1R and N-cadherin are shown (Fig. 8I) and analyzed (Table IV). Of the 21 osteosarcoma cases with elevated miR-194, 16 (76.2%) showed low levels of N-cadherin. High   (Table I).

Discussion
Osteosarcoma is the most frequent primary solid malignancy of bone, which is defined by the presence of malignant mesenchymal cells which produce osteoid and/or immature bone (40). Approximately 20% of patients present with lung metastases at initial diagnosis and in 40% of patients metastases occur at a later stage. Eighty percent of all metastases arise in the lungs, most commonly in the periphery of the lungs, and exhibit resistance to conventional chemotherapy (41).
Uncontrolled cell proliferation and aggressive tumor cell metastasis are two essential steps during cancer progression. Therefore, in the present study, we investigated the effects of miR-194 on tumor growth and metastasis of osteosarcoma. We showed that overexpressed miR-194 significantly suppressed proliferation, migration and invasion of SOSP-9607 and U2-OS cells in vitro. Our mouse model showed that miR-194 also significantly inhibited orthotopic tumor growth and lung metastasis in vivo. We have performed the largest study to date that assessed the expression levels of miR-194 in osteosarcoma by real-time PCR. However, no significant difference of miR-194 expression was found in 107 cancerous and adjacent non-cancerous tissue pairs. Tissue specificity might be one of the reasons which was associated with the differences in miR-194 expression, as observed in cancer miRNA signatures across different organs (42). Furthermore, different osteosarcoma cell lines show different expression profiles of invasion, motility and colony formation, with different mRNAs and miRNA expression (43). We observed that decreased expression of miR-194 was correlated with cancer progression and poor prognosis in osteosarcoma patients, independent of other clinicopathologic factors. Therefore, upregulated miR-194 was very effective in inhibiting tumor growth and metastasis indicating that miR-194 functions as a tumor suppressor gene and as a potential therapeutic target.
Generally, metastatic models are conducted in nude mice by injecting human osteosarcoma cells either intravenously or subcutaneously (32). However, these models are not clinically relevant since osteosarcoma does not occur spontaneously (44). In the present study, we selected a spontaneous metastatic model involving orthotopic transplantation of osteosarcoma cells resulting in spontaneous pulmonary metastases (32). The microenvironment of tibia in nude mice resembled the tumor progression and metastases development clinically.
Cadherins have a role in Ca 2+ -dependent cell-cell interaction (45) as well as acting as metastasis promoting or suppressing proteins in different cancers (46,47). Insulin and insulin-like growth factor receptor (IGFR)-mediated molecular pathways are important effectors of neoplastic transformation in nonsmall cell lung cancer (48) and squamous cell carcinoma of the head and neck (49). IGFIR has a major role in cancer cell proliferation and survival, and confers resistance to cytotoxic, hormonal and targeted therapies (50). Our results indicate that miR-194 interacted with N-cadherin and IGF-IR and negatively regulated their expression at the translational level, which also indicated that miR-194 may suppress tumor growth and metastasis in osteosarcoma cells by downregulating N-cadherin and IGF-IR.
Unlike siRNAs, which silence the expression of a single gene, miRNAs mainly silence the expression of multiple genes simultaneously. It is estimated that an average miRNA may have more than 100 targets (51). It is crucial to identify Table IV. Inverse correlation of expression of miR-194 and N-cadherin and IGF1R in osteosarcoma (using real-time quantitative PCR) and osteosarcoma FFPE tissues (using immunohistochemistry analysis).

Group
High additional target genes that mediate the miR-194-induced regulation of tumor metastasis. Predicting and identifying the miR-194-targeting genes provides an experimental basis for further research on regulatory mechanism of miR-194. By using TargetScan 5.1 and PicTar, we predicted putative genes of miR-194, and obtained several putative targets correlating with tumor growth or metastasis, such as QKI, KIAA1239, EPHA5, NACC2, mCTS1 and SAMD4A. In general, the discovery of miRNA and their functions, has introduced a new dimension to our existing knowledge of signaling molecules and pathways for more precise therapeutic targeting. Further investigation is required for characterization of miR-194 and other miRNAs as prognostic and/or diagnostic markers in human osteosarcoma.
In conclusion, the results demonstrate that miR-194 affected the growth and metastasis of osteosarcoma cells both in vitro and in vivo. Overexpression of miR-194 downregulated the expression of N-cadherin and IGF-IR protein, suggesting that miR-194 functions as tumor suppressor probably by downregulating N-cadherin and IGF-IR in osteosarcoma. Downregulation of miR-194 may be associated with tumor aggressiveness and tumor metastasis of osteosarcoma, suggesting that miR-194 may be an independent prognostic marker for osteosarcoma. Other putative miR-194 target genes that are potentially associated with the growth and metastasis of osteosarcoma cells should be investigated. Finally, miR-194 may prove to be a promising gene therapeutic agent. It could be informative to confirm the putative target genes and further investigate the underlying molecular mechanisms of miR-194 as a tumor suppressor gene in osteosarcoma.