Effects And Mechanisms Of Serum Amyloid A Influences On Osteosarcoma

Background:Osteosarcoma (OS) is the most common primary malignant tumor arising from bone in children and young adults. Osteosarcoma arises predominantly in the long bones and is prone to distant metastasis. Metastatic osteosarcoma is tolerated to conventional chemotherapy and over30%of metastatic osteosarcoma are irresponsive to chemotherapy. Current treatments include neoadjuvant chemotherapy, surgical removal and postoperative chemotherapy. Chronic inflammation is intensively associated with oncogenesis and even the relapse of tumor. Chronic inflammation induced by infection or injuries could lead to malignant of normal cells causing DNA damage, oncogene activation or anti-oncogene inactivation, and thus promote tumorigenesis. Therefore, inflammatory cytokines and inflammatory signaling pathway are closely related to pathogenesis of osteosarcoma.The occurrence of osteosarcoma metastasis was started with tumor cells detaching from primary lesion and then circulated to targets all over the body. Adhesion between cells and the interactions between cells and matrix further influence the formation of distant metastatic lesions. Thus, cellular adhesion molecules play a significant role in osteosarcoma metastasis. Cellular adhesion molecules contain5families of calcium, integrin, selectin, CD44and immunoglobulin superfamily. Angiogenesis in lesions is another vital process of osteosarcoma metastasis. The growth of tumor will be less than1-2mm without angiogenesis. Control of tumor angiogenesis has become a vital approach in control of tumor metastasis.Human serum amyloid protein A (SAA) gene family consists of four discrete loci, including two highly homologous genes SAA1,SAA2and two discrete gene SAA3,SAA4. There is approximately95%of overlapping sequence between SAA1and SAA2in promoter regions exons and introns, and the non-allelic gene loci encodes non-glycosylated SAA protein which is the main protein expressed in the acute phase of the cycle. SAA3gene is considered to be an non-expressed gene, but recent studies have showed that it could be expressed in mammary epithelial cells. SAA4is a constitutive gene, whose product is a a kind of high-density lipoprotein cholesterol in normal non-acute phase.SAA1,SAA2and SAA4are induced to express in smooth muscle cells,human monocyte/macrophage cell line, and also in some tumor cell lines.4SAA genes in mice were gathered on chromosome7and expressed in liver, while SAA3gene only expressed in extra-hepatic tissues in mice.Like other acute phase proteins, SAA is mainly expressed in liver, and therefore, recently many animal and cell experiments have study the expression and regulation of SAA in liver cells.In addition, some researches showed that SAA could be expressed in endothelial cells, smooth muscle cells and monocyte/macrophage cell line in human atherosclerotic lesions.Recently, there is an evidence showed that SAA could be expressed in normal tissue (especially in epithelial and endothelial tissue), lymph nodes, tumor tissue, Alzheimer’s disease and rheumatoid arthritis synovial tissue. SAA expressed by these extra-hepatic tissues is of significant importance, as these SAA protein is not found in acute phase of systematic reaction which may be associated with the expression of related sites. The regulation of SAA gene expression is a complex process, with a variety of signals inside and outside cells involving in it. SAA gene expression is regulated mainly in the transcription level. Turpentine, endotoxin lipopolysaccharide, a variety of proinflammatory cytokines including interleukin1(IL-1) and interleukin6(IL-6), tumor necrosis factor-a, synthetic glucocorticoid dexamethasone, phorbol esters and slightly modified low density lipoprotein cholesterol affect the regulation of SAA expression alone or together. Receptors of IL-1, TNF-a and IL-6is converted to induce the expression of a series of transcription factors ncluding SAA activation sequence binding factor, cytosine-cytosine-adenine-adenine-thymine (CCAAT) enhancer binding protein, nuclear factor kappa B and SP1.In addition, the expression of SAA may also be affected in post-transcriptional level. Studies have shown that sphingomyelin ceramide pathway and macrophage specific cell surface receptor CD36also regulates the post-transcriptional expression of SAA through above transcription factors. By promoting IL-1b,IL-1receptor antagonist, IL-8, IL-10, IL-12, IL-23and TNF-α secreted by granulocyte lymphocyte and monocyte/macrophage, SAA cytokines can show cytokine-like properties. The above findings suggest