The Influence Of Silencing PRSS1 On Gastric Cancer MGC803 Cell Of Migration And Invasion

[Objective]Through silencing PRSS1 gene expression in the gastric cancer MGC803 cell,observed in human gastric mucous poorly differentiated adenocarcinoma MGC803 cell migration and invasion, and to explore the impact PRSS1 gastric cancer MGC803 cell proliferation, migration and molecμlar mechanisms of invasion.[Methods] 1.Through immunohistochemical staining PRSS1 protein expression in the gastric tissue microarray(including normal gastric mucosa, adjacent tissues,well-differentiated,poorly differentiated gastric adenocarcinoma and metastatic lymph nodes).2.Through the using of RNA interference techniques,synthetic pc DNA6.2?-GW/Em GFP-mi Ri-PRSS1 RNAi plasmid,transfected the gastric cancer MGC803 cells,and using the Blasticidin Blasticidin S HCl screened the PRSS1 gene stably lowest expression of transfected MGC803 /mi Ri-PRSS1 cell.And through the using of Real-time PCR and Western blot technique to detect the MGC803/mi Ri-PRSS1 cells the expression of PRSS1 m RNA and PRSS1 protein.3.Through the using of MTT assay,plate colony formation assay,scratch healing assay and Transwell migration and invasion chamber assay the silencing PRSS1 gene the expression of human gastric cancer MGC803 cell,to observe the influence of the silencing PRSS1 gene gastric cancer MGC803 cell in proliferation,migration and invasion.[Results]1.Immunohistochemical staining showed:in the high different tiation,moderately differentiated and poorly differentiated gastric adenocarcinoma and lymph node metastasis of carcinoma,the expression of PRSS1 protein increasing than in the normal gastric mucosa,and in tumor adjacent tissues the expression of PRSS1 protein increasing than in normal gastric mucosa.2.Using RNA interference technology successfully synthesized interference plasmid pc DNA6.2?-GW/Em GFP-mi Ri vector-PRSS1,and transfected into the human gastric cancer MGC803 cells,successfully constructed the PRSS1 protein stability low expression of MGC803/mi Ri-PRSS1 cell line.Detection of RT-PCR and Western blot technology,the expression of PRSS1 m RNA and protein in transfected with plasmid pc DNA6.2?-GW/Em GFP-mi Ri vector-PRSS1 of the MGC803/mi Ri-PRSS1 cells,the expression of PRSS1 m RNA and protein were reduced than non transfected MGC803 cells,and in the negative control group in MGC803/mi Ri cells between the amount of load non transfected MGC803 cells there was no significant difference of the expression of PRSS1 m RNA and protein.3.Effects of silencing the PRSS1 gene on the proliferation of MGC803 cells:1)MTT results showed that:in the without transfection MGC803 cell group of proliferation rate was 0.814±0.003,the transfected into MGC803/mi Ri-PRSS1 cells group of proliferation rate was 0.501±0.002,and the transfected with empty plasmid MGC803/mi Ri cells group of proliferation rate was 0.791±0.002.without transfection MGC803 cell group was significantly higher than that of transfected MGC803/mi Ri-PRSS1 cells was 0.313±0.001(P<0.05).Showed that silencing of PRSS1 expression after that the proliferation activity of MGC803 cells relative reduction(P<0.05).2)The plate colony formation experiment showed that:the without transfection MGC803 cell group and transfected empty plasmid MGC803/mi Ri cells colony formation were 98±5 and 89±5,compared with MGC803/mi Ri-PRSS1 group,respectively 75±3 and 66±4(P <0.05).Transfection of MGC803/mi Ri-PRSS1 cells colony formation were 23±2.Showed that expression of silencing PRSS1 after MGC803 cell colony formation ability of relatively weak(P < 0.05).4.Effects of silencing PRSS1 gene on MGC803 cells migration and invasion:1)The wound healing assay showed:after scratch 24 hours at the end of the migration distance of without transfected MGC803 cell group was 188±8um,the migration distance of transfected MGC803/mi Ri-PRSS1 cell group was 105±3um and transfected empty plasmid MGC803/mi Ri cells group was 179±6um.The without transfection MGC803 cell group and transfected empty plasmid MGC803/mi Ri cells group were transfected into MGC803/mi Ri-PRSS1 cells migration distance of group 83±5um and 74±3um(P < 0.05).Showed that expression of silencing PRSS1 after MGC803 cell migration ability to heal(P<0.05).2)Transwell migration assay showed:that in the non-transfected MGC803 cell group of cell migration was 267±36,transfected MGC803/mi Ri-PRSS1 cell group of cell migration was 60±8,and the transfected with empty plasmid MGC803/mi Ri cells group of cell migration was 245±32.Compared with the non-transfected MGC803 cell group and transfected empty plasmid MGC803/mi Ri cells group,transfection MGC803/mi Ri-PRSS1 cell group cell migration less than 207±28 and 185±24(P<0.05).The results showed that expression of silencing of PRSS1 after MGC803 cell migration ability of relatively weak(P<0.05).3)Transwell invasion assay showed that:in the non transfected MGC803 cell group and transfected empty plasmid MGC803/mi Ri cell group of transmembrane cells was 48±8 and 40±7,and the transfected MGC803/mi Ri-PRSS1 cells group of transmembrane cells was 15±4.Compared with the non transfected MGC803 cell group and transfected empty plasmid MGC803/mi Ri cells group,the transfected MGC803/mi Ri-PRSS1 cell group of transmembrane cells number less than 33±4 and 25±3(P<0.05).That after expression of silencing PRSS1 the MGC803 cells of invasion ability is decreased(P<0.05).5.The low expression of PRSS1 effect on the cell cycle of MGC803 cells.Flow cytometry showed,and transfected MGC803 cells in blank control group and negative control group compared with empty MGC803/mi Ri cells,the percentage of G0/G1 phase cells in MGC803/mi Ri-PRSS1 transfection group experiment is relatively increased,while the percentage of S is relatively lower,the results were statistically significant(P<0.05).That low expression of PRSS1 cells after MGC803 cell cycle arrest relative.[Conclusion]1.The PRSS1 protein high expression in gastric cancer group,and the tumor degree of differentiation of gastric carcinoma, lymph node metastasis and clinical classification stages are closely related.2.The expression silencing PRSS1 after the MGC803 cells with ability of proliferation、migration and invasion were decreased.