Preliminary Studies On Functions Of FBG2, BTG2 And S100A6 Genes In Human Gastric Cancer Cell Lines

Background Gastric cancer is a very important threat to human heath and one of the leading causes of deaths worldwide. In China, incidence and mortality of the disease have been highest in all kinds of cancers in recent one hundred years. Further more its incidence and mortality did not descend with gastroscopy gaining ground and standards of living improving. Some new genes which could be related to gastric cancer have been found and elucidated recently,so human knowledge of the fatal disease has been enriched on some extent. Some researches have been performed and some valuable results have been gained. The different gene expression patterns of gastric cancer vs matching nonneoplastic gastric mucosa previously were reported by using complementary DNA(cDNA) microarry technology by doctor Yang. In the differentially expressed genes, the functions of FBG2, BTG2 and S100A6 were not clear and could play some important roles in occurring and developing of gastric cancer.Aim â‘ To elucidate the different gene expression patterns of FBG2, BTG2 and S100A6 genes in MKN45, SGC7901 and HFE145 gastric cancer cell lines and prepare for function researches. â‘¡ To investigate thefunctions of FBG2, BTG2 and S100A6 genes in gastric cancer by establishing the cell lines which could express the genes highly and analyzing the changes of some pivotal biologic functions. ? To elucidate the functions of interrelated genes in gastric cancer by restraining the expression of those genes in suited gastric cancer cell lines and analyzing the changes of some pivotal biologic functions. Methods (Dlmmunohistochemistry , RT-PCR were used to analyze the gene expression patterns of FBG2, BTG2 and S100A6 genes in MKN45, SGC7901 and HFE145 gastric cancer cell lines. ?FBG2.BTG2 and S100A6 cDNA was gained by RT-PCR using special primers and subcloned into a constitutive vector PCDNA3.1 in order to construction of the genes expression vectors in eukaryotic cell. ?The effective silengcing sequence to S100A6 was selected, designed and synthesized based on the sequence of S100A6 mRNA. They were separately subcloned into the plasmid of IMG800 containing U6 promoter. The RNA interference eukaryotic expression vector specific to S100A6 gene were constructed. ?The constructed genes espression vectors were transfected into MKN45 cells and RNAi vectors were transfected into SGC7901 by using liposome. Then stable expression clones were selected by being screened with G418 and appraised by Immunohistochemistry, RT-PCR and Westen-bloting methods. ?The changes of some pivotal biologic functions of the stable expression clones vs their original gastric cancer cell lines were analyzed. The apoptosis and cell cycles were detected using flow cytometry.The growth and proliferation were analyzed by making cell growth curves, PCNA dyeing and colony formation assay respectively. The ability of infiltration were tested using cancer cell migration assay.Result: ?It was confirmed by Immunohistochemistry and RT-PCR that there were no or very low expressions of FBG2 or BTG2 in MKN45, SGC7901and HFE145 cell lines, a very high expression of S100A6 in SGC7901 and no or very low expressions of S100A6 in MKN45 and HFE145. (2)It was verified by DNA sequencing of the plasmid that the construction of the genes expression vectors of FBG2, BTG2 and S100A6 genes in eukaryotic cell and the RNA interference eukaryotic expression vector specific to S100A6 gene were successful. ?The stable cell lines which could express FBG2,BTG2 and S100A6 genes highly respectively were established and appraised by Immunohistochemistry, RT-PCR and Westen-bloting methods. The stable cell lines whose expression of S100A6 gene were suppressed by 75% in mRNA level or 85% in protein level appraised by Immunohistochemistry, RT-PCR and Westen-bloting methods. ?The changes of some pivotal biologic functions of the stable expression clones vs their original gastric cancer cell lines were analyzed successfully. MKN-FBG2 grew faster than MKN45 and MKN-PC. The cell counts of MKN-FBG2 in the forth, fifth, sixth and seventh days were significantly more than that of others(P<0.05). Cell cycle analysis showed that MKN-FBG2 proliferated faster, proportions of cells in G2-M and S were different significantly(P<0.05). Results of colony formation assay showed that the colony formation rate of MKN-FBG2 was higher than that of control groups (P<0. 05),but the results of cell migration assay were negative. ?MKN-BTG2 grew slower than MKN45 and MKN-PC. The cell counts of MKN-BTG2 in the third, forth, fifth, sixth and seventh days were significantly fewer than that of others (P<0. 05). Cell cycle analysis showed that MKN-BTG2 proliferated slower, proportions of cells in G0-G1 and G2-M S were different significantly(P<0. 05). Results of colony formation assay showed that the colony formation rate of MKN- BTG2 was lower than that of control groups (P<0. 05)and the results of cell migration assay suggested that the cell migration rate of MKN- BTG2 was lower than that of control groups (P<0.05). ?MKN-S100A6 did not grow faster or slower than MKN45 and MKN-PC. The cell counts of MKN-S100A6 in the all seven day were not significantly different with that of others(P<0. 05). Cell cycle analysis showed that MKN-S100A6 proliferated faster, proportions of cells in G2-M were higher significantly than that of control groups (P<0. 05). Results of colony formation assay showed that the colony formation rate of MKN-S100A6 was higher than that of control groups (P<0. 05), but the results of cell migration assay were negative too. The stable RNAi for S100A6 cell lines grow slower significantly than SGC7901 and SGC-IMG. The cell counts in the fifth, sixth and seventh days were significantly different with that of others(P<0. 05). Cell cycle analysis showed that the stable RNAi for S100A6 cell lines proliferated slower, proportions of cells in G0-G1 were higher and proportions in G2-M and S were lower significantly than that of control groups(P<0.05). Cell apoptosis analysis showed that proportions of apoptosis cells were higher than that of control groups(P<0.05). Results of colony formation assay showed that the colony formation rate of S100A6 RNAi cell lines was lower than that of control groups (P<0.05). Further more, the cell migration rate of S100A6 RNAi cell lines was much higher than that of control groups (P<0. 05).Conclusions ?FBG2 can promote the growth and proliferation of gastric cancer cells and help tumor cell maintain malignant phenotype. But it can have a negative influence on infiltration and metastasis of gastric cancer cells. ?BTG2 can restrain the growth and proliferation of gastric cancer cells and restrict tumor cell to maintain malignant phenotype. Further more, it can restrict infiltration and metastasis of gastric cancer cells. (3)S100A6 also can promote the growth and proliferation and restrain apoptosis of gastric cancer cells. It can help tumor cell maintain malignant phenotype andpromote infiltration and metastasis of gastric cancer cells.