Research On The Expression Of SSRP1 And Its Functional Mechanisms In Gastric Cancer

Part One: The expression of SSRP1 in gastric cancer tissues and gastric cancer cell lines and analysis of its relavance with gastric cancer clinicopathological characteristicsObjective:To examine the expression profiles of SSRP1 in gastric cancer tissues and gastric cancer cell lines and a normal gastric epithelia derived cell line;To reveal the correlation of SSRP1 expression levels with clinicopathological characteristics.Methods:1.Search and download the GEO dataset for public datasets containing the mRNA expression profiles of paired gastric cancer tissues and compare the SSRP1 mRNA levels of cancerous tissue with adjacent normal tissue;2.Search for gastric cancer patients' clinical and pathological information in Renmin Hospital of Wuhan University and select paraffin-embeded specimens and make slices.The selected tissues should contain paired gastric cancer tissues and well differentiated,moderately differentiatied and poorly differentiated cancers.Determine the expression profile of SSRP1 expression by immunohistochemistry staining method and analyse SSRP1 expression profiles with clinicopathological characteristis;Collect fresh cancer tissues(paired cancerous and adjacent normal tissue)in gastrointestinal surgery department operation room,and then determine SSRP1 protein expression with Western blot;3.Determine the protein and mRNA expression levels of SSRP1 in three gastric cancer cell lines AGS,HGC-27,SGC-7901 and one normal gastric epithelia cell line by Western blot and RT-q PCR.Results:1.Two mRNA expression datasets containing 45 pairs and 80 pairs of gastric cancer tissues and adjacent normal tissues each were found and downloaded in NCBI GEO Datasets.Analysis of the two datasets revealed that SSRP1 mRNA expression levels were significantly higher in gastric cancer tissues than those in adjacent normal tissues(both P<0.001).2.21 pairs of gastric cancer tissue slices and adjacent normal gastric tissues slices were abtained from the pathology department of Renmin Hospital of Wuhan University.Immunohistochemistry detection of SSRP1 showed that SSRP1 was mainly present in the nucleus in cancerous gastric gland cells,and SSRP1 was barely present in cytoplasma,membrane or the stroma tissue.The expression level of SSRP1 in cancerous gland cells was significantly higher than that in adjacent normal gland cells(P<0.05).The expression of SSRP1 was significantly higher in cancerous tissues than that of adjacent normal tissues as determined by WB analysis of fresh paired gastric cancer tissues.A total of 68 gastric cancer paraffin-embeded slices were collected and IHC results indicated that SSRP1 expression was associated with tumor size,differentiation status,Lauren classification and TNM stage(all P<0.05);3.The mRNA and protein expression levels of SSRP1 in the three gastric cancer cell lines AGS?HGC-27?SGC-7901 were significantly higher than that in the normal gastric epithelial cell line GES-1.Conclusions:The expression levels of SSRP1 were siginificantly higher in cancerous tissues than those in normal gastric tissues,and higher SSRP1 expression indicated a more malignant status.These observations hinted that SSRP1 can be a gastric cancer-related gene.Part Two: The biological function of SSRP1 in gastric cancer cellsObjective:To study the biological function of SSRP1 in gastric cell lines.Methods:1.Search and download public dataset of mRNA expression of gastric adenocarcinoma in NCBI GEO Data Set,predict the function of SSRP1 by GSEA;2.Knockdown of SSRP1 in AGS and HGC-27 with RNAi method and overexpress SSRP1 in SGC7901 with transfection of SSRP1 expression plasmid.Detect the expression level of SSRP1 in the transfected cells by Western blot and RT-q PCR;3.Examing the proliferation activity of SSRP1 knockdown/overexpressed cells or cells treated with SSRP1 inhibittor with CCK-8 kit or Ed U cell proliferation kit;4.Test the colony forming abilities of SSRP1 knockdown or overexpressed cells;5.Use flow cytometry to determine the effect of SSRP1 knockdown or inhibition on cell cycle and apoptosis.Results:1.One mRNA expression dataset consisting of 192 cases of gastric cancer was downloaded and analysed with GSEA.GSEA results indicated that SSRP1 may promote embryonic stem cell phenotype(P<0.001,FDR=0.003,ES=-0.74),early gastric cancer(P<0.001,FDR=0.014,ES=-0.69)and DNA repair(P<0.001,FDR<0.001,ES=-0.63);2.All three si RNAs transfection or expression plasmid overexpression significantly downregulated or upregulated the expression of SSRP1,which were confirmed by Western blot and RT-q PCR;3.CCK-8 assay showed that SSRP1 knockdown or inhibition blunted the proliferation activity of AGS and HGC-27;Cells treated with elevated concentrations of SSRP1 inhibitors presented with weaker proliferative activities,and the weakening extent was positively correlated the expression levels of cell lines,SSRP1 inhibitor had the least influence on proliferation activity of GES-1;4.After knockdown of SSRP1,the colony forming abilities decreased accordingly,and overexpression of SSRP1 promoted the colony forming abilities;5.Flow cytometry showed that cell cycle was arrested in S phase,and apoptosis rate increased significantly in SSRP1 knockdown/inhibited cells.Conclusion:In gastric cancer cells,SSRP1 may play its role by modulating cell proliferation,cell cycle and apoptosis.SSRP1 inhibitor may be useful in eradicating cells that express higher levels of SSRP1.Part Three: Mechanism study of SSRP1 abberant expression and cell death caused by SSRP1 inhibitionObjective:To clarify the molecular mechanism that leads to SSRP1 abberantly higher expression in gastric cancer and to study the mechanism of cell death caused by SSRP1 inhibition.Methods:1.Search and download micro RNA expression dataset of normal gastric epithelia and gastric cancer mucosa from NCBI GEO Data Set,analyse the differential expression of mi R-497 in normal and cancerous gastric tissues.Transfect AGS and HGC-27 cells with mi R-497 mimics,and confirm transfection efficiency by Western blot.Detect SSRP1 expression in our previously constructed mi R-497 knockout mice with immunohistochemistry and Western blot to determine the relationship between SSRP1 and mi R-497 in vivo;Examine the histology changes in mi R-497 knockout mice with HE staining.2.Detect PTEN?AKT?p AKT?P53?c-MYC?Beclin1?ATG5?STING?ISG15?IFIT3?IRF7?IFNB1 protein or mRNA expression by Western blot or RT-q PCR in SSRP1 inhibited cells;Examine P65 expression profiles with immunofluorescence imaging in SSRP1 inhibited cells.Results:1.In a public dataset GSE26595,mi R-497 expression was significantly higher in normal gastric mucosa than the cancerous gastric mucosa.The expression of mRNA of SSRP1 droped down significantly after mi R-497 mimics transfection.mi R-497 knockout was confirmed,immunohistochemistry and Western blot showed that SSRP1 staining is stronger in stomach of mi R-497 knockout mice than the wildtype mice;HE staining found that low grade neoplasia developed in mi R-497 knockout mice;2.In the SSRP1 inhibited cells,SSRP1 protein distribution changed in the nucleus and the expression PTEN?P53?Beclin1?ATG5?Cleaved caspase3?PARP1?STING?ISG15?IFNB1?IFIT3 increased,and the epression of p AKT(ser473)?c-MYC decreased,P65 level decreased in the nucleus.Conclusion:Decreased mi R-497 may lead to higher expression of SSRP1 and promote gastric cancer.SSRP1 inhibitor may act as an anti-cancer agent via regulating PTEN,P53,NF-?B,c-MYC,c GAS-STING and autophagy signaling pathway.