Growth Inhibition By RASSF1A Gene In Gastric Carcinoma Cell Line SGC7901 And Its Related Mechanisms

Background:Gastric carcinoma is one of the most frequent tumors that seriously threaten people's health in China.The cancer develops along a multistage process that is defined by distinct histological and pathophysiological phases.Several genetic and epigenetic alterations mediate the transition from one stage to another and these include mutations in oncogenes,tumour suppressor genes and cell cycle and mismatch repair genes.Molecular genetic studies indicated that loss of 3p was observed in different types of solid tumors.Frequent loss on 3p21-23 was also detected in gastric cancer.RASSF1A(Ras association domain family protein 1,isoform A),one transcript of RASSF1 gene,is a recently identified 3p21.3 tumor suppressor gene.Loss expression of RASSF1A was a frequent event in primary gastric carcinoma.However,the exact role of RASSF1A in gastric tumorigenicity is largely unknown.Objective:In this study,we investigated the effects of RASSF1 gene on biological behaviors of gastric cancer cell line SGC7901 and its mechanisms.Methods:we established gastric cancer cell lines stably overexpressing RASSF1A.Characterization of these cells with regard to proliferation rate and tumorigenicity in vitro and in vivo was performed. NF-κB and AP-1 DNA binding activity were measured by EMSA, expressions of p65,c-Fos,c-jun were measured by western blotting and immunocytochemistry,we used the classic high throughput proteomic technology,two dimensional gel electrophoresis(2-DE),matrix-assisted laser desorption/ionization time of flight(MALDI-TOF)and bioinformatics,to analyze the differential proteomics profiles between vector control and RASSF1A transfectant cells.RT-PCR was used to confirm the differential protein spots.Results:The over-expression of RASSF1A was obvious increased in our established transfected cells.Growth curves indicated that the vector control and parental cells displayed rapid growth rates,whereas the growth rate of the SGC7901-RASSF1A cells was significantly reduced. RASSF1A induced cell cycle into G₁ phase.Compared with parental and vector control cells,the percentage of G₁ phase in RASSF1A transfected cells obviously increased(P＜0.05).And the apoptosis rate of cells expressing RASSF1A had a slight increase.Plating efficiency in parent, vector control and RASSF1A transfected cells were 38.6±1.5%, 39.7±2.1%and 7.3±0.6%,respectively.The mean tumor weights in mice of parental,vector control cells and RASSF1A transfected cell were 1.4±0.26g,1.5±0.32g and 0.6±0.1g,respectively.The EMSA results showed that NF-κB DNA binding activity was significantly lower in SGC7901-RASSF1A cells than in vector control and parental cells,when cells were treated with VCR and Arac,we got the same results.Western blotting and immunocytochemistry data showed that p65 expression in cytoplasim and nuclear extract of SGC7901-RASSF1A cells was significantly inhibited.The EMSA results showed that AP-1 DNA binding activity was significantly lower in SGC7901-RASSF1A cells than in vector control and parental cells.Western blotting and immunocytochemistry data showed that c-Fos expression in nuclear extract of SGC7901-RASSF1A cells was significantly inhibited.While,c-Jun expression in SGC7901-RASSF1A cells had no significant change.The total proteins from RASSF1A transfectant and vector control cells were separated by 2D electrophorosis.869±23and 792±53 spots were detected in RASSF1A transfectant and vector control cells respectively.Locus repeat analysis of protein spots showed that the mean deviation in IEF direction in RASSF1A transfectant and vector control cells were 0.856±0.113 mm and 1.021±0.129 mm respectively whereas the mean deviation in SDS-PAGE direction in these two cells were 0.847±0.125 mm and 1.105±0.127 mm respectively.So the well-resolved and reproducible 2-DE patterns from RASSF1A transfectant and vector control cells were established.Analysis of 2DE patterns revealed that there were 695±18 spots matched between RASSF1A transfectant and vector control cells,and we got 35 different spots.Subsequently 12 protein spots were excised from gels and then digested in gel by trypsin respectively.After that,the digestions were determined by MALDI-TOF mass spectrometry.Lastly, the resulting pepide mass fingerprints(PMF)were used to search the MS Database with the software --- Mascot peptide mass fingerprint.8 protein spots were successively identified,including Galectin-1,TRP-14,ACBP, PSMB5,PSMB4,TIM,Vimentin,CD79.Then we used RT-PCR to confirm the expression of Galectin-1,TRP-14,ACBP in SGC7901 cell. Reaults showed that that Galectin-1,TRP-14,ACBP mRNA was significantly higher in SGC7901-RASSF1A cells than in vector control and parental cells.Conclusion:Our experiment demonstrate that over-expresion of RASSF1A may inhibit the growth of gastric cancer cell SGC7901.NF-κB and AP-1 maybe involved in growth inhibition by over-expresion of RASSF1A gene in SGC7901.Over-expresion of RASSF1A induced up-regulation expression of Galectin-1,TRP-14,ACBP in SGC7901.