Screening And Study Of Peritoneal Metastasis Related Markers Of Gastric Cancer

Partâ… The screening of markers to detectsubclinical peritoneal metastasisof gastric cancerIntroductionWith high incidence, the mortality of gastric cancer is on the first place in ourcountry. The main treatment method of gastric cancer is gastric radical resection, andthe postoperative 5-year survrval rate after surgery is 32% to 40%. Lethal peritonealmetastasis happened in 30% to 50% cases. The process of peritoneal metastasis may be:tumor cells infiltrate and penetrate gastric serosa, then drop into abdominal cavity andadhere to peritoneum, and finally develop into cancer ous mass. And once metastaticfocus comes into being, it is hard to achieve radical cure. Recent years the concept ofneoplasmic micrometastasis rose which meant tumor cells spread into abdominal cavity,lymph nodes, periphery vessels, bone marrow or other tissue, but do not develop intometastatic focus yet. Therefore it is a key stepfor increasing curative effect to detect theexfoliate cancer cells earlier and apply effective blockage.Peritoneal lavage cytology (PLC) has been used routinely to detect exfoliatecancer cells in abdominal cavity. But with certain false-negative rate, PLC is notsensitive to detect small-amount cancer cells, the poor positive rate of which is only14% to 21%.After dropping into abdominal cavity, gastric cancer cells produce kinds ofmolecules because of their biological activities. So it is possible to detect the existenceof cancer cells in abdominal cavity by investigating those molecules.To elevate the detection rate of exfoliate gastric cancer cells in abdominal cavity,many molecular biological methods were used to investigate kinds of moleculesrelating with peritoneal metastasis in peritoneal washing. The focal objects were tumor-specific antigen, extracellular matrix (ECM) and corresponding degradationenzymes, adhesion molecules and cytokines. But the results were not coincident and itwas indefinite which molecule was the best to predict the existence of exfoliate cancercells in abdominal cavity.Among kinds of molecular biological methods, the better sensitivity of RT-PCRmakes it possible to detect peritoneal micrometastasis of gastric cancer.In this study, we selected 8 biological molecules, carcinoembryonic antigen(CEA), heparanase (HPA), dopa decarboxylase (DDC), transforming growth factorβ1(TGF-β1), matrix metalloproteinase-7 (MMP-7), cytokeratin 20 (CK20), basicfibroblast growth factor (bFGF), L-3-phosphoserine phosphatase (L-3-PP), which areall closely related with peritoneal metastasis of gastric cancer. The mRNA expressionof the 8 above molecules in 92 peritoneal washing samples of gastric cancer wereinvestigated by RT-PCR, to find more sensitive molecules to increase the detection rateof exfoliate cancer cells in abdominal cavity.Materials and Methods1. Peritoneal washing samples92 peritoneal washing samples were collected from the patients admitted todepartment of neoplasmic surgery in First Affiated Hospital of China MedicalUniversity. After centrifugalization, half of the sediment was observed cytologically,and the remaining was conserved for RNA extraction. The pathological factors of thecorresponding primary cancer were assessed according Japanese gastric cancerpathology protocol.2. Total RNA extraction in peritoneal washing samplesTotal RNA of each sample was extracted using Trizol reagent. A little of theextracted RNA was used to assess quantity and purity, and the remaining wereconserved at -70℃.3. The mRNA of each molecules was amplified by RT-PCR(1) The total RNA was reverse-transcribed by using a SuperScriptPreamplification System for First Strand cDNA Synthesis Kit. (2) PCR was carried out using primers designed by Primer.(3) Aliquot of the PCR product underwent electrophoresis on 15g/L agarosegel stained with ethidium bromide and was visualized under UV trans-illuminator.4. Statistics Data were analyzed byχ²-test of spss11.5 software.Results1. RT-PCR and PLC resultsIn the 92 peritoneal samples, CEA mRNA positive rate was 65.2% (60/92), HPAmRNA positive rate was 58.7% (54/92), DDC mRNA positive rate was 62.0% (57/92),TGF-β1 mRNA positive rate was 35.9% (33/92), MMP-7 mRNA positive rate was39.1% (36/92), CK20 mRNA positive rate was 42.4% (39/92), bFGF mRNA positiverate was 48.9% (45/92), L-3-PP mRNA positive rate was 23.9% (22/92) and PLCpositive rate was 25% (23/92). There was no mRNA expression in the 10 controlsamples.Among the 8 molecules, the positive rate of CEA mRNA, HPA mRNA, DDCmRNA expression which were 65.2%, 58.7%, 62.0% respectively, were significantlyhigher than the other 5 molecules or PLC (P＜0.05), which indicated their bettersensitivity.In the 21 samples with SE+SI invasion, the samples with positive CEA mRNA,HPA mRNA, DDC mRNA expression were 95.2%, 90.5%, 90.5% respectively; and inthe 23 samples with positive cytology, the samples with positive CEA mRNA, HPAmRNA, DDC mRNA expression were 95.7%, 87.0%, 91.3% respectively; and in the 13samples with positive peritoneal metastasis focus, the samples with positive CEAmRNA, HPA mRNA, DDC mRNA expression were 100%, 92.3%, 100%respectively,which indicated their better specificity.In the 29 