that SAA is complex and involves multiple molecular mechanisms of gene expression, and the serum expression level is related to its biological function.Serum amyloid A (SAA), as the precursor protein of Inflammation related amyloidosis, is an acute phase reactant whose serum level will rise rapidly more than1000times under different injuries such as trauma, inflammation, and neoplasm. SAA is mainly secreted by the liver and is a kind of apolipoprotein related to high-density lipoprotein (HDL). SAA can be expressed in the normal tissue,the atherosclerotic process, Alzheimer’s disease tissue, inflammation tissue and tumor tissue. SAA plays an important role in different physiological and pathological processes such as inflammation, atherosclerosis embolism amyloidosis,Rheumatoid arthritis, and neoplasia.Serum SAA level in health people is low, but under subclinical infection, inflammation or even malnutrition state, the level will fluctuate over time, but it will not increase with age. People of80years old or older had elevated levels of serum SAA which is associated with inflammation and some tumor disease rather than age itself. The difference between metastatic and localized disease mainly depends on the tumor type and histological classification, such as squamous, adenocarcinoma, lymphoma and acute leukemia. Elevated SAA levels can be seen in lung cancer (squamous-cell carcinoma, adenocarcinoma,oat cell carcinoma and anaplastic large cell carcinoma), prostate cancer, colorectal cancer etc.. This shows that SAA can be used as a nonspecific tumor marker and independent diagnostic factors for univariate and multivariate analysis. Researchers believe that SAA can be used as an indicator of renal cell carcinoma distant metastases, rather than the early tumor markers. Rosenthal and Sullivan even suggested it as a marker of disseminated and local or regional biochemical diseases. Biran and his coworkers found that the concentration of SAA is associated with tumor activity, tumor staging and prognosis. Initial SAA concentration is of certain prediction value:the prognosis of patients with less than10mg/ml is better, while high concentrations predict poorer prognosis. SAA serum concentration is more than97mg/m with4times of increased risk of death in gastric cancer patients. Another study involving in different tumor patients showed that SAA rises higher in critically patients, while it is not obvious in patients with breast cancer (phaseⅠ-Ⅲ) in acute phase response. SAA level will increase significantly after receiving recombinant interleukin6(rIL-6) anti-tumor treatment in patients with tumors. The levels of SAA can appear significantly higher in the first week after application of rIL-6in dose-dependent way and reached its peak in the third day. However, during the immunotherapy in the following weeks, SAA levels slowly decline. Application of rIL-3in phase Ⅲ/Ⅳ ovarian cancer patients results in slightly elevated levels of SAA. In colorectal cancer patients with liver metastasis, partial liver resection can result in an increased level of serum SAA, which perhaps is the result of liver proteome change. Patients after laparoscopic assisted distal gastrectomy and open distal gastrectomy can show elevated SAA levels, but patients in laparoscopic assisted distal gastrectomy group is of less SAA levels increase. In patients with gastric cancer, the average concentration of SAA is also high, and the concentration have a certain correlation with tumor stage and positioning, therefore SAA is not only an effective index for evaluating the prognosis of patients with gastric cancer, but also a valuable means of postoperative follow-up. Serum SAA level of multiple myeloma patients is also higher than that of normal, however, it is uncertain for specific marker in tumor patients with febrile neutropenia. SAA can reach more than30times in renal cell carcinoma with the application of microarray gene expression analysis. In antibody microarray analysis in patients with lung cancer, we found that SAA levels rise twice than normal.Serum amyloid A, as a precursor protein associated with inflammation related amyloidosis, is an acute-phase product and could increase1000-fold when stimulated with truma, infection, inflammation and neoplasm. SAA which is mainly secreted by liver is an apolipoprotein associated with high density lipoprotein. SAA could be expressed in normal, atherosclerotic, Alzheimer’s disease, inflammatory and tumor tissues. Besides, SAA play a significant role in different physiologic and pathological process such as inflammation, atherosclerosis, thrombosis and rheumatoid arthritis. SAA1and