Study Of SPON1 Promotes Metastatic Progression In Human Osteosarcoma

Osteosarcoma is the most frequent primary malignant bone tumor in children and adolescents, about 20% of primary bone tumors. Osteosarcoma(OS) defines neoplasms that share the histological finding of osteoid production in association with malignant mesenchymal cells. These tumors are generally locally aggressive and tend to produce early systemic metastases.A distinction is generally drawn between different histologic types of OS(conventional, teleangiectatic, parosteal, periosteal, low-grade central,small cell, not otherwise specified). The conventional type is the most common, and has been subdivided based on the predominant features of the cells(osteoblastic, chondroblastic, fibroblastic),although without clear significant differences of clinical outcome.Osteosarcoma is a rare type of cancer associated with a poor clinical outcome. Due to high degree of malignancy,invasion and metastasis, however, only 60% of patients diagnosed with osteosarcoma will survive for more than 5years, and less than 30% will live beyond 10 years. Pulmonary metastase is present in about 15-25% of patients and this contributes to the failure of chemotherapy and poor prognosis in osteosarcoma. Moreover, nearly 90% of osteosarcoma patients experience metastasis or relapse,even after completely surgical resection of the primary tumor.In the majority of primary OS, the etiology is unknown. Cytogenetic studies have shown various complex changes involving some chromosomes but without any specific pattern.Even though the pathologic characteristics of OS are well established, much remains to be understood, particularly at the molecular signaling level. The molecular mechanisms of osteosarcoma progression and metastases have not yet been fully elucidated. Therefore, it is of great importance to determine the molecular mechanisms that underlie thistype of metastasis.SPON1, also known as F-spondin, is a secreted extracellular matrix glycoprotein that originally isolated from the embryonic floor plate of vertebrates. Initially, treatment with recombinant SPON1 protein promotes neural cell adhesion and neuronal outgrowth, which indicates the functions of SPON1 in the modulation of axonal growth in the embryonic central nervous system. Apart from neuronal tissues, SPON1 expression has recently been detected in several other tissues including ovary,embryonic growth plate cartilage, periodontal tissue and osteoarthritic cartilage. However, whether SPON1 is differentially expressed in tumors, especially in osteosarcoma,remains largely unexplored.Matrix metalloproteinase( MMPs) are a group of dependence peptide protein hydrolase,containing zinc ions and calcium ions,and more than 20 of Members. The main function of MMPs are degradeding some of the important of extracellular matrix protein enzymes, keeping the dynamic balance of extracellular matrix. MMPs family plays an important role in tumor proliferation, angiogenesis, invasion and metastasis. MMP- 9 is an important member of the family of MMPs,and main degradation of Ⅳ, Ⅴ collagen and gelatinof extracellular matrix. MMP- 9 plays an important role in the development and invasion and metastasis of tumor.Ki67 is the first to be discovered cancer protein tyrosine kinase activity,participation multiple intracellular signaling pathways. Ki67 is a sensitive index of the cell proliferation activit,and is expressed in proliferating cells.It Presents a express phenomenon in a variety of malignant tumors.Focal adhesion kinase( FAK) is a kind of protein tyrosine kinase of cytoplasm and Closely related to cell adhesion function.When tumor cell metastasizing, integrin enable heterogeneity adhesion become higher and reduce homogeneity adhesion. It acts a critical factor in the metastatic and invade progression of tumors. FAK is a key molecule in integrin signaling pathways, involved in multiple signal pathways in cancer cells.Src family members are former cancer gene encoding located in thecytoplasm of non-receptor tyrosine kinase, Src molecular weight of 60 k D, is the first to be discovered cancer protein tyrosine kinase activity,participation multiple intracellular signaling pathways.The aim of current study was to determine the expression pattern and cellular functions of SPON1 in osteosarcoma. The cellular migration and invasion affected by SPON1 were analyzed in high(KHOS and KRIB) and low(HOS and U2OS) metastatic potential osteosarcoma cell lines. And to demonstrate the mechanism on this effect induced by SPON1, Fak and Src pathway were inhibited using small interfere RNA(si RNA). We carried out immunohistochemical analysis of SPON1 expression in clinical osteosarcoma specimens and studied the relationship between SPON1 levels and MMP9 and Ki67 expression,respectively. Finally, our results imply that the SPON1/Fak/Src-related pathway might be an effectively therapeutic approach in the management of metastatic osteosarcoma.Part one To explore whether SPON1 is a differentially expressed genes between humanmetastatic osteosarcoma and human non-metastatic bone tumorsObjective : 1 To identify four kinds of osteosarcoma cell line KHOS,KRIB, HOS, U2 OS conform the high and low metastatic osteosarcoma cell line division for the next experiment. 