An Experimental Study Of The Impact Of Biological Behaviors Of GKLF Gene On Human Gastric Carcinoma Cell Line SGC-7901

Part 1 Expression and clinical significance of GKLF and IKLF in human gastric carcinoma tissues and cell linesObjective: To study the expression and clinical significance of GKLF and IKLF in human gastric carcinoma tissues and cell lines.Methods: Expressions of GKLF mRNA and IKLF mRNA in gastric carcinoma tissues and cell lines were detected by real-time fluorescence quantitative PCR.Expressions of GKLF protein and IKLF protein in gastric carcinoma tissues and adjacent normal gastric tissues were detected by immunohistochemistry(IHC) .Expressions of GKLF protein and IKLF protein in human normal gastric epithelium cell line (GES-1) and gastric carcinoma cell lines (MKN-28,SGC-7901,BGC-823,HGC-27)were detected respectively by Western blot and immunocytochemistry(ICC).Results: The expression level of GKLF mRNA in gastric carcinoma tissues was significantly lower than that in adjacent normal gastric tissues, the expression level of IKLF mRNA in gastric carcinoma tissues was significantly higher than that in adjacent normal gastric tissues(P <0.05).The levels of GKLF mRNA and IKLF mRNA in gastric carcinoma tissues were uncorrelated with differentiation.Positive expression rate of GKLF protein in the carcinoma tissues was significantly lower than that in adjacent normal gastric tissues .Positive expression rate of IKLF protein in the carcinoma tissues was significantly higher than that in adjacent normal gastric tissues(P <0.05).The expressions of GKLF protein and IKLF protein were uncorrelated with age,gender,differentiation and Lauren's classification.They were correlated with lymph node involvement and clinical stage(P <0.05).The levels of GKLF mRNA and GKLF protein in four gastric carcinoma cell lines were lower than that in human normal gastric epithelium cell line (GES-1) . The IKLF mRNA and IKLF protein levels in four gastric carcinoma cell lines were higher than that in GES-1(P <0.05).Conclusion:The low expression of GKLF and high expression of IKLF were existed both in gastric carcinoma tissues and cell lines, GKLF and IKLF play roles in the carcinogenesis and progression of gastric carcinoma in a way.To examine the expression of GKLF and IKLF may be useful in judging the degree and prognosis of human gastric carcinoma.Part 2 The transfection of recombinant plasmid pcDNA3.1-GKLF and the establishment of stable transfected cell lineObjective:To establish stable transfected SGC7901-pcDNA3.1-GKLF cell line which expressed high level of GKLF.To observe the impact of biological behaviors of GKLF gene on human gastric carcinoma cell line SGC-7901.Methods: The recombinant plasmid of pcDNA3.1-GKLF was transfected into SGC-7901 cell by lipofectin-mediated method.After screening culture by G418,stable transfected SGC7901-pcDNA3.1-GKLF cell line was established,and the levels of GKLF mRNA and GKLF protein were identified by real-time fluorescence quantitative PCR,Western blot and immunocytochemistry(ICC).Results: The stable transfected SGC7901-pcDNA3.1-GKLF cell line was established.The GKLF gene was expressed successfully in the cell line.Conclusion: The establishment of stable transfected SGC7901-pcDNA3.1-GKLF cell line provide solid foundation for further experimental studies in vitro and in vivo of the impact of biological behaviors of GKLF gene on human gastric carcinoma.Part 3 Experimental study of the impact of biological behaviors on human gastric carcinoma cell line SGC-7901 by GKLF gene transfection in vitroObjective:To observe the effects of GKLF gene transfection on proliferation and invasion of human gastric carcinoma cell line SGC-7901 in vitro,to study the mechanism GKLF gene in human gastric carcinoma.Methods: Proliferation and invasion ability were measured before and after transfection by MTT assay,flow cytometry,colony formation assay and cell invasion assay. The mRNA and protein levels of Cyclin D1,Ki-67,E-cadherin were detected respectively by reverse transcription-polymerase chain reaction(RT-PCR) and Western blot . Results: The proliferative speed of SGC7901-pcDNA3.1-GKLF group was markedly slower than that of SGC-7901 and SGC7901-pcDNA3.1 groups(P <0.05).GKLF gene transfection caused part of the G0/G1 arrest,decreased clone formation rate and the invasion ability(P <0.05).The mRNA and protein levels of Cyclin D1,Ki-67,E-cadherin of SGC7901-pcDNA3.1-GKLF group were lower than that of SGC-7901 and SGC7901-pcDNA3.1 groups ( P <0.05).Conclusion: Transfection with GKLF gene might suppress proliferation and invasion ability of human gastric carcinoma cell line SGC-7901 by downregulation the expressions of Cyclin D1,Ki-67,E-cadherin.Part 4 Experimental study of the impact of biological behaviors on human gastric carcinoma cell line SGC-7901 by GKLF gene transfection in vivoObjective:To observe the effects of GKLF gene transfection on growth and metastasis of human gastric carcinoma cell line SGC-7901 xenograft in nude mice,to explore the potential mechanism of GKLF gene in gastric carcinoma and to bring a new method of gene therapy for human gastric carcinoma.Methods: We carried out an in vivo study with 36 BALB/C-nu/nu nude mice which divided into two parts.Every 18 nude mice were randomly divided into 3 groups.SGC7901-pcDNA3.1-GKLF,SGC7901-pcDNA3.1,SGC-7901 were injected into 3 groups nude mice .Every group nude mice injected with the same cell and volume(2×107 cells/200μl/mouse) .In part one, cells were injected into right costal abdomen subcutis of nude mice.The mice were killed 6 weeks later after injection and the changes of weight,volume,latency,state were observed. The expressions of GKLF,Ki-67,E-cadherin,VEGF and CD34 in xenograft tissues were stained by immunohistochemistry(IHC).The microvessel density (MVD) of xenograft tissues were evaluated.The size of tumour,blood stream signal and abdominal cavity were examined for tumour metastasis by ultrasonic.In part two,cells were injected into the tail vein of nude mice.The mice were killed 6 weeks later after injection and lung were examined for tumour metastasis by microscope. Results:Compared with SGC7901-pcDNA3.1,SGC-7901 groups,for SGC7901-pcDNA3.1-GKLF group,the stage of latency lengthened and growth of xenograft were inhibited,and the volume and weight of xenograft tissues were small(P <0.05).GKLF protein of xenograft tissues in SGC7901-pcDNA3.1-GKLF group increased.The expressions of Ki-67,E-cadherin,VEGF and MVD value decreased.The size of xenograft tissues were small, blood stream signal were in stage I.No metastasis sites of lung and abdominal cavity were seen in all nude mice.Conclusion: GKLF gene might inhibit the growth of tumour and reduce the formation of new blood vessel by downregulating the expressions of Ki-67,E-cadherin,VEGF.So GKLF gene has potential application value in clinical of being target of gene therapy.