SAA2are the main circulating proteins in acute response. SAA is regulated by various factors such as turpentine, LPS, mononuclear cells conditioned medium, proinflammatory factor(IL-1,IL-6), TNF-a, Synthetic glucocorticoid dexamethasone, phorbol ester, and minimally modified low-density lipoprotein under different conditions.A study involving in10types of solid tumors including stomach, colon, pancreas, prostate, breast, ovarian, prostate, lung and sarcoma and3types of hematological malignancies including Hodgkin’s disease, Non-hodgkin’s disease and leukemia showed that the level of SAA is higher in different types of tumors than normal subjects. SAA is considered as a serum biochemical marker distinguishing bone metastasis in prostatic neoplasm patients and is of significant value for initial diagnosis. Similarly, Osteoblast-like osteosarcoma cell line(SAOS-2and MG-63) could express SAA under the stimulation mediated by cytokines. SAA activation factor-1is a transcriptin regulators expressed in osteosarcoma cells no matter stimulated by cytokines or not. SAA is intensively related to osteosarcoma pathogenesis. Therefore, the main purpose of this study is to investigate the influence on osteosarcoma pathogenesis and the possible signaling pathway. http://dict.youdao.com/javascript:void(0);Objective: 1. To investigate the effects of SAA suppression on migration and invasion in U2OS cells;2. To investigate the interaction between SAA and integrinαvβ3;3. To investigate the specific effect of SAA on osteosarcoma and its related signaling pathways.Methods:1.Cell cultureU2OS cells were cultured in DMEM supplemented with10%fetal bovine serum at37℃in a humidified atmosphere of5%CO2.2. FPRL-1siRNA and its transfectionLipofectamine2000was used in the presence of FPRL-1siRNA (100nM) and then the siRNA/lipfectamine2000particles were added into U2OS cells for48h. The cells were collected for western blot and realtime PCR detection to determine the effect of the siRNA sequences.3. Western blot analysisAfter stimulation with SAA or other reagents, U2OS cells were collected for protein extraction. Specific antibodies were used to detect expression of SAA、αvβ3、 p-p38、ERK1/2and JNK, and then analyzed by Photoshop software.First, pour out the culture medium and add RIPA cracking liquid containing PMSF to culture bottle or orifice plate.Scrap with scraper, transfer to the treated clean EP tube. If for suspension cells, collect culture medium containing cells and centrifuge at1000r/min for5min, and then pour out supernatant and add RIPA cracking liquid containing PMSF. The amount of cracking liquid is usually as follow:1ml/bottle for small culture bottle,200ul/holefor orifice plate,80ul/hole for12orifice plate.Then incubate on the ice for15to30min. Centrifuge at13000r/min centrifuge for10min under4℃. Transfer supernatant to the new EP tubes and add protein sample buffer in proportion. Cook at99℃for10min,then store in-80℃.Clean and dry the glass plate,put it in the clip tightly. Make sure that the two pieces of glass plates are under along in a plane. Prepare10%separation gel according to the proportion, and add30%respectively Acr/Bic Tris-HCl (pH8.8) three steamed water10%SDS10%ammonium persulfate TEMED in order. Shock vortex immediately after adding TEMED. Pour into two pieces of glass, pressure air bubbles with isopropyl alcohol. Pour out isopropyl alcohol after about half an hour when the separation gel solidification and rinse with distilled water. Dry the residual water. Prepare5%basal gel according to the proportion. Add30%Acr/Bic Tris-HCl (pH6.8),three distilled water,10%SDS,10%ammonium persulfate,and TEMED. Shock vortex immediately after adding TEMED. Pour into two pieces of glass and insert the corresponding comb.Put gel together with the glass into the electrophoresis tank with electrophoresis buffer, and carefully pull out the comb from the glue.Make sure that the electrophoretic liquid level is above glass. Thaw protein at99℃reheating for5min before adding. Shock blending. Add suitable amount of volume of protein extract to each hole, and add3ul protein marker on both sides of the holes. First electrophoresis at90voltage, after marker indicating with separation, change the electrophoresis to120v. When Bromophenol blue arrive the bottom of separation glue, stop the electrophoresis.According to the experiment purpose, remove excess glue according to the instruction from the Marker, put the target protein glue into the transfer film clip:clip brown-rubber-NC membrane filter paper-filter sponge sponge-clip red face. Make sure no bubble between each layer. Put transfer film clip in