2 To determine the differentially expressed genes(DEGs) between human high- metastatic osteosarcoma cells and low-metastatic osteosarcoma cells from a GEO dataset. 3 To investigate the difference of SPON1 expression between human osteosarcoma tissue specimens and human osteochondroma tissue specimens, and the relationship between SPON1 levels and MMP9 and ki67 expression respectively.Methods:1 Search for differentially expressed genes from GEO database.2 According to the literature,we selected four kinds of osteosarcoma cell lines KHOS,KRIB,HOS and U2OS)to perform Transwell cell Migration and invasion assays.3 The western blotting in OS tissues found that the expression levels of SPON1 in high(KHOS and KRIB) and low(HOS and U2OS) metastatic potential osteosarcoma cell lines.4 Immunohistochemistry detect the differences of SPON1 positive expression between two tissue microarray( seventy two cases of osteosarcoma tissues and twenty-four cases of osteochondroma tissues). The tissue sections were deparaffinized with dimethylbenzene and rehydrated through grade ethanol. After three washes in phosphate-buffered saline(PBS), antigen retrieval was performed in a buffer containing 0.01 M sodium citratehydrochloric acid(p H =6.0) for 15 min by microwave. After rinsing with PBS, the tissue sections were then rinsed in 0.3% peroxidase quenching solution to block endogenous peroxidase, followed by incubation with primary antibody against SPON1(at 1:200 dilution), MMP9(at 1:200 dilution) and Ki67(at 1:100 dilution) overnight at 4 ℃. After washing in PBS for three times, the sections were incubated with HRP-labeled anti-rabbit secondary antibody.The visualization signal was developed with 3,3’-diaminobenzidine tetrahydrochloride(DAB), and all of the slides were counterstained with hematoxylin. The total immunostaining score was calculated as the percentage of the positively stained tumor cells. The percent positivity was scored as follows: 0-10% scored 0; 10%-30% scored 1; 30%-60% scored 2; more than60% scored 3. And scored at 0 and1 was defined as low expression, while 2and 3 was defined as high expression.Results : 1 We analysis GC % content near the high frequency of mutations nucleotide sequences in the GEO database, and observed the differentially expressed genes(DEGs) between human metastatic osteosarcoma cells and non-metastatic osteosarcoma cells from a GEO dataset(GSE49003). In these DEGs, we selected SPON1 for further study. 2Transwell cell migration and invasion assays results show that: According to the literature,we selected four kinds of osteosarcoma cell lines can be used as research subjects of osteosarcoma cell migration and invasion. KHOS and KRIB could be used as human high metastatic potential osteosarcoma cell lines, HOS and U2 OS could be used as human low metastatic potential osteosarcoma cell lines. 3 Through the Western Blotting,We first detected SPON1 in four osteosarcoma cell lines. Expectedly, elevated SPON1 protein was found in high metastatic osteosarcoma cells, KHOS and KRIB, compared with the low metastatic osteosarcoma cells, HOS and U2 OS. 4Immunohistochemical analysis showed that positive expression rate of SPON1 were significantly higher in osteosarcoma compared with osteochondroma samples. Moreover, there was significant positive correlation between SPON1 and MMP9(P < 0.001, R=0.745), but not Ki67(P =0.359) in osteosarcoma specimens.PART2 To investigate the osteosarcoma cell’s migration and invasion and tumor metastasis in vivo affected by SPON1Objective : 1 To investigate the osteosarcoma cell’s migration and invasion affected by SPON1; 2 Observation on the nude mice animal model xenograft tumor growth situation,and SPON1 role in tumor metastasis in vivo.Methods:1 si RNA Transfection, Tumor cell growth curve experiments and MTT test:1.1 According to the design principle of si RNA,we synthesized two kinds of si RNA- SPON1(si-SPON1-1,si-SPON1-2)to knockdown SPON1 gene expression and a negative control si RNA(si- Ctrl)in vitro. In order to knockdown SPON1 gene, we transfected human high metastatic potential osteosarcoma cell lines(KHOS and KRIB) by the si RNA-liposomes carrier.1.2 The interference efficiency of targeted gene was demonstrated by Western Blotting;1.3 Tumor cell growth curve experiments and MTT test were carried out to determine whether the si RNA-lipidosome interference the expression of SPON1 in osteosarcoma cell affect the growth and proliferation of tumor cells.1.4 Transwell cell Migration and invasion assays were carried out to determine whether SPON1 was knockdown in high metastatic osteosarcoma cell affect the migration and invasion of KHOS and KRIB tumor cells.2 Treatment human low metastatic potential osteosarcoma cell lines(HOS and U2OS) with human recombinant SPON1 protein.2.1 Human recombinant SPON1 protein was diluted to 5nmol/L 、10nmol/L 、 20nmol/L 、 50nmol/L, and use Western Blotting experimental identification.2.2 MTT test were carried out to determine whether the different concentrations(0nmol/L、10nmol/L、50nmol/L)of human recombinant SPON1 protein affect the growth and proliferation of HOS and U2 OS tumor cell lines.2.3 Transwell cell Migration and invasion assays were carried out to determine whether the different concentrations( 0nmol/L 、 10nmol/L 、50nmol/L) of human recombinant SPON1 protein affect the migration and invasion