| Background:Osteosarcoma(OS) is the most common bone tumor, accounting for about 15% of all primary malignant bone tumors. The incidence of osteosarcoma rank second following multiple myeloma in primary malignant bone cancers.Occurence of osteosarcoma is 3 cases/million/year, accounting for about 0.2% of all malignant tumors.75% ofosteosarcoma patients are between 15 and 25, which is of higher incidence in men than women with an approximate proportion of 1.5:1.Osteosarcoma is rarely occurred in patients less than 6 years old or over 60.In general, about 80% to 90% of osteosarcoma occurs in long tube bones.85% of limb cancers appear in femoral, tibial,humerus with less than 1% in the hand and foot bones. Osteosarcoma of long bone usually originates from metaphysis.Although the occurence and development of osteosarcoma can be partially explained by the theory of osteosarcoma originating from virus attacking and the theory of radiation.However,the pathogenesis and development mechanism of osteosarcoma is still unclear. In the recent decades,with the further research on osteosarcoma and the development of new drugs, new methods of radiotherapy, chemotherapy and the new operations appear constantly, the treatment concepts of osteosarcoma already cover multiple subjects. Recent studies have found many key genes which are related to osteosarcoma, including P53, FAS, NOTCH and so on, On the basis of these tumor signaling pathways, gene targeting therapy provides new strategies and approaches for the prevention and treatment of osteosarcoma.In the field of diagnosis and treatment of osteosarcoma, despite the new achievements occur every year, but the 5-year survival rate which is used to evaluate the effect of treatment in patients with osteosarcoma is still only 60-70%. So,to find a new marker which can predict the occurrence, development, metastasis and chemotherapy reactivity of osteosarcoma, and can effectively control the proliferation, apoptosis, migration and invasion of osteosarcoma cell, is very significant for understanding the pathogenesis of osteosarcoma, and finding the effective prevention and cure targets.Visfatin was identified as Pre B cell Colony Enhancing Factor (PBEF) or Nicotinamide phosphoribosyl transferase(Nampt) which is secreted by human peripheral blood lymphocytes and participates in the metabolism of nicotinamide adenine dinucleotide(NAD+). Three different biological names and three different functions (the amino acid sequence of three protein is the same) make the protein very special, and the name can be used interchangeably. The molecular weight of Visfatin is 52 kda and active when dimerization with each monomer containing 491 amino acids. In human,it is confirmed that the genelocatingin the long arm of chromosome 7 between 7q22.1 and 7q31.33. Visfatin gene is highly conserved in evolution. Visfatin is mainly expressed in bone marrow, liver and muscle, following by brain, kidney, spleen, assayis and lung. It is more invisceral fat than the subcutaneous fat. Visfatin is considered to be a new pro-inflammatory adipocytokine. It could dose-dependently increase the expression of various cytokines in Mononuclear macrophages, fat cells and tumor cells, such as IL-1□〠IL-1Raã€IL-6ã€IL-10 and TNF-a. These cytokines play a substantial role in a wide range of infectious diseases and tumors. Recent studies demonstrated that the change of intracellular level of NAD+(increase),niacinamide (reduce) and deacetylase sirtuin protein family is important for the survival of certain cells. By the activation of SIRT1, Visfatin can prolong life and promote the maturity of human cells, therefore, can indirectly increasetumor cell life. However, whether Visfatin can play a role in the process of proliferation, migration, and invasion of osteosarcoma cells has not been reported.EMT(Epithelial-mesenchymal transition),as a conversion from human epithelial cells to mesenchymal cells, is a special biological processes. It plays an important role in multiple physiological and pathologica progress involving embryonic development, wound healing, and malignant tumor. In the process of Epithelial-mesenchymal transition, polarized epithelium phenotype cells with various physiological and pathological stimuli, gradually transform into mesenchyme phenotype cells owing high movement characteristics. In this process, the expression of epithelial cells’ phenotypic markers(such as E-cadherin) is gradually reducing,but,the the expression of ectomesenchymal cells’ phenotypic markers(such as the N-cadherin) is gradually increasing. Previously, a number of studies in vitro and in vivo showed that EMT (Epithelial-mesenchymal transition) played an important role during the process of proliferation, migration, and invasion process of malignant