the membrane tank, turn200ma surrounded by ice for1.5h. Block with5%skimmed milk powder for1-2h. Take out the stripe and wash with1TBST5min3times on shaking bed. Then put them into a proper concentration of antbody. Rocking bed for the night at4℃.4. Quantitative real time PCRAccording to FPRL-1cDNA sequence of human in the GeneBank, entrust Shanghai boshang biological company to design primers for FPRL-1:5-upstream CACGGCCACATTACCATTCCT-3; Downstream5-AGCGGTCCAGTGCAATGA-3; primer for αvβ3:upstream5-GCTTCAAGGACAGCCTGATCG-3, downstream5-CTTTATACAGTGGGTTGTTGGCTG-3; Select18s as internal, its upstream primer5-CTTAGTTGGTGGAGCGATTTG-3; downstream5-GCTGAACGCCACTTGTCC-3. Use total RNA extraction kit, according to the recommended use conditions, extrac total RNA from cell. Propose all needed equipment with RNase. Take1ml Trizol mixture into U2OS cells, scrape the cells and blow evenly. Transfer the liquid into new EP tubes after10min standing at room temperature. Add200ul chloroform and vortex shock thoroughly. iPlace them on ice for5min. Centrifuge at a speed of14000RPM for15min at4℃. Transfer the upper0.5ml liquid to RNase processed clean EP tubes, and add same volume of isopropyl alcohol, continue to incubate on the ice for30min. Centrifuge at a speed of14000RPM for15min at4℃. Abandon the supernatant, and add1ml75%ethanol to wash precipitate fully; Centrifuge at a speed of12000RPM for15min at4℃. Remove ethanol, and take lOul treated three steamed DEPC water to dissolve the lower precipitation, measure RNA concentration by ultraviolet spectrophotometer. Save at-80℃.5. Scratch Wound Healing Assay (Migration assay)Cells grow in DMEM medium containing10%FBS. Transfer cells to6holes in the cell culture plate in a certain density, grow after24hours, single cell alignment should reach70-80%. Do not replace the medium. Scratch across monolayer culture cell in the holes with a new1ml spear head gently. Spear and plate at the bottom of the hole is vertical as far as possible. So the gap distance equal to the outer diameter of spear. The distance of the gap can be regulated by choosing different types of spear. Scratches in the same direction is as a straight line. In vertical of the direction of the first scratch appears another Nick. After scratches, each hole appears cross type. Clean plate holes2times with media gently. Add fresh culture medium to each hole. Culture medium contains ingredients, such as inhibiting/promote cell migration and/or proliferation of chemical. Cells grow48hours. Cleaning the cells twice with1XPBS, and then fix the cells with3.7%paraformaldehyde for30minutes. Dye with0.1%of crystal violet (2%ethanol solution) for30minutes, choose different horizons of monolayer cells,take photoes by microscope. Gap distance can be measureed by Photoshop or ImagJ software. In order to reduce the variability of the test results, it is better to select multiple vision observation, every hole do multiple repeat in each group.6. Transwell assay (Invasion assays)Take well grown U2OS cells, clean serum twice with free medium gently; Add the serum free medium,37Κ,5%CO2,cultivate24h～48h. Collect cell supernatant;4℃,12,000RPM centrifugal,10min, take supernatant;0.22um membrane filter bacteria, Partial shipments, save in-20℃.-20℃saved Matrigel melt at2-8℃overnight on the ice. Draw100ul Matrigel and add to300ul serum free medium,fully mix. Take25ul of Matrigel to the upper room and cover the carbon acetate membrane37℃,30min. Matrigel transwell culture plate in37℃can save two weeks. Rinse groups of cells with PBS three times after the stimulation. In conventional methods with serum free medium preparation of single cell suspension,5*105cells/ml, each group of cells is divided into two parts. Placenta from dyeing experiments, cell vitality should be greater than95%. Add100ul cell suspension (5,104) to transwell culture board together with200ul serum free medium. Add the500mu1chemokines to transwell culture plate under room,37℃,5%CO2cultivate for24h. wipe gently with wet cotton swabs the cells on the surface of Matrigel gel and polyethylene carbon ester membrane. Carefully remove the room, tie with lines, and tag, fix with ice precooling methanol for30min. Hematoxylin stain for1min. Gradient ethanol dehydration (80%,95%,100%), xylene transparent. Take off the membrane carefully from the chamber and slide on neutral resin sealing piece. Attached to the poly ester carbon film on the surface of the cells at high magnification (400). Randomly take six view count, averaged them and repeat the experiment two times.7. Statistical analysisData were analyzed with SPSS13.0(SPSS Inc., Chicago IL). Continuous