The Expression Of Smad4 And Smad7 In Gastric Cancer Tissues And Mechanism Study

Research background:gastric cancer which is one of the most common malignancies takes up the second place in the global tumor occurrences and cancer death rates.In our highest occurrence of the malignancy,the arising and development of gastric cancer is a multi steps and multi factors process.Generally speaking,the multiplication and apoptosis of the gastric epithelia mucosae cell keeps a dynamic balance.While this balance maintenance depends on the together modulating control of oncogene,anti-oncogene and some other cell factors.In recent years,the intra-cellular signal transduction has already became a focused biological problem the intra-cellular signal transduction modulates biological behaviors of thegrowth,development,abruptly and death of organisma multicellularis,which is closely related with occurrences of cancers the intra-cellular signal transduction of the transforming growth factor-β(TGF-β)mediated by Smads is one of the most important point of this.The Smad protein group are signal transducer and it direct participate of signal transduction of TGF-βsuper family,which is the well known exclusive intra-cellular TGF-βreceptor kinase substrate and it can convert TGF-βsignal into the intranuclear through cell membrane. Smads can transduction TGF-βsignal after phosphorylation and its role include cell multiplication,differentiation and apoptosis.TGF-β/Smad4 signal transduction access is tumor repress access composed of TGF-βsuperfamily,TGF-βreceptor,Smad protein family and its intranuclear transcription regulation factor.Any of abnormal situations of the access can cause the TGF-β/Smad signal conduction disturbance and then result in the occurrence of tumors.Recently Smad4 is only one of the common accesses type of Smad that affirmed among mammals and it plays a central action during TGF-βsuper family signal transduction and gene regulation situations.Smad7 belongs to inhibitory type Smad(Ⅰ-Smad). At present,people only know the role of Smad7 is TGF-βand BMP signal transduction which come from receptor activation of Smad protein mediated,which was listed into oncogene.Now it is confirmed:The losing of life Smad4 in pancreas cancer cell,its lacking of expression is closely related with pancreas cancer development,histolytic classification.Smad4 expression is concerned with the differentiation degree of gastric cancer tissues.The lower of the gastric cancer differentiation,the less of its expression.It is a not good clinic prognosis.Smad4 is lower than the same specimen cancer tissue during liver caner tissue expression,while the Smad7 protein is higher than the same specimen cancer tissue during liver caner tissue expression. The functional losing rate of Smad4 takes up 30 percent in colon carcinoma.After the plasmid of Smad4 of the colon cancer cell transfect and express in vitro,the discovery of transfection cells is higher than the contrasted cells.The percentage of G0+G1 cell increased,while the S cell percentage decreased and its cell losing rate also increased.The expression of Smad7 in gastric cancer tumorigenesis and progression, especially after the in vitro to gsatric cancer cell transfection of the Smad4 and Smad7 plasmid.There is a little research report to effect of gsatric cancer cell.This research to make use flow cytometry and Western-blotting identified objective gene expression and MTT probe the influence of transfection into of the Smad4 and Smad7 to gastric cancer in vitro and also discusses its effect mechanism to the tumorigenesis and progression of gastric cancer.Part 1 Expression of Smad4 and Smad7 in different gastric tissuesObject:To investigate the expression of Smad4 and Smad7 in different gastric tissues.And to explore their effect in gastric cancer.Methods:The expression of Smad4 and Smad7 was examined by using immunohistochemistry(SP method)in different gastric tissues, which were confirmed as gastric cancer radical excision specimens by pathology.Gastric cancer specimens have 78 cases.Lateral spherical aberration tissues of gastric cancer have 78 cases.Results:1.The positive rate of Smad4 expression was 69.23%in lateral spherical aberration tissues of gastric cancer,and 44.87%in gastric cancer tissues(x~2=9.44,P＜0.01).The positive rate of Smad4 expression was 26.32%in poorly differentiated gastric cancer,The positive rate of Smad4 expression was 62.50%in Well and moderately differentiated gastric cancer Compared to well and moderately differentiated gastric cancer,the expression of Smad4 was significantly reduced in poorly differentiated gastric cancer,The positive rate of Smad4 expression poorly differentiated gastric cancer Compared to well and moderately differentiated gastric cancer have significant difference(P＜0.01),whereas difference between well,moderately differentiated gastric cancer(62.50%) and lateral spherical aberration of gastric cancer tissues(69.23%)didn't have significance(P＞0.1).There was also a significant difference in the EphrinA-1 expression between lymph node metastasis positive and lymph node metastasis negative tumors(23.25%Vs 71.43%,x~2= 18.10,P＜0.01). In 38 cases of poorly differentiated gastric cancer,five cases were completely absence expression of Smad4 and one case was strong expression of Smad4.2.The