SPAG9 Promotes The Proliferation And Invasion Of Prostate Cancer

Background and objective:Prostate cancer is a common malignant tumor of urinary system. In developed countries, the incidence of Prostate cancer ranks first and mortality ranks second. In China, although the incidence is lower than that of western countries, but the incidence increased gradually due to the changes of standard of living and environment in recent years.At present, the main treatment for prostate cancer is multimodal combined treatment, including operation, radiotherapy, endocrine therapy, and so on. But due to male hormone androgen resistance, the efficacy of operation and radiotherapy for patients with advanced prostate cancer is not significant. Prostate cancer is susceptible to recur, invade and metastasize, therefore seeking for related tumor markers of prostate cancer becomes the key point for early detection、targeted therapy 、relapse prevention of prostate cancer. Sperm associated antigen 9(SPAG9) is one of the members of the family of cancer testis antigen(CTA) and it specifically expresses in the testis. Recent studies show that SPAG9 can play an important regulatory role in proliferation, invasion, metastasis and other biological behaviors of tumors. Mainly acting as a scaffold protein, SPAG9 can assist the activation of JNK signaling pathway, thereby it can promote proliferation, invasion and metastasis of tumors and inhibit tumor apoptosis. The effects of activation of JNK signaling pathway on tumor promotion have been reported. Thus, it can be speculated that up-regulating of SPAG9 may change the original mode of gene regulation and then promote proliferation, invasion and metastasis of tumors. This study aims to investigate the effects of SPAG9 on proliferation, invasion, metastasis and other biological behaviors of prostate cancer.Method:1. Immunohistochemistry was used to detect the SPAG9 expression levels of clinical prostate cancer specimens, which was analyzed for correlation with age, GS score, tumor stage, lymph node metastasis, distant metastasis, blood PSA value and other pathological parameters of patients with prostate cancer.2. The expression levels of SPAG9 in prostate cancer cells were detected by Western blot. The high-expressed cell lines were selected to be interfered and low-expressed to be over-expressed.3. We constructed the over-expressed plasmid in the PC3 cells and interfered plasmid of SPAG9,respectively. When the overexpression of and the interference of DU145 cells were set up, we used qRT-PCR and Western blot to verify the changes of SPAG9 expression in transfected cells.4. CCK-8 testing was used to detect the effect of SPAG9 on the proliferation of prostate cancer cells.5. Flow cytometry and western blot were used to detect the effect of SPAG9 on the cell cycle of prostate cancer cells.6. We adopted transwell assays and scratch tests to detect the influences of SPAG9 on migration capacity of prostate cancer cells in vitro.7. Tumorigenesis in nude mice was applied to verify the effects of SPAG9 on the proliferation of tumor.8. The changes which were caused by SPAG9 of ERK, JNK and P38 proteins in MAPK signaling pathway were detected by Western blot.Results:1.By immunohistochemical staining, we found that SPAG9 was highly expressed in Pr ostate carcinoma tissues,while down-regulated in Prostate paraneoplastic tissues. In additio n, SPAG9 was correlated significantly with GS score and tumor stage through statistical an alysis(P<0.05).2.we found that the expression of SPAG9 was highest in DU145 cells and was lowest in PC3 cells by screening prostate cancer cells with western blot.3.Using Q-PCR and western blot,we verified that RNA and protein expression of SPAG9 in PC3 cells were up-regulated after over-expression and were down-regulated in DU145 cells after interference.4. CCK-8 testing showed that SPAG9 promoted prostate cell proliferation.5.Flow cytometry counting showed that over-expression of SPAG9 accelerated the cell cycle in PC3 cells and interference of SPAG9 could lead to cell cycle arrest in DU145 cells.6.Transwell migration experiment confirmed that the transmembrane capacity of PC3 cells which were overexpressed with SPAG9 was significantly higher than control group; and the transmembrane capacity of DU145 cells which were interfered with SPAG9 was lower than control group; Scratch experiment verified that the ability of cell migration of PC3 cells with overexpressed SPAG9 was higher than control group.7. We found that interference of SPAG9 could inhibit the tumor formation in nude mice.8. Western blot confirmed that phosphorylation of JNK, ERK and P38 protein expression were up-regulated in PC3 cells when SPAG9 overexpressing,while these were down regulated in DU145 cells when interference of SPAG9.Conclusion1. The SPAG9 was highly expressed in prostate cancers and it could be regarded as a indicator for early diagnosis and prognosis.2. SPAG9 could promote proliferation, cell cycle and invasion metastasis of prostate cancer cells.3. SPAG9 may play the role through ERK 、JNK and P38 signaling pathway.