data were presented as mean±SEM. Differences among groups were calculated with oneway analysis of variance. P<0.05was considered statistically significant.Results:1. SAA Induced the Migration and Invasion of U2OS CellsWound healing assay demonstrated that SAA of10μg/ml displayed a significant effect on the healing over the scratch compared with control group(P<0.05), indicating that SAA induced the migration of U2OS cells.The result of Transwell assays displayed that10μg/ml of SAA could significantly promote the invasive activity of U2OS compared with control group. The data above intensively suggested that SAA induced the migration and invasion of U2OS cells.2. SAA up-regulated αvβ3integrin expression at both protein and mRNA levelsWestern blot results showed that αvβ3expression demonstrated no significant change when stimulated with SAA at the concentration of0.1μg/ml. But when the stimulating concentrations were elevated to1and10μg/ml, the expression of αvβ3integrin was significantly increased compared with the control group, and reached its maximal activity at10μg/ml. The cells were stimulated with SAA (10μg/ml) for different times (0,3,6,12and24h). After stimulation with SAA (10μg/ml) for3hours the relative expression of αvβ3integrin changed slightly. However, when the stimulating time prolonged to6,12and24h, the relative expression of αvβ3integrin was significantly increased compared with control group.The relative expression of αvβ3integrin mRNA was significantly increased after stimulation with SAA at the concentration of1,10μg/ml. In addition, it was also increased gradually compared with control group after stimulation with SAA (10μg/ml) for1,3,6and12hours, just like the protein level. The protein and mRNA had a similar change tendency and the transcriptional activation appeared ahead of protein synthesis which suggested that this transcriptional activation was required for αvβ3integrin protein synthesis. The data above intensively suggested that SAA induced αvβ3integrin production in concentration-and time-dependent manners both at protein and mRNA levels.3. Treatment with LM609abolished SAA-induced cell migration and invisionThe data suggested that there was no difference among control group, LM609group and LM609+SAA group, which intensively proved that preincubation of LM609(20μg/ml) for2h significantly abolished SAA-induced increases in U2OS cells migration. Similar results were observed when the transwell assay was conducted which indicated that SAA-induced increases in U2OS cells invasion was also abolished after blocking αvβ3integrin pathway with LM609. These results indicated that αvβ3integrin was required for SAA-induced U2OS cells migration and invasion.4. ERK1/2Mediates SAA-induced αvβ3integrin productionTo determine the relation between MAPKs and αvβ3integrin production, we firstly assessed the relative expression of phospho-JNK, phospho-p38and phospho-ERK1/2respectively after U2OS cells stimulated with SAA (10mg/ml) for5,15,30,60and120min. The relative expressions of the three members were all significantly up-regulated compared with control group(P<0.05). The activity of the3members all rapidly increased after5-30min stimulation, while gradually decreased after1-2h stimulation. However, when inhibited JNK, p38and ERK signaling pathways respectively by their inhibitors:SP600125(50μmol/L), SB203580(10μmol/L) and PD98059(20μmol/L) for1h before SAA treated for another24h, we found that only PD98059(ERKl/2specific inhibitor) could dramatically inhibit SAAinduced αvβ3integrin production. The results strongly indicated that all of the3members of MAPK family could be activated by SAA, but only ERK1/2signaling pathway participated in the reaction in U2OS cells.5. FPRL-1was involved in SAA-Induced αvβ3integrin productionThe results demonstrated that FPRL-1siRNA could effectively down-regulate the expression of FPRL-1siRNA both at protein and mRNA levels (p<0.05) Meanwhile, pertussis toxin (an antagonist of G protein-coupled receptor) and WRW4(a novel specific inhibitor for FPRL-1) were also chosen to detect whether SAA-induced αvβ3integrin production in U2OS cells was mediated by FPRL-1(p<0.05). The data indicated that the SAAinduced αvβ3integrin secretion could be inhibited significantly by all of the3reagents (Fig5C and D), which supported that FPRL1is required for SAA-Induced αvβ3integrin production.Conclusion:1. SAA promote the migration and invasion of U2OS cell via αvβ3integrin;2. SAA induced αvβ3integrin production in concentration-and time-dependent manners both at protein and mRNA levels;3. SAA induced αvβ3integrin production via FPRL-1/ERK1/2pathway.