Expression Of A New Cancer-oocyte Gene SAS1B In Human Gastric Cancer And Thyroid Cancer And Its Biological Significance

The SAS1B protein(Sperm acrosomal SLLP1 binding protein,SAS1B)1 is a newly discovered gene which was first discovered on the surface of mature oocyte cells and later found in female-related cancers,such as uterine cancer and ovarian cancer.Cancer chemotherapy is the most common treatment in the clinic,but it is non-specific killing.While killing cancer cells,normal cells are also eliminated.If chemotherapeutic drugs only recognize cancer cells and kill them specifically,it will undoubtedly bring good news to cancer patients,but there has been no important breakthrough in the research for finding specific antigens on the surface of cancer cells.If SAS1B protein is only expressed on the surface of cancer cells,this will inevitably provide a powerful target for cancer treatment,but the function of SAS1B gene is currently unclear.In this study,CRISPR/Cas9 gene modification,gene cloning,flow cytometry,real-time fluorescent quantitative PCR,Western Blot,immunohistochemical staining,protein purification,antibody preparation,ELISA and other techniques were used to test human tumor tissue,gastric cancer cell lines,mice embryos and adult tissues.It is a study of multi-angle,all-round and in-depth investigation for SAS1B gene which aiming to reveal the biological significance of SAS1B gene and providing a solid basis for the development of anti-cancer drugs,early diagnosis of tumors and reproductive medicine.(1)The prokaryotic expression plasmid pET-22b-SAS 1B was successfully constructed by gene cloning technology,BL21(DE3)cells was transformed,and the recombinant protein of SAS1B was successfully prepared by AKTA pure 25 protein purification instrument and Millipore 10 kDa ultrafiltration tube.Rabbits were immunized with purified SAS1B protein to prepare SAS1B polyclonal antibodies;antibody titers were identified by indirect ELISA;antibody reactivity was detected with SAS1B recombinant protein by Western Blot method;the expression of SAS1B protein in gastric and thyroid cancer tissues was detected by immunohistochemistry and its sensitivity to gastric and thyroid cancer was evaluated.SAS1B in sera of patients with thyroid and gastric cancer was detected by indirect ELISA method.The results showed that the prepared rabbit anti-human SAS1B polyclonal antibody had a serum titer of 1:25600.The SAS1B gene in gastric cancer and thyroid cancer tissues was expressed in the cytoplasm and membrane of cancer cells,and SAS1B was not expressed or lowly expressed in adjacent tissues(P<0.05),.The expression of SAS1B protein in serum of patients with gastric cancer and thyroid cancer was significantly higher than that sample of the control group.The expression of SAS1B protein in serum of patients with thyroid cancer was significantly different from that in the control group(P<0.05),ROC curve shows that SAS1B antibody is very good in the diagnosis of gastric cancer and thyroid cancer Sensitivity.The research results further suggest that the SAS1B expression products mainly exist in the cell membrane and part of the cytoplasm,and have the characteristics of secreted proteins.The SAS1B serological examination results undoubtedly provide a more economical and convenient early screening detection method for clinical gastric cancer and thyroid cancer.At the same time,the specific expression of SAS1B in gastric cancer and thyroid cancer tissues suggests that SAS1B is a very promising target.(2)CRISPR/Cas9 gene editing technology was used to construct pYSY--Cas9--SAS1B-gRNA1-eGFP and pYSY-Cas9-SAS1B1-gRNA2-Puromycin plasmids,and gene cloning was used to construct pcDNA3.1+/C-(K)-SAS1B Overexpression plasmid.Using cell transfection technology to study gastric cancer SGC 7901 cells,it was found that SAS1B gene can up-regulate the expression of centrosome replication kinase Aurora-A and the expression of Bard1(P<0.05),which is closely related to proliferation,which accelerates the DNA replication process including centrosome replication and promotes cell proliferation.Gastric cancer cells knocked out of the SAS1B gene down-regulated the expression of Aurora-A and Bardl(P<0.05),which caused the cell cycle to be blocked in S phase,resulting in the death of gastric cancer cells.The results further suggests that SAS1B gene can be used as a new specific target for the diagnosis and treatment of gastric cancer cells,and it is also a key target for the development of new clinical anti-tumor drugs.(3)The SAS1B gene was expressed during mouse embryo development.During mouse embryonic development,the expression level of SAS1B gene was higher than those in E7.5,E8.5,and E9.5(P<0.05).During mouse embryo development,Bardl gene and Aurora-A kinase showed an upward trends,especially the rise of Bardl was very obvious(P<0.05).SAS1B gene expression in adult mouse tissues was restricted.In the study,SAS1B protein expression was found in the kidney and testis,but no SAS1B gene expression product was detected in the liver,small intestine,spleen and lung.Studies have shown that the SAS1B gene is a novel embryo-cancer antigen.In summary,the cancer-oocyte gene SAS1B in gastric cancer and thyroid cancer groups was expressed higher than the adjacent control tissues.SAS1B protein had secretory function.The serum SAS1B concentration in gastric cancer and thyroid cancer patients was higher than that in the normal control group.When Bardl gene was up-regulated by SAS1B gene,centrosome amplification kinase Aurora-A was highly expressed to promote tumorigenesis.Bardl gene was down-regulated after the SAS1B gene knocked out,which resulted in the suppression of Aurora-A expression,the cell cycle was blocked in the S phase,and cell death was induced.Animal experiments had confirmed that the SAS1B gene could be expressed in mouse embryos,and adult tissues had restricted expression characteristics.It further illustrated that the SAS1B gene was a specific biomarker for tumor cell and was of great significance for the early diagnosis and therapy for gastric and thyroid cancer.The findings suggest huge business opportunities.