Study On Optimization And Screening Of Metastasis Related Genes In Gastric Carcinoma

ObjectiveGastric cancer is very frequent in China and often in advanced stage at first presentation. It has a characteristic for its low early diagnosis rate, high metastasis rate and poor prognosis. Rencently with the further study on mechanism of invasion and metastasis, metastasis is considered as a multigene participant, multi - step complex process, including adhesion, degradation, motility, angio-genesis and lymphogenesis and escaping from immune surveillance. The expression abnormality of metastasis related gene takes an important role in metastasis control. Studying further and finding out the interaction among themselves would contribute to understand the mechanisms of tumor metastasis. However, up to now most literatures have reported the separated or isolated studies of single or several genes, which is short of the whole estimation about them.Gene chip is one of the greatest technology progress since 1990s, and has been paid widespread attention to in last few years. Because of the parallel quick analysis, small samples, little contaminant and reliable results, expression gene chip has been used to study in many domains and made indelible dedication to expression profiling analysis. The expression gene chip could correlate several genes to tumor, so it could help us to value the whole status and role of multi -genes in tumor metastasis.During the course of the onset and development of some disease only a few fraction of genes ( several decades or hundreds ) varied in its expression profiling in recent literature reports. However, with respect to the high density cDNA mi-croarray, due to the expensive price, fixed target genes and lack of individuation there must be lots of waste source and difficulty to be used widely. In recent years, there are some literatures about the optimization of cDNA microarray, but the conclusion is not accordant. So we constructed a stable low - density cDNAmicroarray technology, and studied the most suitable target gene length, concentration, spotting solution, hybridizing dynamics, reproducibility and reliability systemicly. A stable low -density cDNA microarry was built, on which all target genes had a close relation to metastasis of gastric cancer in past research. It was used to examine the genes'expression in gastric carcinoma in order to value them the whole status and role in metastasis.Materials and methodsExperiment object SGC7901 gastric cancer cell line; the blood serum of hepatitis B patients; 52 gastric cancer patients who underwent gastrectomy in Surgical Oncology Department, the First Hospital of China Medical University between September 2001 and September 2002. Chemotherapy and/or radiotherapy werent executed before surgery. Tissue Samples (primary lesion, coupled normal mucosa and metastasis lymph node) were snap - frozen in liquid nitrogen, then stored at 70 C. Each sample was examined histologically by using H&E - stained cryostat sections. Clinical and pathological features were recorded in details.1. Total RNA extraction, quantity and quality monitoringTotal RNA from cell line and fresh gastric carcinomas were extracted with Trizol (Invitrogen) reagents according to the protocol supplied by the manufacturers. UV300 spectrophotometer was used to determine OD^/OD^ ratio and 1% formaldehyde denaturing agarose gel electrophoresis was used to assess the purity, quality and integrity of total RNA.2. Purification of target genesThe primers for RT - PCR and PCR were designed with Primer Premier 5. 0,and the product length was between 189 and 1078 base pairs. The products were obtained by RT - PCR and PCR, and purified by ethanol and then dissolved in spotting buffers.3. Fabrication of the low - density cDNA microarrayThe low - density cDNA microarray was prepared by printing targets onto the amino -slides (CEL) using Micro Grind II gene chip spotting equipment(England).Beta actin and GAPDH were selected as internal control, hepatitis B as negative control and spotting solution only as blank control. Such genes as beta actin, glyceraldehyde phosphate dehydrogenase ( GAPDH ) , heparanase, matrix metalloproteinase 2(MMP -2) and matrilysin(MMP -7) , vascular endothelial growth factor C ( VEGF - C ) , S100A4, hRadl7, human telomerase reverse transcriptase (hTERT) , transforming growth factor beta(TGF -beta) , CD44s, Integrin beta, E - cadherin (CDH1) , KAI - 1 ( CD82 ) , tissue inhibitors of met-alloproteases 1 and 2(TIMP1,2) , pTEN, nm23Hl and hepatitis B were selected as targets. Afer watering, baking and blocking, the microarray was prepared for hybridization.4. Probe preparationFor each labeling reaction, 100 jxg high - quality total RNA was used. First -strand cDNA synthesis was primed with oligo dT(18) primer, and simultaneously incorporated fluorescently labelled deoxyribonucleotides ( Cy5 - dUTP, Pharmacia). Reactions were quenched by the addition of 0. 