Cell Cycle Arrest And PCDH17mRNA Expression And Promoter Demethylation By Genistein In Gastric Cancer Intrauterine Growth Retardation(IUGR)Modifies The Histone Code Along The Mouse Hepatic IGF-1Gene

Background:Gastric cancer is the second most common malignant neoplasm and the second leading cause of cancer death throughout the world. However, the mechanisms that underlie the carcinogenesis of gastric cancer are still poorly understood. Since chemotherapy limits its therapeutic results for the high incidence rate of severe side effects, there has been growing interest in developing more effective chemotherapeutic agents for gastric cancer.Carcinogenesis is a multistep process that results from the accumulation and interplay of genetic and epigenetic changes. Increased DNA methylation of CpG islands in the promoter region of genes is well established as a common epigenetic mechanism for the silencing of tumor suppressor genes (TSGs) in cancer cells.Protocadherins are a subfamily of the cadherin superfamily, but little is known about their functions. Several PCDHs including PCDH8, PCDH10and PCDH20, are frequently silenced in carcinomas of the breast, nasopharynx, and lung, respectively, suggesting their function as TSGs. Since PCDH17is in the same subgroup as PCDH8and PCDH10, we focused on it as a candidate TSG. Since the PCDH17promoter is a TATA-less promoter containing CG-rich sequences that may be susceptible to DNA methylation and gene silencing, we hypothesized that PCDH17may be susceptible to promoter methylation and gene silencing.Epigenetic silencing of a gene can be reversed by drugs such as5-aza-2'-deoxycytidine (5Aza-Cdr) that forms a covalent complex with the active sites of methyltransferase resulting in generalized demethylation. Unfortunately, the applicability of5Aza-Cdr is hampered by its high toxicity and instability in physiological solutions. Recently, genistein-a soy derived isoflavone has attracted much attention of the medical scientific community. Genistein has been reported to upregulate mRNA expression in many TSGs. However, it is still unclear if it could upregulate transcriptional PCDH17by promoter demethylation in gastric cancer.Objective:To determine the toxicity of genistein and its anti-tumor effect on gastric cancer cells, illustrate the relavence of PCDH17mRNA expression and gastric caner, as well as the effect of genistein on PCDH17mRNA expression, To investigate the relavence of PCDH17promoter methylation and mRNA expression, and determine the effect of genistein on PCDH17promoter methylation in gastric cancer cells. This study can not only identify the PCDH17's functions, investigate its epigenetic mechanism in gastric carcinogenesis, but also provide the experimental evidence in seeking new treatments for gastric cancer.Methods:Part I. Genistein's effect on cell proliferation and cell cycle in gastric cancer cell line AGS and the non-malignant immortalized gastric cell line Ges-1. The cell lines were cultured as monolayers in RPMI Medium1640with10%fetal bovine serum,50μg/ml penicillin,50μg/ml streptomycin and maintained in an incubator with a humidified atmosphere of95%air and5%CO2at37℃. Both cells were treated with varying concentrations of genistein (0,10,25and50μMl) dissolved in dimethyl sulfoxide and5-Aza-C (0,2.5,5and10μM). Cells treated only with vehicle served as control. Cell proliferation was examined by cell viability assay and fluorescence-activated cell sorting was performed to analyze cell cycle and apoptosis,Part Ⅱ. Detection of PCDH17mRNA levels in gastric cancer and study of genistein induced mRNA expression in gastric cancer cells. Fresh tumor and paired normal gastric tissue specimens were obtained after informed consent from patients who underwent gastric resection for gastric cancer. No patient received radioactive therapy or chemotherapy before operation. Fresh gastric tissues were homogenized in1ml TRIzol reagent. RNA was prepared by TRIzol reagent. cDNA was synthesized from2μg total RNA using High-Capacity cDNA Reverse Transcription Kit. Target primers were designed using Primer Express software. RT-PCR quantification was then performed using glyceraldehyde-3phosphate dehydrogenase (GAPDH) as an internal control. The comparative Ct method was used to calculate the relative changes in gene expression in the7900Fast