Effects Of5-Aza-2’-deoxycytidine And Trichostatin A On Expression, Methy-Lation Of Reprimo Gene And The Growth Of Human Gastric Cancer Cell Line SGC-7901

Objective: Gastric cancer (GC) is the fourth most common cancer and the secondmost common cause of cancer related death worldwide. The worldwide incidence ofGC has declined rapidly over the recent few decades because of the recognition ofcertain risk factors, but GC is still the predominant form of cancer and remains asignificant burden in developing world, particularly China. GC is thought to arisethrough the accumulation of many genetic and epigenetic alterations, leading to gainof function in oncogenes and loss of function in tumor suppressor genes. A lot ofevidences now suggested that, in addition to genetic alterations, epigenetic alterationsplay a critical role in the development and progression of GC. DNA methylation andhistone acetylation are the main aspects of epigenetic alterations, and are consideredto be the main mechanisms of tumor suppressor gene inactivation. In this study, weaim investigate effects of5-Aza-2’-deoxycytidine (5-AzadC) and trichostatin A (TSA)on the expression of Reprimo gene and the growth of human gastric cancer cell lineSGC-7901.Methods:(1) Human GC cell line SGC-7901was cultured in RPMI1640. Dividecell line into four groups:①Control group without drug;②5-AzadC(5μmol/L)group was used for72h in the treatment;③TSA（300nm/L）group was used for72h in the treatment；④5-AzadC(5μmol/L) was used for24h followed by TSA(300 nm/L) for an additional24h in the combined treatment.(2) Human GC cell line SGC-7901was treated with concentration gradient of5-AzadC (0.025,0.25,2.5,25,250μmol/L) and TSA (0.0015,0.015,0.15,1.5,15μmol/L). The proliferation of the cells was detected by MTS assay after24,48,60,72h cultured.(3) Methylation-specific Polymerase Chain Reaction (MSP): DNA methylation statusof Reprimo gene promoter in human GC SGC-7901cell line was assayed by MSPafter being treated for72h.(4) Reverse Transcription Polymerase Chain Reaction (RT-PCR): The mRNAexpression of Reprimo gene in human GC SGC-7901cell line was detected byRT-PCR after being treated for72h.(5) Western Blot：The protein expression of Reprimo gene in human GC SGC-7901cell line was detected by Western Blot after being treated for72h.Results:(1)5-AzadC and (or) TSA all inhibited the growth of SGC-7901cells afterbeing treated for24,48,60,72h. The inhibitory rate in the union medication group wassignificantly higher than that in the independent medication group (P<0.05). Theinhibitory rate was increased in a time-dependent and concentration-dependent manner(P<0.05).(2) CpG islands hypermethylation of Reprimo gene promoter were assayed in humanGC SGC-7901cell line by MSP (methylation strip only). After5-AzadC or TSAintervened, CpG island of Reprimo gene promoter happened nonmethylated(methylation and nonmethylated strip). After combinations intervened, CpG islands ofReprimo gene promoter happened only nonmethylated (nonmethylated strip only). (3) RT-PCR was used to assay mRNA expression of Reprimo gene in human GCSGC-7901cell line after5-AzadC and (or) TSA intervened. We found that single druggroup was more significant increased than control group (P<0.05). The combinationgroup was more significant increased than single drug group (P<0.05).(4) Western Blot was used to detect protein expression of Reprimo gene in human GCSGC-7901cell line after5-AzadC and (or) TSA intervened. The single drug group wasmore significant increased than control group (P<0.05). The combination treatmentgroup was more significant increased than single drug group (P<0.05).Conclusion:(1)5-AzadC and TSA intervention all can inhibit human GC SGC-7901cell growth. The combined treatment group can synergistically inhibit cell growth.(2)5-AzadC and TSA can induce GC cells methylation Reprimo gene demethylation.The combined treatment can significantly enhance the expressions of Reprimo gene.(3) Methylation of gene promoter may be one of the main reasons leading to geneinactivation in human GC SGC-7901cell line.