tumors.In the process of tumor invasion and metastasis, tumor cells hide their differentiated epithelial characteristics including intercellular adhesion and polarity, and thus gain mesenchymal characteristics such as motility, invasive and more important stem cell properties.The process of EMT(Epithelial-mesenchymal transition)is reversible so as to differentiate into epithelial phenotype via mesenchymal epithelialtransition.Abnormal environment conditions such as hypoxia, low pH or malnutrition can cause a series of reactions within the tumor cells, including metabolic adaptation, epigenetic change and differentiation related to EMT.This will eventually leads to metastasis to distant tissues and organs, which can provide necessary nutrition support to maintain rapid growth. The above studies of epithelial mesenchymal transition can provide a method for cancer prevention and treatment. However, its role in the occurrence and development process of osteosarcoma is still under initial research.To sum up, invasion and migration is an important part of the progress of osteosarcoma, is an important factor leading to poor prognosis of osteosarcoma, therefore the research on invasion and migration mechanism is of great significance to the prevention and treatment of osteosarcoma.A wide variety of cancers are accompanied with the rise of Visfatin expression level.So far, no direct evidence demonstrates that Visfatin is involved in migration and invasion of osteosarcoma cells. Therefore, we aimed to assess whether Visfatin affacts migration and invasion of osteosarcoma cells,and to find out the mechanisms.Objective:1. To investigate the effects of Visfatin on migration and invasion in U2OS cells;2. To investigate the effects of Visfatin on epithelial-mesenchymal transition;3. To investigate the specific effect of Visfatin on osteosarcoma and its related signaling pathways.Methods:1. Cell cultureDulbecco’s modified Eagles medium supplemented with 10% fetal bovine serum was used to culture the U2OS cells. The U2OS cells were grown in a 37℃ incubator with 5% CO2. To determine theeffect of Visfatin on EMT markers expression, U2OS cellswere maintained in DMEMsupplemented with 1% FBS for 12h and subsequently culturedin the absence or presence of Visfatin or other reagents for specified time.2. Migration AssayCell migration was studied usingscratch wound healing assay.U2OS cells were cultured in six-well plates and grown in a confluent monolayer. Straight scratches of the same width were made in the monolayer of U2OS cells with a pipette tip. After incubation with the reagents for specified time, photo images were taken to measure the wound healing under a microscope.3. Invasion assayCell invasion was studied using matrigel-coated Transwell assay. Modified Boyden chambers with 8-μm pore filter inserts were coated with matrigel (50μg/well). U2OS cells were cultured in 24-well plates and the upper chamber contained cells in DMEM plus 1% FBS, while the lower chamber contained DMEM plus 10% FBS. Cells were re-suspended in the upper chamber at 37℃ and 5% CO2. After 24h-incubation, cells that had invaded onto the lowersurface of the matrigel-coated membrane were fixed with methanol for 30 min and stained with hexamethylpararosaniline, while the cells remained on the upper surface were wiped away.4. Western blot analysisAfter stimulation with Visfatin and other reagents, U2OS cells were collected and the protein wasextractedin ice-cold RIPA buffer with 1mM phenylmethyl-sulfonyl fluoride for 30min. Concentration of the extracted protein was measured with BCA protein assay kit. Cell lysates were electrophoresed on a 10% SDS polyacrylamide gel and transferredonto nitrocellulose (NC) membranes. Blots were blocked and incubated withprimary antibodies overnight and followed by incubation with secondaryantibodies for 2h at room temperature. At last, the blots were visualized with electrochemiluminescence (ECL) detection system.5. Real-time quantitative PCR (qPCR) analysisTotal RNA was isolated and extracted using Trizolreagent according to the manufacturer’s directions from the cells which were cultured followed the specified experimental programs. The concentration of total RNA was quantified by spectrophotometry. RNA samples were reverse-transcribed into cDNA using M-MLV Reverse Transcriptase System. Total cDNA was amplified and detected using Light-Cycler-FastStart DNA Master SYBR Green â… .18s was chosen as the reference gene and the primer sequences for real-time PCR analyses were as follows:E-cadherin forward primer:5’-ACCAGAATAAAGACCAAGTGACCA-3’, and reverse primer, 5’-AGCAAGAGCAGCAGAATCAGAAT-3’; N-cadherin forward primer, 5’-CACTGCTCAGGACCCAGAT-3’, and reverse primer, 5’-TAAGCCGAGTGATGGTCC-3’; Snail forward primer,5’-TTGGATACAGCTGCTT