The Exterimental Research Of MMP-9 Gene Inhabitation On Gastic Adenocarcinoma Growth And Invasive

Primarily gastric carcinoma is one of the most common malignant tumors in our country, which mortality is up to 25.53/100000,accounting for 23.93%of totaldeath population of malignant tumors.The gastric carcinoma incidence is very high in our country.At present, the available combined therapeutic approaches are used to treat gastric carcinoma,such as surgical resection,postoperative radiotherapy,chemotherapy and biotherapy.Although the development of endoscopic technique have delevated the detection rate and cure rate of pristine gastric carcinoma,the prognosis is still very poor for patients with progression gastric carcinoma.Therefore,how to elevate the therapeutic effect and improve prognosis have bcome the emphasis of researching gastric carcinoma.Ivasion and migration are the main metabasis factors for patients with gastric carcinoma,but the molecule mechanisms of ivasion and migration have not been clear.To search and study the function and expression variance of some genes and productions related to ivasion and migration of gastric carcinoma in the progress not only can deepen our understanding of gastric carcinoma ivasion and migration,but aslo provid theoretical fundament for early diagnosis and targeting therapeutic of gastric carcinoma.RNA interference technology(RNAi) has applied rapidly to detect the gene function related gastric carcinoma and research gene therapy because of its especial principle and its superiority to other gene therapy since RNAi was discovered.MMP-9 which regulates the cells growth,invasion and miagration,is closely related to the adhesion of tumor cells,angiogenesis,the degradation of extracellular matrix and so on.The present study focused on the abnormal expression of MMP-9 in gastric adenocarcinoma,and RNAi targeting MMP-9 was used to observe its inhibitory effect on MMP-9,and then observe its effect for the malignant phenotype of human gastric adenocarcinoma SGC7901 cells and therapeutic efficacy of nude mice subcutaneous SGC7901 gastric adenocarcinoma model.The study was divided into 3 sections: In the first part,Using tissue microarray techniques and immunohistochemistry assay to survey the expression of MMP-9,VEGF,PCNA and CD31 in gastric adenocarcinoma malignant progression.In the second part,siRNA targeting MMP-9 was transfected into SGC7901 cells mediated by oligofectamine in vitro.Realtime PCR was used to decteted the expression of MMP-9 mRNA after transfection.Western blotting and immunofluorescence staining were used to detedted the expression of MMP-9 and other main downstream members including VEGF,PCNA and CD31 after transfection.And the Transwell,Matrigel 2D,3D, "scratch",and MTT experiments were used to analyze the changing of growth,invasion and migration of SGC7901 cellsIn the third part:Subcutaneous SGC7901 gastric adenocarcinoma model was established in nude mice.Inject in situ siRNA maxture into endermic tumors,when the tumors were 50-100mm~3.Observe tumor growth for 4 weeks continually,and detecte the expression changing of VEGF,PCNA and CD31 by immunohistochemistry after transfection.Results:The first part:We fond the expressions of MMP-9,VEGF,PCNA and CD31 were higher in gastric adenocarcinoma than normal tissue and paracancer tissue by tissue microarray techniques and immunohistochemistry assay(MMP-9:nomal tissue 30.0%,adjacent non-cancer tissue 64.4%,gastric adenocarcinoma tissue 82.2%;VEGF:nomal tissue 30.0%,adjacent non-cancer tissue 62.2%,gastric adenocarcinoma tissue 73.3%;PCNA:nomal tissue 10.0%,adjacent non-cancer tissue 71.1%,gastric adenocarcinoma tissue 84.4%;CD31:nomal tissue 30.0%,adjacent non-cancer tissue 60.0%,gastric adenocarcinoma tissue 75.6%,P＜0.05).The expression intensity of MMP-9 increased when the malignant degree of gastric adenocarcinoma increased(nomal tissue 30.0%, well-differentiated gastric adenocarcinoma 70.0%,moderately-differentiated gastric adenocarcinoma 80.0%,poorly-differentiated gastric adenocarcinoma 88.0% P＜0.05);The expression of MMP-9,VEGF,PCNA and CD31 were showed positive relationship with each other by rank correlation analysis(P＜0.05).The second part:MMP-9 mRNA expression was degraded in SGC7901 cells after transfected with siRNA targeting MMP-9 by realtime PCR(F=29.58 P=0.001 ).Western blot, immunohistochemistry and immunofluorescence indicated that the expressions of VEGF,PCNA and CD31 were degraded after silencing MMP-9 by RNAi(Western blot:MMP-9:control group 1.12,nonsense siRNA group 1.10,MMP-9 siRNA group 0.38;VEGF:control group 1.03,nonsense siRNA group 1.06,MMP-9 siRNA group 0.36;PCNA:control group 1.06,nonsense siRNA group 1.06,MMP-9 siRNA group 0.35;CD31:control group 1.12,nonsense siRNA group 1.09,MMP-9 siRNA group 0.34 P＜0.05).Transwell and Matrigel 3D experiments indicated the invasion ability of SGC7901 cells transfected with siRNA targeting MMP-9 was attenuated(the number of invasion cells in Transwell:control group 82.1,nonsense siRNA group 78.36,MMP-9 siRNA group 21.97;the cell diameters after treatment in Matrigel 3D:control group 91.65μm,nonsense siRNA group 90.93u.m,MMP-9 si RNA group 33.81μm P＜0.05). Scratch and Matrigel 2D experiments showed that the migration ability was also weakened after transfection(the number of migration cells in Scratch:control group 295.38,nonsense siRNA group 293.29,MMP-9 siRNA group 82.14 P＜0.05).And MTT showed that the proliferation was inhibited remarkably after silencing the expression of MMP-9.The third part:The results of western blotting and immunohistochemistry in vivo were similar to those of in vitro.Furthermore,the tumor volumes in treatment group were smaller than other two groups.The growth curve of subcutaneous tumors indicated that the growth of tumors were inhibited markedly after injecting siRNA maxture(the tumor volumes after treatment:control group 5016.04 mm~3,nonsense siRNA group 4887.36mm~3,MMP-9 siRNA group 2400.08 mm~3 P＜0.05).Conclusion:1.Overexpressions of MMP-9,VEGF,PCNA and CD31 were widespread existence,and they taked part in the development of gastric adenocarcinoma.Meanwhile,the correlation of thesel proteins aslo demonstrated this conclusion.2.siRNA targeting MMP-9 transfection mediated by oligofectamine efficiently can silence the expression of MMP-9 in human gastric adenocarcinoma SGC7901 cells by RNAi technology,and also can inhibit the expression of VEGF,PCNA andCD31,which resulting in decrease of cell proliferation activity and attenuateion of invasive and migration ability.3.The established subcutaneous SGC7901 gastric adenocarcinoma models in nude mice treated with siRNA targeting MMP-9 mediated by oligofectamine can also silence the expression of MMP-9,inhibit the expression of VEGF,PCNA and CD31,accordingly inhibit the proliferation,invasion and migration of SGC7901 gastric adenocarcinoma.The results are coincidence with these in vitro.