5 mol/L EDTA and RNA template was hydrolyzed by the addition of 1 mol/L NaOH followed by heating at 68 C for 30 min. Reactions were neutralized with 1 mol/L Tis - HC1, and the labelled cDNA was precipitated by ethanol and then dissolved in hybridization buffer.5. Optimization of low density cDNA microarrayHouse keeping genes such as beta actin and GAPDH were selected to stud-y. Design three target gene length( 200, 500 and 1000 base pair or so) , three kinds of spotting buffer(3 x SSC, 50% DMSO and 0. 5 mol/L carbonate buffer ( pH =9.0) ) , three sorts of concentration of target gene(0. 5,1.0 and 1.5 g/L respectively) , different hybridizing time( 1, 6, 12, 18, 24 hours) and different hybridizing temperature( 37, 50, 60, 68 centigrade). Compare the hybridization results.6. Reproducibility and reliability verificationSGC7901 cell line was examined by the identical low density cDNA microarray two times according to the optimized condition to value the reproducibility, and further examined by RT -PCR to value the reliability.7. Multi - genes'expression in gastric cancer by low density cDNA microarrayThe multi - genes'expressions in 18 fresh gastric carcinomas, coupled normal mucosa and 15 lymph node metastasis lesions were examined by the low density cDNA microarray. The correlation between gene expression and clinico-pathological factors was analyzed.8. Verification by RT - PCRIn order to know the reliability of the low - density cDNA microarray, five genes were selected randomly, such as GAPDH, heparanase, MMP - 7, KAI - 1, nm23Hl, and the mRNA expressions in above samples were examined by reverse transcription polymerase chain reaction ( RT - PCR) . Compare the two results to know the concordance.Results1. Optimization of the low density cDNA microarrayHybridization signals were seen for house keeping genes, but were not seen for hepatitis B gene ( negative control) and spotting solution only ( blank control). Among three kinds of spotting solution, 50% DMSO was the best, and there was no significant difference among three target gene length and solutions, but the higher was the concentration, the stronger was the signals.There was no signals when hybridizing 1 hour, but signals were seen at 6, 12, 18, 24 hours, and the longer was hybridizing time, the stronger was the signals, 18 hours was the best. At different hybridizing temperature, faint signals were seen at 371 , good signals were seen at both 50X1 and 601 , but 60 Tl was the best. When hybridizing time was too long (24 hours) and hybridizing temperature was too high (68°C) , the hybridization solution often dried, and the background was bad as if many stars on the chip.The signals had a good reproducibility during the two hybridizations, and the correlation coefficient was 0. 588. When compare the results between microarray and RT - PCR, the correlation coefficient was 0. 778. In comparison with RT-PCR, the specificity was 100% (20/20) , and sensitivity was 80%(16/20) for the low density cDNA microarray.2. The expression of muti - genes and the correlation to biobehavior in gastric cancerThe expressions of MMP-7^heparanase^S100A4^hTERT^hRadl7 in gastric carcinoma primary lesions were higher than coupled normal mucosa, whereas the expressions of nm23Hl and CDH1 were lower; The expressions of MMP -7Nheparanase^S100A4NhTERT in lymph node metastasis lesions were higher than coupled normal mucosa, whereas nm23Hl and CDH1 were lower. The expressions of hTERT in lymph node metastasis lesions were higher than coupled primary lesions, whereas hRad 17 and integrin beta were lower. There was no significant difference among other genes.When studying the multi - gene expression in gastric cancer primary lesion, we found that among serosa involvement group the expression of MMP - 7, heparanase, S100A4 increased more serious than non -serosa involvement, but the expression of nm23Hl, CDH1, KAI - 1 decreased more serious. Among the area of serosa involvement heavier group ( more than 20cm2) the expression of MMP-7, heparanase increased obviously, but nm23Hl, CDH1, KAI -1 expression decreased obviously. With respect to the number of lymph node metastasis , two groups were divided: no more than 6 was regarded as the fewer and lighter; no less than 7 as the more and heavier. The expression of MMP -7, hTERT in the heavier group were higher than that in the lighter. There was no significant correlation between the genes'expression and other clinincopathologi-cal factors, such as surgery type, growth pattern, differentiation degree, serosa type, histology type, lymphatics cancer embolus and vein cancer embolus, and so on.When studying the multi - gene expression in lymph node metastasis lesions, we found that among serosa involvement group the expression of CDH1 decreased more serious than non - serosa involvement. Among the area of serosa involvement heavier group (more than 20cm2 ) , the expression of CDH1 decreased obviously. The expression of MMP -7, S100A4, hTERT in the heavier lymph node metastasis group were higher than that in the lighter, wheras nm23Hl and CDH1 were lower. There was no significant correlation among oth-...