The Study Of Expression And Roles Of Semaphorin 5A, An Axon Guidance Factor, In Human Gastric Cancer And Its Molecular Mechanisms

Gastric cancer (GC) is one of the most common malignancies with high mortality in the world. One of the main reasons for this is that the exact molecular events responsible for the development and progression of gastric cancer are still largely unclear. Therefore, it is of great clinical value to identify such endogenous factors, as should lead not only to a better understanding of the processes of tumor cell progression and metastasis, but may also find valuable diagnostic markers as well as novel therapeutic strategies.Semaphorin 5A, a member of Semaphorin family, was initially identified as axonal guidance factor responsible for the guidance of growing axons to their targets during the development of the central nervous system. However, a growing body of evidence to date indicates that they are expressed in a variety of tissues, and have a broader spectrum of functions outside the nervous system. Noticeablly, certain Semaphorins have been found to play a regulatory role in tumorigenesis and in the process of tumor formation. By the way of comparison, few investigations and studies have been done on the potential roles and expression of Semaphorin 5A from Semaphorin family in human tumors including gastric cancer until now. Therefore, we design the study to examine the expression and functional roles of Semaphorin 5A in gastric cancer and its mechanisms.ObjectiveTo detect the expression of Semaphorin 5A in gastric cancer and explore its roles in the metastasis and invasion of gastric cancer so as to add more weight to the understanding of mechanisms of gastric carcinogenesis, and provide theoretical and experimental evidence for the gene therapy of GS through silencing Semaphorin 5A gene.Methods(1) The expression of Semaphorin 5A was detected by RT-PCR and Western blotting assay in gastric cancer cell. Immunohistochemistry, real-time RT-PCR and Western blotting assay were used to examine the expression of Semaphorin 5A in primary gastric cancer and corresponding normal gastric mucosa tissues, and to analyze its correlation with clinicopathological data.(2) pGen-1-SEMA 5A targeted Semaphorin 5A and pGen-1-Scrambled vector as negative control were constructed by using recombinant techniques such as restrictive cleavage, sequence and the like, transfected into gastric carcinoma cell line SGC7901 with liposome, respectively. After the acquirement of positive colonies with G418. Expression of Semaphorin 5A protein and mRNA in the transfected and parental SGC7901 cells was examined by Western blotting and RT-PCR, respectively.(3) The effect of siRNA-Semaphorin 5A on the biological effects of gastric cancer cell such as growth, apoptosis, invasiveness and such on were examined by MTT, anchorage-independent growth, flow cytometry as well as wound healing and transwell assays.(4) The expression of MMP2, MMP9, uPA, ERK phosphorylation were examined by using ELISA, RT-PCR assays in si-Sema 5A and SGC7901. Invasiveness ability in si-Sema 5A and SGC7901 cells with or without anti-MMP2, MMP9, uPA antibody and PD98059 was compared to determine whether Semaphorin 5A promotes the invasion and metastasis of gastric cancer by regulating MMP9 and uPA expression via the MEK/ERKs signal transduction pathway.(5) The expression of TGF-β1, PAI-1, Smad2 phosphorylation were examined by using ELISA, RT-PCR or Western blotting assays in si-Sema 5A and SGC7901, to determine whether Semaphorin 5A activate TGF-β1. Invasiveness ability and pERK caused by siRNA-TGF-(31 in si-Sema 5A and SGC7901 were compared to determine whether Semaphorin 5A promotes the invasion and metastasis of gastric cancer depending on activating TGF-β1 to regulate MMP9, uPA expression via the MEK/ERKs signal transduction pathway.Results(1) The semaphorin 5A was ubiquitously expressed in all gastric cancer cell lines examined and its expression was higher in SGC7901, MKN45 than that in AGS, SNU-1.The expression level of semaphorin 5A was significantly higher in tumor tissues than those in normal mucosa tissues, which was close correlation with lymph nodal metastasis and TNM and differentiation, but not age, gender, the depth of invasion.(2) siRNA-Semaphorin 5A inhibited the growth, anchorage-independent growth, invasion, and enhanced apoptosis of gastric cancer.(3) When compared to SGC7901, the expression of MMP9, uPA, ERK phosphorylation were downregulated but no difference in the expression of MMP2 in si-Sema 5A. Anti-MMP9, uPA antibodies remarkably inhibited the invasive ability of SGC7901 as compared with si-Sema 5A. Except for reducing the invasive ability of SGC7901, PD98059 significantly downregulated the expression of MMP9, uPA in SGC7901 compared to si-Sema 5A.(4) As compared with SGC7901, the expression of TGF-β1, PAI-1, Smad2 phosphorylation were downregulated, siRNA-TGF-β1 decrease ERKs phosphorylation in si-Sema 5A.Conclusion(1) Axon guidance factor Semaphorin 5A was overexpressed in gastric cancer tissue and gastric cancer cells, its expression was correlated positively with the development and progression of gastric cancer.(2) Semaphorin 5A promoted the invasion and metastasis of gastric cancer depending on activating TGF-β1 to regulate MMP9, uPA expression via the MEK/ERKs signal transduction pathway.