| Section 1.Screening of differentially expressed genes related to epithelial-mesenchymal transition in peritoneal metastasis of gastric cancerObjective:As the third most malignant cancer in the world,the incidence and mortality of gastric cancer are extremely high.Gastric cancer is a major threat to the health of patients.The most difficult problem in the treatment of gastric cancer is metastasis of gastric cancer cells.Peritoneal metastasis is the most common in patients with gastric cancer metastasis.At present,the molecular mechanism of peritoneal metastasis in gastric cancer is still unclear,therefore it is very important to explore the mechanism of peritoneal metastasis in gastric cancer.It has been found that posttranscriptional regulation plays an important role in peritoneal metastasis of gastric cancer.Therefore,we aimed to screens differentially expressed genes at the translation level during peritoneal metastasis of gastric cancer in this section,in order to find molecular targets for peritoneal metastasis therapy.The results of the first part of the experiment showed that the expression level of STEAP1 in tumor tissue of patients with gastric cancer peritoneal metastasis and HMrSV5 cells transfected with miR-3978 antagonist was significantly up-regulated,and STEAP1 is the most prominent gene.STEAP1 was found to be significantly up-regulated in cancers such as prostate cancer,bladder cancer,and Ewing sarcoma,and STEAP1 could regulate the cell proliferation,invasion and migration ability of cancer cells in vitro,and regulate tumor growth ability in vivo.However,whether STEAP1 was involved in the regulation of cell proliferation,invasion and migration ability of gastric cancer cells was still unclear Therefore,this study was to explore the influence of STEAP1 expression level on the tumorigenesis of gastric cancer and the sensitivity of tumor cells to chemotherapeutic drugs.Methods:In this study,20 patients with peritoneal metastasis of gastric cancer were collected for the isolation of polyribosomes from cancer tissues and adjacent normal tissues.The levels of differently expressed genes related to epithelial-mesenchymal transformation(EMT)in polyribosomes between gastric cancer tissues and adjacent normal tissues were analyzed by qRT-PCR.Then human peritoneal epithelial cell line HMRSv5 transfected with mir-3978 antagonist was used to simulate peritoneal metastasis.Polyribosomes were extracted from HMRSv5 cells transfected with mir-3978 antagonist and HMRSv5 cells without transfection of mir-3978 antagonist.Then,qRT-PCR was used to analyze the expression level of genes related to epithelial-mesenchymal transition in polyribosomes.Gastric cancer cell line MKN45 were transfected with siRNA-STEAP1 to decrease the expression of STEAP1 in gastric cancer cells.After the expression level of STEP1 was down-regulated,MTT assay was used to detect the proliferation of gastric cancer cells,Trans well cell migration assay was used to detect the migration ability of gastric cancer cells,and Transwell cell invasion assay was used to detect the invasion ability of gastric cancer cells.Rat xenografts were established by MKN45 cells which were stably transfected with shRNA-STEAP1.MTT assay showed that the sensitivity of MKN45 cells to docetaxel after the expression of STEAP1 decreased.Results:The expressions of 49 genes have significant differences in between tumor tissues and normal tissues in patients with gastric cancer.The expression levels of 30 genes in gastric cancer patients with peritoneal metastasis were significantly higher than that in normal tissues,and 19 genes in gastric cancer patients with peritoneal metastasis were significantly lower than that in normal tissues.The top ten genes with significantly up-regulated gene expression level were STEAP1,SPP1,VPS 13A,MMP2,BMP1,ZEB1,TBP,ESR1,ZEB2 and MMP9.STEAP1 is the most prominent gene in cancer tissues.The top ten down-regulated genes were CDH1,KRT14,CALD1,FGFBP1,EGFR,CAV2,IL1RN,SPARC,GNG11 and DSP.Compared with HMRSv5 cells that were not transfected with the antagonist of microRNA-3978,59 genes related to epithelial-mesenchymal transformation were significantly changed in HMRSv5 cells transfected with the antagonist of microRNA-3978,of which 42 genes were significantly up-regulated and 17 genes were significantly down-regulated.The top ten up-regulated genes were STEAP1,VCAN,SPP1,VPS 13 A,KRT19,FN1,TMEM132A,TGFB3,ZEB2 and MMP9.STEAP1 is the most prominent gene in HMRSv5 cells transfected with mir-3978 antagonist.The top ten down-regulated genes were KRT14,CDH1,FGFBP1,EGFR,CAV2,CAMK2N1,ERBB3,CALD1,SNAI2 and SNAI1.Forty-one genes were differentially expressed in tumor tissues of gastric cancer patients with peritoneal metastasis and HMrSV5 cells transfected with miR-3978 antagonist.After transfection of siRNA-STEAP1 into gastric cancer cell line MKN45,qRT-PCR results found that the expression of STEAP1 was significantly down-regulated.The down-regulation of STEAP1 expression could significantly inhibit the proliferation,migration and invasion of MKN45 cells in vitro.The rat's xenograft model was established by MKN45 cells which were stably transfected with two kinds of shRNA-STEAP1.After the expression of STEAP1 was significantly down-regulated,the tumor growth was effectively inhibited.In HMRSV5 cells,up-regulation of STEAP1 expression enhanced the cell viability,that is,the sensitivity of HMRSV5 cells to docetaxel decreased after the expression of STEAP1 increased.In MKN45 cells,down-regulation of STEAP1 expression decreased the viability of MKN45 cells,that is,the sensitivity of HMRSV5 cells to docetaxel increased after the expression of STEAP1 decreased.Conclusion:1.There were 49 EMT related genes differentially expressed in the polyribosomes of peritoneal metastasis patients with gastric cancer,including 30 up-regulated genes and 19 down-regulated genes.STEAP1 is the most prominent gene in gastric cancer tissues2.There were 59 EMT related genes differentially expressed in the transfected miR-3978 antagonist-transfected HMrSV5 cell polyribosome,including 42 up-regulated genes and 17 down-regulated genes.STEAP1 is the most prominent gene in HMRSv5 cells transfected with mir-3978 antagonist.3.There were 41 differentially expressed genes in tumor tissues of gastric cancer patients with peritoneal metastasis and HMrSV5 cells transfected with miR-3978 antagonist,indicating the similarity between the two chosen experimental models.4.In gastric cancer cell line MKN45,the down-regualtion of STEAP1 decreased cell proliferation ability,migration ability and invasion ability of MKN45 cells in vitro.5.The tumor growth of xenografts tumor established by MKN45 cells was significantly inhibited by down-regulation of STEAP1 expression.6.Upregulation of STEAP1 expression decreased the chemosensitivity of human mesothelial cell line HMrSV5 to docetaxel.Downregulation of STEAP1 expression significantly increased the chemosensitivity of MKN45 cells to docetaxel.7.STEAP1 may be a potential biological target for the treatment of gastric cancer metastases. |
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