Real-Time PCR System.We further evaluated the effects of genistein(0uM,10μM,25μM and50μM,72h) on PCDH17mRNA level and compared the results with that of5Aza-C(0μM,2.5μM,5.0μM and10.0μM,72h) in AGS and Ges-1, respectively.Part Ⅲ. Methylation pattern of PCDH17promoter in gastric carcinoma and paired normal gastric tissue, and genistein induced PCDH17promoter demethylation in gastric cancer cells. Genomic DNA extraction from clinical samples and cell lines was conducted by using a combination of TIANamp Genomic DNA KIT and RNase A. Genomic DNA was treated with sodium bisulfite to convert unmethylated cytosines to uracil, which then converts to thymine, leaving5-methylcytosines unchanged using EZ DNA Methylation-Gold Kit. We elected to analyze CpG sites that were upstream of the longest known5'UTR of PCDH17transcripts and designed bisulfite PCR primer to amplify the promoter region. The PCR products from bisulfite treated genomic DNA were cloned into the vector pSC-A. Ten colonies from each PCR cloning were inoculated into SeqPrepTM96plates. The plasmid DNA was prepared by using SeqPrepTM96Plasmid Prep Kit and sequenced.We further evaluated the effects of genistein(25uM,72h) on promoter hypermethylation and compared the results with that of5uM5-Aza-Cdr(5μM,72h).Results:Part I. Genistein's effect on cell proliferation and cell cycle in gastric cancer cell line AGS and the non-malignant immortalized gastric cell line Ges-1. Genistein(25uM for72h) resulted in a significantly higher number of AGS cells in the G2-M phase (38%) compared with vehicle treated control (17%), whereas5Aza-C(5μM for72h) induced higher apoptosis (12%) in AGSs. No obvious cell cycle and apoptotic changes were observed in non-malignant-immortalized Ges-1cell under either agent' affection, genistein and5Aza-C significantly decreased cell viability in a time-dependent manner. Moreover, the anti-proliferative effects of both agents were observed earlier and more obvious in AGS than in Ges-1. Part Ⅱ. Detection of PCDH17mRNA levels in gastric cancer and study of genistein induced mRNA expression in gastric cancer cells. No obvious difference was observed in gastric cancer cell line AGS and non-malignant-immortalized Ges-1. The PCDH17transcription levels were significantly different in different individual, and29of35samples(82.86%) has relative decreased PCDH17mRNA expression in gastric cancer when compared with paired normal. Furthermore, hige-middle and milled-low differentiation groups had higher decreased percentage than low differentiation group. TNM Ⅰ+Ⅱ group had higher decreased percentage than TNM III+IV group.Genistein significantly upregulated the relative expression level of PCDH17in a dose-dependent manner over vehicle control in gastric cancer cells AGS. Genistein (25μM,72hrs) treatment increased the PCDH17mRNA expression level by15to16-fold in AGS compared with vehicle control while5Aza-C(5μM,72hrs) treatment increased by8to9-fold. However, no obvious upregulated PCDH17mRNA level was observed in non-malignant-immortalized Ges-1cell under either agent's affection.Part III. Methylation pattern of PCDH17promoter in gastric carcinoma and paired normal gastric tissue, and genistein induced PCDH17promoter demethylation in gastric cancer cells. DNA sequencing results revealed that the promoter of the PCDH17gene in tumor samples was hypermethylated in some target CpG sites in comparison with paired normal tissue samples, such as CpG site-870,-731and-458in the pair of No.1sample. Besides, the distribution of its target sites in different individuals was also different.Under genistein and5Aza-C treatment, methylation of several CpG sites in PCDH17promoter was changed in AGS cells.25μM Genistein significantly demethylated CpG site of-678,-614,-458,-207and-129, and5μM5Aza-C significantly demethylated the PCDH17promoter at site-84and-74. However, no significant demethylation was observed in Ges-1cells.Conclutions:(l)genistein has antiproliferative effect on cancer cell growth through induction of cell cycle arrest. Moreover, the anti-proliferative effects was early and more obviousely in AGS than in Ges-1, indicating that genistein targeted tumor cells rather than normal cells.