of HOS and U2 OS tumor cell lines.3 In vivo tumorigenesis and metastasis:KHOS cells stably interfered SPON1 and control cells were collected and resuspended in 100 m L cold PBS. Then 5 ×105 KHOS cells were mixed with the same volume of matrigel and injected into the proximal tibia of each anesthetized nude mice(n =5 animals/group). Six weeks after inoculation,orthotopic tumors and mouse lungs were harvested. The orthotopic tumors were weighed and the number of pulmonary metastatic tumor nodules was counted under an under a dissecting stereomicroscope. Finally, lung tissues were fixed with 10% neutral-buffered formalin, embedded in paraffin,sectioned at 5 mmand stained with H&E(hematoxylin and eosin). Observe the xenograft tumor growth determine the effects of SPON1 on tumor metastasis in vivo.Results:1 Westernblot experiment results show that: Transfection of two SPON1 si RNAs(si-SPON1-1, si-SPON1-2) specific silenced SPON1 gene expression of human high metastatic potential osteosarcoma cell lines(KHOS and KRIB),and resulted in markedly decrease in SPON1 expression. 2 Tumor cell growth curve experiments and MTT test results show that: 2.1 The si RNA-lipidosome interference in osteosarcoma cell didn’t affect the growth and proliferation of KHOS and KRIB tumor cells. 2.2 The different concentrations( 0nmol/L 、 10nmol/L 、 50nmol/L) of human recombinant SPON1 protein didn’t affect the growth and proliferation of HOS and U2 OS tumor cells. 3 Transwell cell Migration and invasion assays results show that:3.1 At human high metastatic potential osteosarcoma cell lines(KHOS and KRIB),knockdown of SPON1 resulted in significantly decreased migrated and invaded cells compared with negative control cells. 3.2 At human low metastatic potential osteosarcoma cell lines(HOS and U2OS),recombinant SPON1 protein pronounced promoted cell migration and invasion in a dose-dependent manner. 4 KHOS cells stably interfered SPON1 and control cells were injected into the proximal tibia of each anesthetized nude mice. Six weeks after inoculation, orthotopic tumors and mouse lungs were harvested.Through pathological section, we confirmed the situ tumors were osteosarcoma, and rat lung tumors are metastatic osteosarcoma. The results show that:stably suppression of SPON1 in KHOS cells did not affect tumor growth in xenograft. However, all the mice in the control group had gross more pulmonary metastatic lesions compared with the SPON1 knockdown group.PART3 SPON1’s affection to FAK \ Src signaling pathway in osteosarcoma cellsObjective:To investigate the SPON1’s affection to FAK \ Src signaling pathway in osteosarcoma cells.Methods:1 Western blotting detected the phosphorylation levels of Fak and Src in specific SPON1-gene-silenced KHOS cells. And detected the phosphorylation levels of Fak and Src in human low metastatic potential osteosarcoma cell lines HOS cells which cultivated by human recombinant SPON1 protein. 2 To further confirm the role of FAK and Src signaling in SPON1-induced metastatic progression, cell migration and invasion assay were performed on human high metastatic potential osteosarcoma cell lines(KHOS and KRIB)after Fak and Src were silenced by si RNAs. Treat FAK\Src silenced KHOS cells with human recombinant SPON1 protein and observe its migration and invasion.Results : 1 Knockdown of SPON1 resulted in decreased phosphorylation levels of Fak and Src in HKOS cells,while SPON1 protein treatment remarkably promoted the phosphorylation levels of Fak and Src in HOS cells.The above results suggest that the metastatic progression of human osteosarcoma cell lines KHOS and HOS related with SPON1, FAK and Src. 2After Fak and Src were silenced by si RNAs, HKOS and KRIB cells’ s ability of migration and invasion decreased obviously. The different concentrations(0nmol/L、10nmol/L、50nmol/L) of human recombinant SPON1 protein didn’t affect the migration and invasion of FAK\Src silenced KHOS cells.Indeed, the enhanced effects of SPON1 on osteosarcoma cell migration and invasion were completely blocked by the silencing of Fak or Src in both HKOS and KRIB cells.Conclusion:1 SPON1 is a differentially expressed genes between human highmetastatic osteosarcoma cells and low-metastatic osteosarcoma cells, and is a differentially expressed genes between humanmetastatic osteosarcoma and human non-metastatic bone tumors. we speculated that SPON1 might function as a modulator in the metastatic potential of osteosarcoma cells. By immunohistochemistry,we found up-regulated SPON1 levels are closely correlated with MMP9 expression, which involved in the dissemination of tumor cells from the primary tumor.2 The metastatic behavior of human high metastatic potential osteosarcoma cells(HKOS and KRIB)was remarkably inhibited by silencing SPON1 both in vitro and in vivo. Similarly, treatment with SPON1 protein in the human high metastatic potential osteosarcoma cells( HOS and U2OS),cell invasiveness was significantly promoted. Based on these results, we speculated that SPON1 might function as a modulator in the metastatic potential of osteosarcoma cells.3 We found that the phosphorylation levels of Fak and Src was remarkably enhanced by SPON1, thus promoting cell invasiveness. These results indicate that Fak and Src signaling is a critical mediator of SPON1-derived metastatic progression.