samples with M+SM+MP invasion, the expression specificity of CEAmRNA, HPA mRNA, DDC mRNA were 86.9%, 86.9%, 93.1% respectively.2. The relationship of CEA mRNA, HPA mRNA and DDC mRNAexpression with the clinicopathological features of peritoneal metastasis.The relative quantity of CEA mRNA, HPA mRNA and DDC mRNA expression was related significantly with invasive depth and serosal types (P＜0.05).The relative quantity of CEA mRNA and DDC mRNA expression was relatedsignificantly with growth modes (P＜0.05). And the relative quantity of CEA mRNAexpression was related significantly with gross types (P＜0.05).Conclusions(1) RT-PCR is sensitive to detect exfoliate cancer cells in peritoneal cavity ofgastric cancer;(2) As 3 markers related with peritoneal metastasis of gastric cancer, CEA,HPA and DDC are more sensitive and specific than PLC, to predict peritonealmetastasis and peritoneal subclinical metastasis of gastric cancer. Partâ…¡The study of relationship of HPA and DDCwith invasion and metastasis of gastric cancerIntroductionGastric cancer is a kind of neoplasm with highest incidence and mortality in ourcountry and metastasis is the main lethal cause after radical resection. It is essential tomake clear molecular mechanism of gastric cancer metastasis and find new therapeutictarget gene.Present studies and author's previous experiments indicated that heparanase (HPA)and dopa decarboxylase (DDC) were closely related with metastasis of gastric cancer. Itcould help finding new therapeutic target gene to investigate the relationship of HPAand DDC with metastasis of gastric cancer.RNA interference (RNAi) is a new genetic method, which can degradate certainintracellular mRNA effectively and specificly and has been paid much attention inresearching rumor specific genes.In this study, siRNA-HPA and siRNA-DDC were synthesized in vitro, andtransfected into gastric cancer cell BGC823. The expressions of HPA and DDC aftertransfection were investigated by RT-PCR and invasive ablility of cancer cells aftertransfection was observed by transwell assay, to assess the role of HPA and DDC ininvasion and metastasis of gastric cancer.Materials and Methods1. Cell cultureThe gastric cancer cell line BGC823 and NIH3T3 cells was maintained inRPMI-1640 containing 10% fetal bovine serum (FBS), gentamycin and penicillin at37℃, 5%CO₂ atmosphere.2. Small interference RNA (siRNA) synthesis in vitro,The primers were designed by internet tool from Ambion company. Double strand RNA (dsRNA) was constructed by Ambion SilencerTM siRNA Construction Kit invitro.3. siRNA transfectionsiRNA was transfected into BGC823 cells using TransMessenger transfection Kit.4. RNA extraction and RT-PCRTotal RNA from each group of cells was independently extracted using TrizolReagent and was reverse-transcribed for cDNA. PCR product underwentelectrophoresis on 15g/L agarose gel stained with ethidium bromide and was visualizedunder UV trans-illuminator. PCR product underwent electrophoresis on 1% agarose geland was visualized under UV trans-illuminator.5. Western blottingCells were harvested, protein samples were lysed 10 min, and then centrifuged12000rpm/min, 1h at 4℃, transferred the supernatant to a new trip on ice and thencalculated the amount of protein. Equal amounts of protein were electrophoresed at40V 90min and then 80V 120min; transferred to a NC membrane, the membrane wasincubated in bloking solution contained 5% defatted milk powder 1h, then probed withrabbit anti-human antibody (1:500) overnight, and incubated with mouse anti-rabbitALP-IgG(1:1000)2h, the membrane was then stained by ECL solution. The intergrateddensity value of result was analyzed by tluorchem software.6. Invasion assayA total of 2×10⁵ cancer cells were added into the upper part of Millicell model with8μm diameter, matrigel-coated polycarbonate membrane (8μm pore size). Then thecells on the upper surface of the filter were wiped off using a cotton swab, and the cellsthat had invaded the underside of the filter were fixed in 95% alcohol for 30 min andstained by HE, and counted under bright-field microscopy at x400 magnification in fiverandom fields of view.7. StatisticsData were analyzed by ANOV and t test of spssl 1.5 software. Results1. The mRNA expresson in cells BGC823 after siRNA transfectionThe results showed that the mRNA expression of HPA and DDC were suppresseddramatically after 4 days of interference compared with the control groups.2. The protein expresson in cells BGC823 after siRNA transfectionThe results showed that the protein expression of HPA and DDC were suppresseddramatically after 8 days of interference compared with the control groups.3. The change of invasion ability of cancer cells by siRNAtransfectionCells penetrating filter membrane were counted. The number of transfection groupwas significantly lower than the control group (P＜0.05), which indicated that inhibitedHPA and DDC expression resulted in decreased invasion ability of cancer cells.Conclusions(1) siRNA-HPA and siRNA-DDC can inhibited significantly HPA and DDCexpression in human gastric cancer cell line BGC823 respectively;(2) siRNA-HPA and siRNA-DDC transfection can effectively inhibit theinvasive and metastatic ability of gastric cell line BGC823.