Role Of SPAG9 In Promoting Human Osteosarcoma Cells Motility, Invasion And Angiogenesis

Background and purposeOsteosarcoma is the most common malignant tumor of bone. It is regarded as a hotspot and difficulty in osteosarcoma clinical treatment because of its high malignant degree, metastasis rate and fatality rate. With the development of osteosarcoma therapy over the past several years, including neoadjuvant and adjuvant chemotherapy coupled with surgical resection, patient outcomes have been obviously improved. However, there still exist two major problems which are difficult to solve:the recurrence and metastasis. Metastasis (mainly pulmonary metastasis) is the leading cause to result in about 80%-90% of death in patients with osteosarcoma. Consequently, it is extremely urgent to find effective diagnostic tools and treatment for osteosarcoma.Cancer testis (CT) antigens is a class of plasma membrane antigens, which is specifically expressed in the normal testis and embryo, and show aberrant expression in various malignancies. CT antigen is a well-accepted tumor biomarker. At present, CT antigen is used as tumor marker in the diagnosis and monitoring of tumor. A large number of studies have confirmed that CT antigen gene products are involved in cell differentiation, signal transduction, transcription, translation and chromatin remodeling signaling pathway. Therefore, CT antigen is considered as a novel biomarker for the occurrence and metastasis of tumors. Sperm associated antigen 9(SPAG9) is a new discovered type of CT antigen. It is highly expressed in many human tumors, suggesting that SPAG9 may act as an oncogene in human cancer cells. But the role of SPAG9 in human osteosarcoma and its mechanism is still unclear. In this study, we will use SPAG9 as the research object and the research object of this study, to find the role of SPAG9 in the osteosarcoma cell line U2OS, and to explore its mechanism in the osteosarcoma.To investigate the expression of SPAG9 in human osteosarcoma tissues, and its function and possible mechanism. Compare and analyze the SPAG9 molecules of biological behavior of osteosarcoma from molecular level, so as to provide new research ideas and experimental basis for developing osteosarcoma metastasis mechanism, also provide a theoretical basis for early diagnosis, metastasis monitoring and molecular target in clinical.Methods:1. Real time PCR and Western blot were used to determine the expression of SPAG9 in human osteosarcoma tissues.2. The effective SPAG9 small interfering fragment was confirmed by SPAG9 design. SiRNA-Ctrl and siRNA-SPAG9 small interfering RNA fragment were used to interfere the gene of U2OS cell line. Western blot was used to detect SPAH9 protein expression.3. Cell proliferation assay was used to detect the effect of silence of SPAG9 on U2OS cell proliferation.4. Wound healing assay was employed to investigate the effect of si-SPAG9 on cell motility.5. The effects of SPAG9 on the migration and invasive abilities of human osteosarcoma cell line U2OS were respectively investigated by Transwell assay.6. Endothelial cell tube formation assay was performed to count the number of capillary-like tubes formed by HUVECs in the supernatants of transfected cells.7. Gelatin zymography was performed to visualize the MMPs activities in human osteosarcoma cells.8. Western blot was employed to detect the protein levels of MMPs and VEGF.9. Real time PCR was used to a determine the mRNA expression of MMPs and VEGF after the knockdown of SPAG9.10. ELISA was performed to determine VEGF concentration in the supernatants of transfected cells.Results1. Western blot and real time PCR of human osteosarcoma tissues showed that there was a significantly higher expression of SPAG9 in the carcinoma tissues compared with paired non-cancerous tissues (P<0.05).2. We designed three couple small interfering fragments for SPAG9 and real time PCR data showed that SPAG9 siRNA-2 dramatically increased SPAG9 protein and mRNA expression of U2OS cells (P<0.05).3. Cell proliferation assay was used to detect the effect of silence of SPAG9 on U2OS cell proliferation. The data showed that silence of SPAG9 had no effect on cell proliferation (P>0.05).4. In wound healing assay, knocking down of SPAG9 decreased cell migration abilities of U2OS cells by 17% after 12 hours(P<0.05), and 26% after 24 hours (P <0.05).5. In Transwell assay, knocking down of SPAG9 decreased cell migration abilities of U2OS cells by 66%(P<0.05).6. Cell invasion assay showed that it also decreased invasive abilities of U2OS cells by 69%(P<0.05).7. Compared with those of control cells, decreased SPAG9 expression reduced the average number of complete tubular structures by 55% formed by HUVECs (P<0.05).8. The HUVECs cell proliferation displayed that decreased SPAG9 expression inhibited its cell variability (P<0.05).9. Gelatin zonography showed that inhibition of SPAG9 significantly suppressed the MMP2 and MMP9 activities by 59% and 61% in cancer cells (P<0.05).10. Western Blot showed that inhibition of SPAG9 significantly suppressed the MMP2 and MMP9 protein level by 47% and 52%, and VEGF by 54%(P<0.05).11. In the real time PCR assay, the data indicated that the mRNA expression of MMP2 and MMP9 was decreased by 37% and 58%(P<0.05).12. ELISA analysis displayed that VEGF concentration in the supernatants of knocking down cells was significantly reduced compared to the control cells in the osteosarcoma cancer cells (P<0.05).ConclusionSPAG9 protein levels were increased in human osteosarcoma cancer cells. Knocking down SPAG9 decreased the mRNA and protein expression of MMP2 and MMP9, reduced the enzyme activity of MMP2 and MMP9. The inhibition of MMP2 and MMP9 resulted in suppression of osteosarcoma cancer cell motility, and invasion abilities. Knocking down SPAG9 suppressed the secretion and expression of VEGF and inhabited the ability of angiogenesis in osteosarcoma cancer cells.