(2)PCDH17gene transcription level varied widely due to individual differences, while it is transcriptionally downregulated in gastric cancer, suggesting it as a tumor suppress gene. Furthermore, its decreased expression is more happened in early gastric cancer, which suggest it might be an indicator in diagnosis of early gastric cancer.(3)The silencing of PCDH17expression through hypermethylation of the promoter leads to loss of its tumor-suppressive activity, which may be a factor in the carcinogenesis of gastric cancer.(4)The transcriptional silencing of the PCDH17gene is due to promoter hypermethylation that can be reversed by genistein and5Aza-C treatment.(5)Genistein showed similar effects to that of5Aza-C, and our results indicate that genistein is a novel, advantageous therapeutic agent for treating gastric cancer.Therefore, genistein may be a promising agent for the treatment and prevention of gastric cancer.Taken together, these results not only provide insights for identifying PCDH17's function and gastric carcinogenesis, but also has great significance on genistein's ancicancer mechanisms. Part II Intrauterine growth retardation(IUGR) modifies the histone code along the mouse hepatic IGF-1geneBackground:Intrauterine growth restriction decreases serum insulin growth factor-1(IGF-1) levels, and liver makes the most IGF-1. IGF-1is an epigenetically regulated gene that has two promoters, alternative exon5splicine, and multiple termination sites. It can produce different transcripts, including exon1derived transcripts, exon2derived transcripts, IGF-1A transcripts(excluding exon5) and IGF-1B transcripts(including exon5). Variation mRNA species affect transcription, translation, and post-translational processing. It is not clear if IUGR would affect hepatic IGF-1expression and alter the epigenetic characteristics of the IGF-1gene along its length.Objective:We hypothesized that IUGR in the mouse would affect hepatic IGF-1expression and alter the epigenetic characteristics of the IGF-1gene, including the promoter methylation and histone modification along its length.Methods:IUGR was induced by giving pregnant females thromboxane A2(TXA2) receptor agonist loaded micro-osmotic pump at embryonic day13. Vehicle loaded micro-osmotic pumps given group were used for sham control. Real time RT-PCR was used to measure IGF-1mRNA expression at day of life (DOL)0and21. We measured1) total IGF-1; IGF-1transcripts from2) promoter1(P1);3) promoter2(P2),4) IGF-1A variant (excludes exon5), and5) IGF-1B variants(includes exon5). Bisulfite sequencing and chromatin immunoprecipitation (ChIP) were used to quantify IGF-1epigenetic characteristics.Results:(1)IUGR's effect on mouse hepatic IGF-1transcripts level:At DOLO, early IUGR significantly decreased hepatic mRNA levels of the P1and IGF-1B in both genders, as well as total IGF-1. IGF-1A was also significantly decreased in male IUGR livers.At DOL21, IUGR significantly decreased mRNA levels of P1, P2, and IGF-1A, as well as total IGF-1mRNA, in males only. IUGR did not affect female hepatic IGF-1mRNA levels.(2)IUGR's effect on Mouse hepatic IGF-1promoter1and promoter2methylation: At DOLO, IUGR significantly increased CpG methylation at-78site in males, increased CpG methylation at-142site and decreased at-110site and-328site in females.At DOL21, IUGR increased CpG methylation at-328site,-347site and-93site in females.(3)IUGR's effect on mouse hepatic IGF-1histone code:At DOL0, acK9was upregulated by IUGR at exon5and distal3'UTR in males, while in females we found P2, exon3and exon5had increased me3K9, proximal3'UTR had increased acK14, and distal3'UTR had decreased acK9, me3K3and increased me3K9.At DOL21, me3K9was significantly decreased at proximal3'UTR in IUGR males. We also found increased me3K4and K9at P1, decreased acK14and increased me2K4at exon5, increased me3K9at proximal3'UTR, and increased me3K4at distal3'UTR in DOL21females by IUGRConclutions (1)IUGR decreased hepatic IGF-1gene expression and altered epigenetic characteristics, and males are more affected than females.(2) Promoter DNA methylation and histone covalent modification interacted with each other, and different histone modification interacted synergistically and antagonistically, which regulated the IGF-1gene transcription epigenetically.(3)IUGR caused histone code changed at different ages, suggesting that different histone modifying complexes are vulnerable to perturbation at different stages.