Investigation Of Fatty Acid Synthase (FASN) Promote Migration And Invasion Of Osteosarcoma Cell And Its Molecular Mechanisms

Background and ObjectiveOsteosarcoma is highly invasive and metastatic which counts for35%in all malignant bone tumors, especially with a high-occurrence in children and teenages. In the whole world, the five-year survival rate in patients with osteosarcoma remains stagnant, though the diagnosis and treatment of osteosarcoma develop quickly. Pulmonary metastasis is mainly responsible for death of patients with osteosarcoma. By transferring to lung through blood and forming cell cloning in early stage, pulmonary micrometastasis of osteosarcoma occurs in almost80%patients with osteosarcoma when they got their definite diagnosis. Now molecular mechanism of osteosarcoma metastasis has brought into focus which may be crucial to the diagnosis, treatment, prevention and the increasing survival rate in patients with osteosarcoma.Now it has been confirmed that matrix metal loproteinases （MMPs） are obbligato in degradation of basement membrane and epimatrix, among which MMP-2and MMP-9are mostly correlated with tumor invasion and metastasis. MMP-2and MMP-9overexpress in osteosarcoma and help osteosarcoma cells transfer to other tissues including lung by degrading some components of basement membrane and epimatrix. At the same time, MMP-2and MMP-9promote the growth of osteosarcoma and the forming of metastatic foci by inducing the neogenesis of blood capillary. Various studies indicate that activation and transposition of NF-κB gene, an upstream gene of MMPs, are closely related with tumor invasion and metastasis promoted by MMPs. And, it is confirmed that Akt is an important regulative factor in gene transcription which NF-κB depend on and essential for IκB degradation and NF-κB activation mediated by IKKs.PI3K/Akt signaling pathway is on of main intracellular signaling pathway by HER2-mediated. HER2is a receptor tyrosine kinase and closely related with osteosarcoma invasion and metastasis, which can activate itself by self-phosphorylation without extracellular ligand and promote tumor invasion and metastasis by activating PI3K/Akt pathway and NF-κB transcription pathway.In recent years it has been confirmed that fatty acid metabolic pathway plays an important role in metastasis of malignant cells. FASN, an oncogene playing a role in fatty acid metabolic pathway, overexpress in many kinds of malignant tumor cells without regulation of normal cell signal and spontaneously synthesize plenty of lipid to construct the biomembrane in increasing tumor cells. Inhibition of FASN can lead to apoptosis and inhibition of proliferation in tumor cells. Now FASN caused high attention on its role in tumor invasion and metastasis. The expression of FASN in head and neck squamous-cell carcinoma is positively correlated with pulmonary metastasis of tumor cells, which makes the high expression of FASN is an important trait of pulmonary metastasis in head and neck squamous-cell carcinoma. Recently, some studies indicated that inhibition of FASN caused inhibition of tumor cells invasion and metastasis, but the definite molecular mechanism has not been reported. By now, whether FASN is overexpressed in osteosarcoma and correlated with its metastasis and what is the molecular mechanism have not been reported. It was found recently that overexpression of FASN in human breast epithelial cells led to mass synthesis of intracellular endogenous fatty acid, which then caused dimerization of HER2as internal stimulative signal. HER2was activated by its dimerization and self-phosphorylation and then activated its downstream PI3K/Akt and NF-κB pathway. Therefore, we speculate that FASN can promote osteosarcoma invasion and metastasis by activating HER2/PI3K/AKT/NF-κB signaling pathway. To test our assumption, miRNA expression vector targeting FASN gene was constructed using pcDNA6.2-GW/EmGFP-miR and then transfect osteosarcoma cells to inhibit FASN expression. Wound healing and Transwell invasion experiments were performed to examine changes of osteosarcoma cells invasion and migration. RT-PCR and Western blot were performed to examine expression changes of some factors in HER2/PI3K/AKT/NF-κB signaling pathway.Objective1. FASN protein expression was detected using immunohistochemistry in Osteosarcoma tissue paraffin specimen. The relationship between FASN expression and clinical pathological parameters, which can be as the histological evidences for FASN