The Research On Expression And Promoter Methylation Of RASSF1A Gene In Gastric-Carcinoma

BackgroundThe pathogenesis of tumor is a process of multiple factors, multiple steps andmany stages, which concerned with abnormalities of many oncogenes, tumorsuppressor genes(TSG), mismatch repair genes(MMR), cellular adhesive factorsetc. Tumor metastasis is a complex process of multiple steps, in which abnormalities ofmany gene structure and function play an important role. RASSF 1 is a tumorsuppressor gene, RASSF 1 encodes more than seven isofomes including RASSF1A,RASSF1B and RASSF1C. K-ras is a tumor gene which included H-ras, K-ras andN-ras that located in different code of gene. However, different studies found that indifferent kind of tumor cells the effects of RASSF1, K-ras were different.Aberrant methylation of promoter CpG island is now recognizing as an importantmechanism for gene inactivation as an alternative to gene mutation or deletion intumorigenesis. Many tumors show simultaneous methylation of multiple genesinvolved in the tumorigenesis, which including lung cencer, breast cancer, colon anduterine cervical cancer. Gastric carcinomar is one of the tumors with high frequencyof aberrant methylation. It is suggested also as the major target tumor suppressor at3p21. 3 based on its freguent epigenetic silencing and loss of heterozygosity (LOH) inlung cencer.Our purpose was to investigate the loss expression ofRASSF1A, RASSF 1 B andexpression of K-ras in gastric carcinomar and benign gastric disease. The RASSF1Aexpression is lost or down-regulated in primary tumors by aberrant promoter hypermethylation. The RASSF1A may play a critical role in the maliganant progressionof gastric cancer.Material and Methods1. Material :32 samples after surgical operation with gastric-carcinoma, 32adjacent non-carcinomatous gastric tissues were collected from the PLA generalHospitol of shenyang during 2004-2005, and 51 case with benign gastric diseaseincluding chronic shallow gastritis 30 and chronic atrophic gastritis 21 were collectedfrom the Endoscopy Center of the First Affiliated Hospital, Chinese MedicalUniversity during 2004-2005. All the patients didn't undergo chemotherapy orradiotherapy before surgical operation. The mean age is 56 years(range 26-78years). The gastric-carcinoma consist well-differentiated adenocarcinoma are 12 cases,middel differentiated 9 cases, poorly differentiated 11 cases. All samples werecollected fresh and cut into pieces, snap frozen in liquid nitrogen, and kept frozen at-80^C.Working fluid of RT-PCR were bought from ZhongShan JinQiaobiotechnology corporation, BeiJing. Primers of RASSF1A, RASSF 1B and K-raswere searched according to USA Liberury gene Bank and designed by Primers 5.0software. The primers were synthesized by SanBo YuanZhi biotechnology limitedcorporation, BeiJing. Taq enzyme of RT-PCR, dNTP and marker were all boughtfrom LianBao biology coporation, DaLian.2.Methods: RNA were extracted according to TRIZOL Reagentdirections. RNA purity and concentration were measured with ultravioletspectrophotometer. RNA purtity: OD260/OD280≈1.65.cDNA inverse transcription: RNA 2μl. Reaction system: 10Xbuffer 10μl,MgSO₄(25mM)4μl, AMV(22u/μl)lμl, dNTP (10mM)lμl, oligodT(50μM)lμl,Rnase-inhibitor(40u/μl)0.5μtl, ddH₂O 0.5μl, do the process as follows: ows: Inversetranscription reaction: 65℃for 1 minute, 30℃for 5 minutes, 65℃for 30 minutes,98℃for 5 minutes. 5℃for 5 minutes. PCR reaction system: Totol volum 25μl. cDNA 3μl, 10Xbuffer 2.5μl, dNTP(2.5mM)2μl, Taq-E(5u/μl)0.2μl, ddH₂O17, lμl, primer ofRASSF1A, RASSFIBand K-ras 0.1μl (5pmol/μl). Run amplification as follows: pre-apomorphosis at 94℃for 3 minutes, apomorphosis at 94℃for 45 seconds, anneal at 55.8℃for 1minute, elongation at 72℃for 1 minute, repeat 35 times, finally elongation at 72℃for 7 minutes.β-actin was inner-controlo Product of gene amplification went onelectrophoresis by 2 % agarose gel, and then took pictures.DNA Preparation: Genomic DNA was extracted from the scaples using theclassical method of phenol/chloroform and by precipitation with ethanol.Methylation-specific PCR (MSP): 5μg of DNA was denatured with 3M NaOH15.5μl, followed by treatment with 24.5μl hydroquinone and 3M Sodium bisulfite andincubation for 16-20 hours at 50℃. After purification using a JETSORB gelextraction kit,the DNA was treated with 3M NaOH 5.5μl and precipitated with threevolumes of 100% ethanol and a one-third volume of 6 M NH4Ac at-20℃. Theprecipitated DNA was washed with 70% ethanol and dissolved in distilled water. Thetested genes included primer sequences of RASSF1A, RASSF1B, for methylated orunmethylated reactions, are RASSF1A-m-F 5'-GTG TTA ACG CGT TGC GTATC-3'; RASSF1A- m-R 5'-AAC CCC GCG AAC TAAAAACGA-3, long 128bp,RASSF1A-u-F 5'- TTT GGT TGG AGT GTG TTA ATG TG-3'; RASSF1A u-R 5'-CAAACC CCA CAA ACT AAAAAC AAA-3', long 109bpo Totol reaction volum 25μlcontained DNA 5μl, 10Xbuffer 2.5μl, dNTP (2.5mM)2μl, Taq-E(5u/μl)0.2μl ,ddH₂O13.3μl, primer of RASSF1 A, RASSF1B lμtl(5pmol/μl). The reaction werehot-started at 95℃for 5 minutes, apomorphosis at 95℃for 60 seconds, anneal at 55℃for 1 minute, elongation at 74℃for 2 minute, repeat 30 times, finally elongationat 74℃for 5 minutes. Product of gene amplification went on electrophoresis by 2%agarose gel, and then took pictures.3. Statistical treatment: All statistical calculationa were made using the SPSS 9.0Software. Student's test or analysis of variance test andײ test were used to compare. Results1.The loss expression ofRASSF1A, RASSF 1 B in gastric carcinomar and benigngastric disease.2.Relationship between loss expression of RASSF1A, RASSF1B anddifferentiation degree.3. The positive expression of K-ras in gastric carcinomar and benign gastricdisease.4.RASSF1A mRNA aberrant promoter hypermethylation in gastric carcinomar andbenign gastric disease. Conclusions1. The loss expression of RASSF1 A, RASSF1B in non-carcinomatous tissueswere 6.25%, 6.25% and 59.3%, 28.2% in gastric carcinomar. RASSF1A in gastriccarcinomar is lost or down-regulated in primary tumors.2. Expression of RASSF1A, RASSF1B has no correlation with differentiate degreeof gastric cancer.3. The expression of K-ras has positive 9.3 %in non-carcinomatous tissues, and68.7% in gastric carcinomar.4. Expression of K-ras has no correlation with differentiate degree of gastriccancer.5. The expression aberrant promoter hypermethylation of RASSF1A ingastric-carcinoma was higher than in adjacent non-carcinomatous tissues. Itsuggested that APH of RASSF 1A is main reason in gastric-carcinoma silence.6. The promoter methylation of RASSF1A in gastric-carcinoma has no statisticalrelation with differentiation grade, lymph node metatasis and the size of tumors.