TGAG-3’, and reverse primer,5’-ATTGCATAGTTAGTCACACCTC-3’; 18S forward primer:5’-CTTAGTTGGTGGAGCGATTTG-3’,reverse primer: 5’-GCTGAACGCCACTTGTCC-3’.6. Statistical analysisEach data in the study was evaluated with SPSS 18.0. The normally distributed data, which also owned the characteristicof homogeneity of variance,were analyzed by Student T Assay or One-way ANOVA, the others were analyzed byWilcoxon Assay or multiple independent samples Kruscal-Wallis Rank-sum Assay. Statistical significance was confirmed as P< 0.05.Results:1. The effects of Visfatin on the migration of osteosarcoma cellsThe cultured Osteosarcoma cells were randomly divided into Control group and Visfatin group. Respectively the two groups of cells were performed wound healing assay (scratching assay). The cells of Visfatin group were stimulated by 500 ng/ml restructured Visfatin, while the cells of Control group were given normal saline with the same volume as Visfatin. After 24 hours, the picture of scratch parts was taken and analysed. Respectively, the percentages of cell relative migration distance of Control group and Visfatin group were (100±7)%, (255.67±43.50)%, there was a significant difference (t=6.12, P<0.01)2. The effects of Visfatin on the invasion of osteosarcoma cellsThe cultured Osteosarcoma cells were also randomly divided into Control group and Visfatin group. Respectively the two groups of cells were taken Transwellassay. The cells of Visfatin group were stimulated by 500 ng/ml restructured Visfatin, while the cells of Control group were given normal saline with the same volume as Visfatin. After 12 hours and 24 hours’ stimulation, the obtained cells were dyed by gentian violet and were counted under a microscope, then the results were analysed. After 12 hours, respectively, the percentages of relative invasive cell number of Control group and the Visfatin group were (100±7)%, (121.33±16.56)%,there was no significant difference between them (t=2.01, P=0.11). After 24 hours, respectively, the percentages of relative invasive cell number of Control group and the Visfatin group were (100±7)%, (214±24.27)%, there was a significant difference between them (t=7.82, P<0.01)3. The effects of Visfatin on EMT markers expression in osteosarcoma cellsWe thus examined the effects of Visfatin on EMT markers and its mRNA expression in osteosarcoma cells with Western blot and real-time PCR. The osteosarcoma cells were respectively stimulated by various concentrations of Visfatin (0,5,50and 500ng/ml) for 24h,then we got the multiples of E-cadherin, N-cadherin, E-cadherin mRNA and N-cadherin mRNA respectively were0.99±0.11,0.88±0.09,0.58±0.10,0.38±0.08; 0.99±0.07,1.51±0.13,2.60±0.29,3.28±0.59;0.99±0.11,0.82±0.07,0.48±0.10,0.39±0.09; 0.99±0.07,1.48±0.12,4.20±0.64,5.45±0.90± By statistics analysis, we found that, with the increase of Visfatin’s concentration, the expression of E-cadherin gradually reduced, there was significant difference respectively (F=27.07, P<0.01), the expression of N-cadherin gradually increased, there was significant difference respectively (x2=20.57, P<0.01), the expression of E-cadherin mRNA gradually reduced, there was significant difference respectively (F=27.51, P<0.01), the expression of N-cadherin mRNA gradually increased, there was significant difference respectively (X2=20.75, P<0.01)Subsequently, Visfatin of 500ng/ml was added into the osteosarcoma cells and cultured for various times (6,12,24,48h), then we got the multiples of E-cadherin, N-cadherin respectively were 0.99±0.11,0.95±0.06,0.72±0.07,0.51±0.05,0.38±0.08; 0.99±0.07,1.05±0.09,1.80±0.12,3.01±0.31,3.5±0.43.Then, Visfatin of 500ng/ml was added into the osteosarcoma cells and cultured for various times (3,6,12,18,24h), then we got the multiples of E-cadherin mRNA and N-cadherin mRNA respectively were 0.99±0.11,0.95±0.06,0.75±0.13,0.52±0.07, 0.33±0.06,0.31±0.09;0.99±0.11,1.03±0.09,2.38±0.38,4.62±0.64,6.94±0.43,6.72±0.47. By statistics analysis, we found that, with the increase of culture time, the expression of E-cadherin gradually reduced, there was significant difference respectively (F=38.17, P<0.01), the expression of N-cadherin gradually increased, there was significant difference respectively (F=126.53, P<0.01), the expression of E-cadherin mRNA gradually reduced, there was significant difference respectively (F=35.37, P<0.01), the expression of N-cadherin mRNA gradually increased, there was significant difference respectively (F=134.35, P<0.01)4. The effects of Visfatin on Snail expression and effects of Snail siRNA on EMT marker expressionVisfatin of 500ng/ml was selected to stimulate osteosarcoma cells for various times (3,6, 9 and 12h). Then we got the multiples of Snail, and Snail mRNA respectively were 0.99±0.11,1.02±0.16,2.25±0.44,2.36±0.46,2.38±0.58;0.99±0.11,1.16±0.19,4.18±0.67, 