positive rate of Smad7 expression was 17.95%in lateral spherical aberration of gastric cancer tissues,which most of them were weak expression and only 2 cases were positive expression.The positive rate of Smad7 expression was 89.74%in gastric cancer tissues(x~2=80.89, P＜0.01).Positive reactivity with anti-Smad7 antibody was more frequently observed in poorly differentiated gastric cancer than well, moderately differentiated(97.37%vs 82.50%%,P＜0.05).Difference between well,moderately differentiated gastric cancer and lateral spherical aberration of gastric cancer tissues had significance(82.50% vs 17.95%,P＜0.01).There was also a significant difference in the Smad7 expression between lymph node metastasis positive and lymph node metastasis negative tumors(97.67%vs 80.0%,x~2=6.55,P＜0.05).The expression of Smad7 was mainly positive or strong in poorly differentiated gastric cancer specimens,whereas was weak or positive in well and moderately differentiated gastric cancer specimens.Conclusions:1.The expression of Smad4 was low in gastric cancer tissue.Which Compared to lateral spherical aberration of gastric cancer tissues,the expression of Smad4 had a significant difference(P＜0.01).The positive rate of Smad4 expression related to differentiated degree and metastasis of gastric cancer.2.The expression of Smad7 was high in gastric cancer tissue.Which Compared to lateral spherical aberration of gastric cancer tissues,the expression of Smad7 had a significant difference(P＜0.01).The positive rate of Smad4 expression related to differentiated degree and metastasis of gastric cancer.Part 2 Effects of Smad4 and Smad7 on gastric cancer cellObject:To investigate the effect of plasmid transferred Smad4 and Smad7 into gastric cancer cell in vitro and And to explore their effect mechanism in gastric cancer tumorigenesis and progression.Methods:Gastric cancer cell lines(SGC7901)at log phase growth were cultured in vitro.Lipo-Smad4 or Lipo-Smad7 Complex were transferred into gastric cancer cell in the dosages of 1μg;4μg and 8μg respectively and continued to cultivante for 24h,48h and 72h.The morphological features of cell were observed under fluorescence microscope.Apoptosis of the cells in different groups was detected by flow cytometry.To make use Western-blotting identified objective gene expression.To make use MTT calculate relative survival rate of cell obser effect to gastric cancer cell multiplication.Results:1.Protein strap of Smad4 or Smad7 gene expression were occurrenced in plasmid of Smad4 or Smad7 were transferred into Gastric cancer cell lines(SGC7901).It was verification that Smad4 or Smad7 gene was expressed Gastric cancer cell. 2.Plasmid of Smad4 was transferred into gastric cancer cell in the dosages of 1μg,4μg and 8μg respectively and continued to cultivante for 24h,48h and 72h.Apoptosis rate of gastric cancer cell was 10.2%, 15.6%,21.7%after Plasmid of Smad4 was transferred into gastric cancer cell in the dosages of 1μg and cultivante for 24h,48h and 72h.Apoptosis rate of gastric cancer cell was 16.8%,21.8%,28.9%after Plasmid of Smad4 was transferred into gastric cancer cell in the dosages of 4μg and cultivante for 24h,48h and 72h.Apoptosis rate of gastric cancer cell was 22.3%,31.7%,43.6%after Plasmid of Smad4 was transferred into gastric cancer cell in the dosages of 8μg and cultivante for 24h,48h and 72h.Apoptosis rate of gastric cancer cell was enhanced following Smad4 concentration and time.The highest of apoptosis rate was the gastric cancer cell cultivanted for 72h which transferred dosages is 8μg.3.Plasmid of Smad7 was transferred into gastric cancer cell in the dosages of 1μg,4μg and 8μg respectively and continued to cultivante for 24h,48h and 72h.Apoptosis rate of gastric cancer cell was29.7%, 26.5%,16.2%after Plasmid of Smad4 was transferred into gastric cancer cell in the dosages of 1μg and cultivante for 24h,48h and 72h.Apoptosis rate of gastric cancer cell was 24.8%,22.3%,14.8%after Plasmid of Smad4 was transferred into gastric cancer ceil in the dosages of 4μg and cultivante for 24h,48h and 72h.Apoptosis rate of gastric cancer cell was21.6%,19.4%,13.3%after Plasmid of Smad4 was transferred into gastric cancer cell in the dosages of 8μg and cultivante for 24h,48h and 72h.Apoptosis rate of gastric cancer cell was descending following Smad7 concentration and time.The lowest of apoptosis rate was the gastric cancer cell cultivanted for 72h which transferred dosages is 8μg.4.To use MTT calculate relative survival rate of cell obser toxic effect to gastric cancer cell in different concentration and time of Smad4 or Smad7.When concentration of Smad4 was 1μg,4μg,8μg,relative survival rate of gastric cancer cell was 32.31%,45.20%,67.39%and When concentration of Smad4 was 1μg,4μg,8μg,relative survival rate of gastric cancer cell was 46.41%,38.52%,32.29%。Conclusions:1.Apoptosis rate of gastric cancer cell was increased obviously after transfection of Smad4 in different concentration and was increased progressively following increased of Smad4 concentration and time.It offer dependence of concentration and time.2.Apoptosis rate of gastric cancer cell was descending after transfection of Smad7 in different concentration and was decreased progressively following increased of Smad4 concentration and time.It offer dependence of concentration and time.