| Semaphorins are a family of membrane-bound proteins that were initially recognized as a kind of axon guidance factors in nervous system, but were deregulated in patients with cancer, affecting tumor progression. Semaphorin 4A (SEMA4A) belongs to this family, it exists primarily as membrane-bound protein, but its extracellular portion can be processed into soluble form by proteolytic cleavage. SEMA4A was preferentially expressed on immune cells and involved in immune responses. However, its expression and function in tumor cells remained unknown. Tumor-stroma interaction in the microenvironment is an essential event during prostate cancer (PCa) metastasis. The function of SEMA4A that released into microenvironment is also undetected. In this study, we found that SEMA4A was highly expressed in prostate cancer and correlated with Gleason scores and distant metastasis. The in vitro and in vivo experiment demonstrated that SEMA4A could promote the invasion and metastasis of tumor cells though regulating the epithelial-mesenchymal transition. Importantly, SEMA4A could induce 1L-10 production in stromal cells, and IL-10, in turn, could up-regulate the expression of SEMA4A in tumor cells. All these indicate the existence of a positive feedback loop between them to promote tumor metastasis. Furthermore. SEMA4A and IL-10 were validated as the co-targeted genes of STAT3 and they also functions through STAT3 signaling. In our study, the expression and function were systematically studied and found that, as an oncogene. SEMA4A could promote invasion of PCa cells. In addition, SEMA4A mediates the interaction between tumor and stromal cells and collaborates with IL-10 to exacerbate the progression of prostate cancer.Objects:1. To analyze the characteristics of SEMA4A expression in Chinese PCa patients.2. To study the function and underlying mechanism of SEMA4A in PCa cells.3. To explore the function and underlying mechanism of SEMA4A in prostatic tumor microenvironment.Methods:1. The characteristics of SEMA4A expression in Chinese PCa patients.1.1 Immunohistochemistry (IHC) assay was used to detect the expression of SEMA4A in PCa and BPH tissue. The correlations with other pathological indicators were also analyzed to clarify the clinical significance of its abnormal expression.1.2 The expression of SEMA4A in PCa was assessed with GEO database.1.3 RT-qPCR and Western Blot were performed to detect the mRNA and protein levels of SEMA4A expression in PCa cell lines, respectively.2. The function and underlying mechanism of SEMA4A in PCa cells.2.1 Small interfere RNA (siRNA) was used to silence SEMA4A, while recombinant human protein SEMA4A (rhSEMA4A) was used to imitate the soluble form of SEMA4A.2.2 MTS assay and transwell assay were used to identify the capability of proliferation and migration after certain treatments, respectively.2.3 RT-qPCR and Western Blot were used to detect the changes of EMT markers on mRNA and protein levels, respectively. And the effective receptor was identified.3. The function and underlying mechanism of SEMA4A in prostatic tumor microenvironment.3.1 Human cytokine array panel A array kit and ELISA assay were used to determine the effect of SEMA4A on WPMY-1 and its cytokine secretion. The effective receptor was further identified.3.2 Transwell assay was performed to test the effect of IL-10 on cell migration.3.3 Indirectly co-culture and transwell assay were combined to test the effect of tumor environment on cell migration. In addition, siRNA was applied to down regulate the expression of SEMA4A and neutralizing antibody was applied to block the effect of IL-10, we identified the function of SEMA4A and IL-10 in this system.3.4 Inject cancer cells with or without stromal cells, as well as with or without SEMA4A into nude mice and compare the number of lung metastatic foci.3.5 Western Blot was conducted to detected regulation of STAT3 on the expression of SEMA4A, and Chromatin Immunoprecipitation (ChIP) assay was performed to determine the binding site of p-STAT3 in SEMA4A promoter.3.6 STAT3 selective inhibitor Stattic was used to investigate possible approach of SEMA4A function in tumor microenvironment.Results:1. Compared with normal prostatic and BPH tissues, SEMA4A overexpressed in Chinese PCa tissues. In addition, expression of SEMA4A was exclusively detected on the membrane of cancer cells, but not in cytoplasm of cancer cells or in stromal cells. Furthermore, SEMA4A expression was correlated with Gleason scores and tumor metastasis.2. SEMA4A promoted PCa cells migration in vitro, but did not affect the proliferation ability. Further, SEMA4A could promote EMT progression of PCa cells, and that might be a critical mechanism of tumor cells migration.3. SEMA4A bound to NRP-1 and promoted IL-10 secretion of stromal cells, in turn, IL-10 could affect cancer cells and up-regulated the expression of SEMA4A. SEMA4A and IL-10 cooperated to promote migration of PCa cells. In addition, IL-10 could activate STAT3 signaling in cancer cells and the activated STAT3 (p-STAT3) might bind to SEMA4A promoter directly, regulating the transcription and expression of SEMA4A. SEMA4A/IL-10/STAT3 formed positive feedback loop in prostatic tumor microenvironment, and promoted EMT progression and metastasis in PCa.Conclusions:SEMA4A could promote PCa progression and played a critical role in regulating tumor microenvironment. |
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