4.06±0.60,4.37±0.82. By statistics analysis, we found that, with the increase of culture time, the expression of snail significantly increased, here was significant difference respectively (F=10.32, P<0.01), the expression of Snail mRNAalso significantly increased, here was significant difference respectively (F=29.06, P<0.01)After that, in order to ascertain the irreplaceable position of Snail in promoting EMT, osteosarcoma cells were randomly divided into three groups, Control group, Visfatin group, Visfatin+Snail siRNA group. After 24h, we got the multiples of E-cadherin and N-cadherin respectively werel±0.08,0.37±0.09,0.69±0.08; 1±0.07,2.74±0.16, 1.64±0.15. The expression of E-cadherin of the three groups were significantly different (F=44.33, P<0.01),so were the expression of N-cadherin (F=134.54, P<0.01)5. The effects of Visfatin on the nuclear translocation of NF-κBThe osteosarcoma cells were stimulated by 500ng/ml of Visfatin. After the predetermined culture times (3,6,9,12h), the expressions of p65 peptide of NF-κB in nulcei of osteosarcoma cells were:0.99±0.11,1.87±0.29,2.25±0.57,5.36±0.06, 5.52±0.64, respectively. The expressions of p65 in cytoplasm were 0.99±0.11,0.67±0.09, 0.45±0.09,0.40±0.06,0.30±0.11, respectively. With time prolonging, Visfatin could statistically increase the expression ofp65 in nuclei (F=55.02, P<0.01) and simultaneously decrease its expression in the cytoplasm (F=26.79, P<0.01)6. The effects of Bay11-7082 on the exprssions of EMT markers and SnailIn order to further determine the molecular mechanisms underlying Visfatin-induced promotion and suppression of EMT markers, we chose 500ng/ml of Visfatin to stimulate the osteosarcoma cells for 24h and BAY 11-7082 (20μM) to pre-treat the cells for 2h. The osteosarcoma cells were randomly divided into three groups:control, Visfatin and Visfatin+Bay11-7082 group. After 24h of culture, the expressions of E-cadherin protein were 1.00±0.07,0.27±0.08 and 0.72±0.17 in control, Visfatin and Visfatin+Bayl 1-7082 group, respectively. There were significant differences among the three groups(F=29.70, P<0.01). The expressions of N-cadherin were 0.99±0.05,4.45±0.48 and 2.36±0.34, respectively, which were statistically different (F=157.14, P<0.01). Subsequently, the expression of Snail was examined after the same treatment. The expressions of Snail were 0.99±0.05,5.09±0.85 and 2.22±0.41, respectively. There were also significant differences among three groups (X2= 15.16, P<0.01)7. The effects of Visfatin/Bayll-7082/Snail siRNA on migration and invasion of osteosarcoma cellsWound healing assay and Transwell assay were respectively performed to evaluate the migration and invasion after osteosarcoma cells were treated with various concentrations of Visfatin (0,5,50and 500ng/ml) for 24h. After stimulation, the percentages of relative migration distance of osteosarcoma cells were (100±7)%, (103.67±7.57)%, (176±22.11)% and (255.67±43.50)%with various concentrations of Visfatin, which were increased significantly in a concentration-dependent manner (F=26.01, P<0.01). The percentages of relative invasive cell number were (100.50±4.63)%, (103.67±5.05)%, (167.17±5.85)% and (213.83±15.51)%, respectivly. The invading cells transferred from the upper surfaces to the lower surfaces were significantly promoted by Visfatin in the similar tendency (F=221.61, P<0.01)In order to further ascertain the signal pathway underlying the effects of Visfatin on osteosarcoma cells migration and invasion, Snail siRNA and BAY 11-7082 (20μM) were repectively used to pre-treat the cells for 2h. The osteosarcoma cells were randomly divided into four groups:control, Visfatin, Visfatin+Bay11-7082 and Visfatin+Snail group. Wound healing assay and Transwell assay were also performed in the four groups. The percentages of cell relative migraiton distance were(100±7)%, (263.67±36.69)%, (152.67±10.02)% and (146.67±16.80)%. The percentages of migration was highest in the Visfatin groups and lowest in the control group, there was significant difference respectively (F=32.50, p<0.01). The results were similar between Visfatin+Bayl 1-7082 and Visfatin+Snail groups. The percentages of relative invasive cell number were(100.50±4.64)%,(206.00±15.63)%,(136.33±10.19)% and (127.67±11.20)%, respectivly.The tendency similar with migration assay was found in the Transwell assay (F=97.80, P<0.01)Conclusion:1. Visfatin could promote the migration and invasion of U2OS cell in concentration-dependent and time-dependent manner;2. Visfatin could change the expressions of EMT markers in concentration-dependent and time-dependent manner;3. Visfatin promotes the migration and invasion of osteosarcoma via NF-κB/Snail/EMT pathway. |
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