Feasibility Study For Application Of ODC Gene Expression In The Diagnosis And Therapy Of Gastric Carcinoma

The ornithine decarboxylase(ODC),the first rate-limiting enzyme required for polyamine synthesis,which can catalyze the ornithine into putrescine.By regulating the level of polyamine,ornithine decarboxylase play an important role in the differentiation and proliferation of cell and the generation and development of cancer.Gastric cancer is one of the most frequently diagnosed cancer,which has the highest death rate in our country.According to the unknown etiopathogenesis,many factors can lead to gastric cancer.Frequently,it has been into the advanced stage when the patient came to visit the doctor,and the death rate is high.Now the first-line therapy is radical surgery with adjuvant chemotherapy.But overall 5 year survival rate for this disease is around 40%.The combination of chemotherapy and radiotherapy has some kind of therapeutic effect,but is not satisfactory well enough.Therefore,it is important to find a novel non-surgical intervention for treating gastric cancer.Previous studies have demonstrated the correlation between gastric cancer and aberrant polyamine biosynthesis pathway,ornithine decarboxylase(ODC),the rate-limiting enzyme required for polyamine synthesis,has been approved to have a high level expression in breast cancer. But there are no related reports on the relationship between the ODC and gastric cancer. In order to explore the feasibility for the utilization of ODC gene as a target of gastric cancer's diagnosis and therapy.This article reveal the ODC gene expression in gastric cancer with mRNA and protein level,to observe the relationship between ODC gene expression and the phathogenesis of gastric cancer.Moreover,this study use antisense vector for ODC to transfect the gastric cancer cell lines in order to obtain more evidence about the relationship between ODC and gastric cancer. Part 1 Research of the ODC gene expression in gastric cancerMethods1.RT-PCRWe sampled tumor and normal tissue from examples of 21 fresh gastric cancer,The distance from normal tissue to tumor was beyond 7 centermetre.Extract total RNA from tumor and normal tissue according to the illustration of Trizol agent kit.The RNA was rerverse transcribed according to the illustration of cDNA first chain construction agent kit with random Primers OligodT.The RT reaction was used as the template DNA for the PCR.Ten mieroliter of total PCR mix was electrophoresed on 1%agarose gel.β-actin was used as internal contrast.Electrophoretozones were investigated by using Ultraviolet light analysis apparatus,and photographed by using gel imaging analysis system. Semiquantitative methods are used to determine the relative expression level of the 21 examples between latero-caneer the normal tissues contrast to the cancer tissues.The correlation of ODC gene expression to clinical pathological stages was determined. Statistical treatment:Repeat test every sample for three times,take the mean±standard deviation((?)±s) as result.Use the statistic soft pack SPSS10.0 to deal the data by matched-pairs T-test and two samples-test.2.Western-blotTissues were homogenized in RIPA buffer then electrophoresed on a 12%SDS-PAGE gel. Separated proteins were transferred onto a nitrocellulose membrane.Nonspecific binding sites were blocked.The blots were incubated in a solution containing primary anti- ODC antibodies.Reactive proteins were visualized with a chemiluminescence detection system.Results1.The expression of ODC gene mRNA in lesion of gastric carcinoma was significantly higher than that in contiguous normal mucosa(P＜0.01).The expression of ODC gene mRNA of well-differentiated gastric carcinoma was significantly higher than the poorly-differentiated(P＜0.01).2.Western blotting:showed that ODC protein is expressed at low levels in the normal gastric tissue but is markedly up regulated in most of the tumor tissues.ConclusionODC gene play an important role in gastric carcinogenesis and the up-regulated expression of ODC mRNA was related to the differentiation of gastric cancer,indicate that elevated ODC expression is associated with the invasive and aggressive behavior of gastric cancer.These results supports previous observations and it also enhances our understanding of the molecular mechanisms responsible for gastric tumor development and progression.These findings may help to design new therapeutic strategies for the treatment of GC and its metastases.Part 2 Research of inhibition of ODC expression in the gastric cell lines and the capability of proliferation and invasion of gastric cell linesMethods1.Transfect transiently ODC antisense vector-pcDNA-ODCr to the cell lines (SGC-7901 and GES-1),then use western blotting to measure the expression of ODC and make the comparison between the experimental group and control group.2.Measure the reproductive activity of two cell lines with MTS3.Measure the capability of invasion of the two cell lines.Result1.After the transfection of pcDNA-ODCr,the expression of ODC in gastric cell lines is significantly reduced.2.The reproductive activity is sharply reduced and the rate of inhibition for the two cell lines is 60%and 57%.3.The transfection of pcDNA-ODCr effectively impairs the invasion ability of cell lines. ConclusionThe pcDNA-ODCr can down regulate the ODC gene expression and inhibit the proliferation and invasion of the gastric cell lines.So the down regulation of ODC has the potential value for the gene therapy study.On the whole,all the experimental results above suggest the close relationship between the gene expression of ODC and gastric cancer.The dpression of ODC gene expression can inhibit the ability of growth and invasion of gastric tumor cell.This implies that it is feasible for ODC gene using as a target of gastric cancer's diagnosis and therapy.