involving osteosarcoma metastsis and the underlying mechanism, was determined.2. To investigate whether inhibition of FASN causes the inhibition of osteosarcoma cell migration and invasion in vitro, miRNA expression vector targeting FASN gene was constructed and transfected into U2-OS cells to examine changes of cell migration and invatsion. The positive experimental result can inversely indicate that FASN overexpression promote metastasis and invasion of osteosarcoma cells.3. To investigate whether inhibition of FASN causes inhibition of HER2/PI3K/AKT/NF-κB signaling pathway, expression of some proteins will be detected in U2-OS cells transfected by miRNA expression vector targeting FASN gene. The positive experimental results can inversely indicate that FASN overexpression promotes migration and invasion of osteosarcoma cells by activating HER2/PI3K/AKT/NF-κB signaling pathway.Materials and MethodsPart Ⅰ. The expression of FASN protein in osteosarcoma tissues and its clinical significance136osteosarcoma paraffin specimens from patients diagnosed by clinical, iconographical and histopathological method and21osteochondroma paraffin specimens were used for immunohistochemistry analyse. The average age of osteosarcoma patients including84males and52females was19.4years old with a range from7to74years old. There were44patients with pulmonary metastasis among136osteosarcoma patients. All patients received X-ray and CT scanning in tumor tissue, chest radiograph, and systemic bone scanning. The pulmonary metastasis survey was performed with plain films and chest computed tomographic （CT） scans at first diagnosis. All the patients had not a history of prior therapies with anticancer drugs or radiotherapy. FASN expression level in all136osteosarcoma patients including78osteoblastic sarcoma patients,34fibroblastic osteosarcoma, and24chondroblastic osteosarcoma was determined by immunohistochemistry analyse, based on which the relationship between FASN expression and clinical pathological parameters was determined.Part Ⅱ The effect of silence FASN gene by RNA interfering on U2-OS cell invasion and migration 1. Contruction of recombinant plasmid containing miRNA targeting FASN gene.The human cDNA sequence encoding FASN protein （NM₀04104.4） was obtained from Genbank, based on which4pairs of miRNA oligo and a control single-strain DNA oligo were designed and synthesized. After annealing into4double-strain miRNA oligo, recombinant plasmid was constructed using BLOCK-iTTM Pol II miR RNAi Expression Vector Kit with EmGFP by inserting the4double-strain miRNA oligo into pcDNATM6.2-GW/EmGFPmiR, the miRNA expression vector（kown as:X197-1-1、X197-2-1、X197-3-2、X197-4-1）. Competent DH5a cells were then transformed by the4miRNA recombinant plasmids targeting FASN gene and cultured in plates.4clones were respectively chosen from each plate and the sequence of recombinant plasmid was determined. Cultured U2-OS cells were then transfected with the recombinant plasmids containing insertion sequences. The transtected efficiency of all the4recombinant plasmids was measured, and the recombinant plasmid, which with the best transfecting ratio, was determined for the further more experiment. Also, the optimized transfecting condition was determined.2. The effect of inhibition FASN on U2-OS cell migration and invasion in vitroThe recombinant plasmid, which was determined in previou experiment, was transfected into U2-OS cells in the optimized conditions （plasmid:Lipofectamine2000=1:3）. Wound healing and Transwell invasion were performed to measure migration and invasion cells treated with the recombinant plasmid, and compare with the migration and invasion of cells transfected with the empty vector or untransfected cells.Part Ⅲ The effect of Silencing of FASN on HER2/PI3K/AKT/NF-κB signaling pathway.The recombinant plasmid, which was determined in previou experiment, was transfected into U2-OS cells in the optimized conditions （plasmid:Lipofectamine2000=1:3）. The expression level of FASN mRNA was detected by RT-PCR, and western blot were performed to detect protein expression change of some factors in HER2/PI3K/AKT/NF-κB signaling pathway in cells, and compare with the migration and invasion of cells transfected with the empty vector or untransfected cells.Statistical analysisAll the data was analyzed using spss13.0software. The count data was analyzed using non-parametric Wilcoxon rank sum test analysis. All Measurement data were expressed as X±S.D. Statistical significance