Effect Of FOXC1on Proliferation Of Human Gastric Adenocarcinoma Cell SGC-7901and Biological Behaviours Of Gastric Adenocarcinoma

Objective: To investigate the role of FOXC1in human gastricadenocarcinoma cell SGC-7901by constructing the recombinant plasmidpshRNA-FOXC1, and to detect the expression of FOXC1in gastricadenocarcinoma tissues and analyze the relations between FOXC1andbiological behaviours of gastric adenocarcinoma.Methods:1. Three DNA oligonucleotides containing small hairpinstructure were designed and synthesized. The recombinant plasmids wereobtained by annealing and being cloned into vector pGensil-1. Theplasmids pshRNA-FOXC1_a, pshRNA-FOXC1_band pshRNA-FOXC1cwereisolated, then verified by double-enzyme cleavage method and DNAsequencing, and were transfected into SGC-7901cell by liposome2000.The transfection efficiency was detected under fluorescent microscopy.2. The expression of FOXC1at the levels of mRNA and protein inhuman gastric adenocarcinoma cell SGC-7901and gastric mucosalepithelium cell GES-1was detected by RT-PCR and Western blot respectively.3. The cells were divided into five groups: pshRNA-FOXC1_agroup,pshRNA-FOXC1_bgroup, pshRNA-FOXC1cgroup, pGenesil-1group andSGC-7901group. The expression of FOXC1at the levels of mRNA andprotein in each group was detected by RT-PCR and Western blotrespectively. Cell proliferation was detected by MTT assay, and the cellcycle was analyzed by flow cytometry.4. The expression of FOXC1in gastric adenocarcinoma tissues wasdetected by immunohistochemical method. The relations between FOXC1and biological behaviours were analyzed.Results:1. The results of double-enzyme cleavage method and DNAsequencing showed that the recombinant plasmids pshRNA-FOXC1_a,pshRNA-FOXC1_band pshRNA-FOXC1cwere successfully constructed.The green fluorescence protein was detected under fluorescent microscopy.2. The expression of FOXC1was both detected in SGC-7901cell andGES-1cell. The results of RT-PCR showed: FOXC1mRNA in SGC-7901cell （1.37±0.16） was significantly higher than that in GES-1cell （0.70±0.06）（P<0.05）. The results of western blot showed: FOXC1protein inSGC-7901cell （0.70±0.03） was significantly higher than that in GES-1cell（0.15±0.05）（P<0.01）.3.After transfection, FOXC1mRNA in pshRNA-FOXC1_agroup （62.38±5.61）, pshRNA-FOXC1_bgroup （64.07±4.01） and pshRNA-FOXC1cgroup （62.14±7.07） was markedly lower than that inpGenesil-1group （84.36±6.99） and in SGC-7901group （84.61±7.18）respectively （P<0.05）. The results of Western blot showed: FOXC1protein in pshRNA-FOXC1_agroup （38.44±1.90）, pshRNA-FOXC1_bgroup （39.20±0.27） and pshRNA-FOXC1cgroup （38.25±2.03） wasmarkedly lower than that in pGenesil-1group （63.82±3.08） and inSGC-7901group （65.03±2.52） respectively （P<0.01）.4. The results of MTT showed: after transfection,the inhibition ratio ofcellular proliferation at24h,48h and72h in pshRNA-FOXC1_agroup,pshRNA-FOXC1_bgroup and pshRNA-FOXC1cgroup wassignificantly higher than that in pGensil-1group respectively （P<0.05）.The percentage of cells at G_1and G₂in pshRNA-FOXC1_agroup,pshRNA-FOXC1_b group, pshRNA-FOXC1c group wassignificantly more than that in SGC-7901group respectively （P<0.05）, thepercentage of cells at S in pshRNA-FOXC1_a group, pshRNA-FOXC1_bgroup, pshRNA-FOXC1c group was significantly less than that inSGC-7901group respectively （P<0.05）. The percentage of cells at G₁,G₂and S in pGensil-1group and SGC7901group had no statisticallysignificant （P>0.05）.5. In25gastric adenocarcinoma tissues, the expression rate of FOXC1protein was62.5%. FOXC1protein was detected in cell nucleus and cytoplasm. The expression rate of FOXC1in lymph node transfer group（75%） was markedly higher than that in no lymph node transfer group（33.3%）（P<0.05）.Conclusion: FOXC1is highly expressed in human gastricadenocarcinoma cell SGC-7901. After reducing FOXC1expression inSGC-7901cell, the the cell cycle is blocked at G₁, G₂and the cellularproliferation is inhibited. FOXC1may play an important role in gastricadenocarcinoma with lymph node transfer.