for the difference between the groups was assessed using one-way ANOVA test. Values of P<0.05were considered to indicate a significant difference.ResultsPart I. Expression of FASN protein in osteosarcoma and its clinical significance1. Differences of FASN expression in osteosarcoma and osteochondroma.The FASN protein expression level was significant higher in osteosarcoma patients than in osteochondroma （P=0.001）. It showed that FASN protein overexpression in osteosarcoma and may be a molecular target for treating osteosarcoma.2. The relationship between of FASN expression and pulmonary metastasisThe FASN protein expression level was significant higher in osteosarcoma patients with pulmonary metastasis than those patients without pulmonary metastasis （P=0.002）. The results indicated that FASN may be involved in pulmonary metastasis of osteosarcoma.3. There is no significant correlation between FASN expression and age, gender, pathological type and Ennecking staging. However, FASN expression in osteosarcoma tissues with the diameter larger than8cm is significantly higher than in those with the diameter less than8cm, which indicates that FASN may be involved in osteosarcoma cell proliferation.Part II. Silence of FASN gene by RNA interfering inhibit U2-OS cell migration and invasion in vitro.1The construction and identification of recombinant plasmids containing miRNA targeting FANS gene.（1） Sequencing testified that the double-strain miRNA oligo was exactly inserted into expression vectors without point mutation, indicating that miRNA recombinant plasmids targeting FASN gene were constructed successfully.（2）4recombinant plasmids can effectively transfect osteosarcoma cells in24hours, among which X197-1-1has the higest transfecting ratio.（3） The U2-OS cell was treated with difference ratio of plasmid to Lipofectamine2000for24hours, respectively. The results showed that there was the highest transfecting ratio in cell treated with plasmid in ratio of plasmid to Lipofectamine2000is1to3.0.（4） FASN mRNA level and FASN protein level in cells transfected by X197-1-1recombinant plasmid were significantly lower than in cells transfected by negative plasmid or untransfected, which indicated that miRNA recombinant plasmid targeting FASN gene can inhibit FASN gene expression effectively.2Silence of FASN gene inhibit U2-OS cell migration and invasion in vitro.（1）. The migration rate of U2-OS cells transfected by X197-1-1recombinant plasmid was （10±4）%and significantly lower than that in both negative control group and blank control group which was （84±5.4）%and （87±8.5）%respectively. The results showed that inhibition of FASN gene can cause inhibition of U2-OS migration in vitro.（2）. The number of transmembrane cells in U2-OS cell transfected with X197-1-1recombinant plasmid targeting FASN gene was22.2±5.6（400x） and significantly less than that in both negative control group and blank control group which was91±11.3and93.7±10.3respectively. The results indicated that inhibition of FASN gene could inhibit U2-OS cell invasion in vitro.Part III. Silence of FASN inhibit HER2/PI3K/AKT/NF-κB signaling pathway1. The expression level of pHER2protein in osteosarcoma cells transfected by X197-1-1recombinant plasmid containing miRNA targeting FASN gene for24hours was decreased significantly when compared with that in cells untransfected or transfected with empty plasmid. The data indicated that inhibition of FASN gene could decrease phosphorylation of HER2in U2-OS cell.2. Expression level of pAKT, NF-KBp65, MMP2and MMP9was decreased significantly in cells transfected with X197-1-1recombinant plasmid containing miRNA targeting FASN gene for24hours when compared with that in cells untransfected or transfected empty plasmid. The results revealed that inhibition of FASN gene could decrease phosphorylation of AKT, and further more inhibit nuclear transfer of NF-κB and the expression of MMP-2, MMP-9protein in U2-OS cell.Conclusions1. FASN protein overexpresses in osteosarcoma tissues, and its expression level is closely correlated with osteosarcoma pulmonary metastasis.2. miRNA recombinant plasmid targeting FASN gene was constructed successfully in the study.3. Silence of FASN gene can cause inhibition of U2-OS migration and invasion in vitro. 4. Silence of FASN gene could down-regulate the phosphorylation of HER2and AKT, and further inhibit nuclear transfer of NF-κB and protein expression of MMP2and MMP9. All these cause inhibition of U2-OS migration and invasion.