| Gastric cancer is a common dige stive tract malignant tumor,Although early cure rate of gastric cancer is more than 90% by radical surgery,but gastric cancer morbidity symptoms are hidden,most patients are already in advanced stage or late stage,Postoperative recurrence,invasion and metastasis are important prognostic factors.Therefore,the key biological factors in the development,invasion and metastasis of gastric cancer are searched,that is of great significance for further understanding of gastric cancer and treatment of gastric cancer.Kallikrein(Kallikrein,KLK)is a group of serine protease,mainly in the tissue which secret steroid or is hormone dependent such as the prostate,breast,ovary,testis,and plays its biological function by the series reaction to stimulate or terminate physiological or pathological process.KLK6 is one of the members of the KLK family,the study found that its expression is up-regulated in breast cancer,ovarian cancer,endometrial cancer,prostate cancer,colon cancer,esophageal cancer,gastric cancer,pancreatic cancer and other tumors in recent years,may be involved in tumor growth,invasion and metastasis.The formation of new blood vessels contributes to the growth of tumors.VEGF(Vascular endothelial growth factor,VEGF)family is a powerful growth factor and can produce a variety of biological effects,can promote endothelial cell growth in vitro,can induce angiogenesis in vivo,It can promote the migration and metastasis of tumor cells by regulating extracellular matrix,altering vascular permeability and increasing angiogenesis.MVD(microvessel density,MVD)can reflect the growth of tumor blood vessels quantitatively,which is a good indicator of angiogenesis.MMP-9(matrix metalloproteinase-9,MMP-9)is the biggest molecular weight of MMPs family of enzymes,its main role is to degrade the extracellular matrix(ECM)type IV collagen,make the tumor cell break through the normal basal membrane of the primary site,simultaneously degrade blood vessels and lymphatic vessels basement membrane,directly infiltrate tumor cells and participate in the invasion and metastasis of tumor.The above four biological indexes were related to the proliferation,growth and metastasis of tumor.At present,There are few reports about KLK6 in gastric cancer.The mechanism and function of its promoting angiogenesis in gastric cancer have not been reported,its involvement in gastric cancer cells,invasion,metastasis is not clear.This paper plans to study from the following two parts,First,The collection of gastric cancer and normal gastric mucosa tissues was collected to detect the expression of KLK6,VEGF,MMP-9 mRNA and protein in gastric cancer,and the relationship between the correlation of each index and the clinical pathology and prognosis of gastric cancer.In vitro cell culture experiment was carried out to select four gastric cancer cells and a normal cell line,to detect the expression of KLK6,VEGF and MMP-9 in each cell line,and to understand the effect of KLK6 on the cell biology of gastric cancer,and to analyze the relationship between the expression of KLK6 and angiogenesis.Part I Expression of KLK6 in gastric carcinoma and its relationship with angiogenesis and prognosis Methods1.The expression of KLK6,VEGF and MMP-9 proteins in 326 cases of adenocarcinoma of stomach and corresponding normal gastric mucosa were detected by immunohistochemistry.CD34 marked microvessel density(MVD)was counted in per high-power field,and then converted into qualitative data.The expression of klk6,VEGF,MMP-9,MVD in gastric cancer and their relationship with clinicopathological parameters of gastric cancer were compared and analyzed.2.A total of 10 cases of gastric adenocarcinoma tissues and corresponding normal gastric mucosa tissues were random selected to detect expression of KLK6,VEGF and MMP-9 mRNA and protein by real-time fluorescent quantitative qPCR and Western blotting assay,the experimental results of immunohistochemical techniques were further verify3.A total of 326 patients with gastric cancer were followed up.The survival time,the expression of KLK6,VEGF and MMP-9 and the prognosis of gastric cancer were statistically analyzed.The survival curve was drawn and the relationship between the indexes and prognosis was analyzed.Results1.Compared with normal gastric mucosa,the expressions of KLK6,mRNA and protein in gastric adenocarcinoma tissues were significantly increased(P<0.05).2.High expression of KLK6 was significantly correlated with lymph node metastasis and TNM staging in gastric cancer patients(P<0.05).3.The survival rate of patients with high expression of KLK6 in gastric carcinoma was significantly lower,and KLK6 was an independent prognostic factor in gastric cancer patients.4.The results of correlation analysis showed that the expression of KLK6,VEGF,MMP-9,MVD in gastric adenocarcinoma tissues was significantly correlated(P<0.05).KLK6 may promote tumor angiogenesis and invasion,and metastasis of tumor cells.Part II Effect of up-regulated expression of klk6 on biological behavior of gastric cancer cells and possible mechanism Methods1.A normal gastric mucosal cell and four gastric cancer cells were selected.The expressions of KLK6,VEGF,MMP-9,mRNA and protein in each cell line were detected by RT-PCR and Western blotting methods,and the correlation was analyzed.2.KLK6 positive gastric cancer cells were selected by flow cytometry,and the proliferation ability of gastric cancer cells was detected by MTT and plate cloning.Transwell test,scratch healing test and adhesion test were used to detect the invasion and migration ability of gastric cancer cells.3.Western blotting method was used to detect the expression of VEGF protein in KLK6 positive gastric cancer cells and KLK6 negative gastric cancer cells,and to analyze the correlation between them.Results1.Compared with normal gastric mucosa,the expression of KLK6,VEGF,MMP-9,mRNA and protein in each gastric cancer cell line increased,among which SGC7901 gastric cancer cells were much higher than those of the other three groups(P<0.05).2.KLK6 overexpression significantly increased the proliferation,adhesion,invasion and metastasis of gastric cancer cells.3.The results of Western blotting displayed that the expression of VEGF in KLK6 positive gastric cancer cell was significantly higher than that in KLK6 negative gastric cancer cells,and the difference between the two groups was statistically significant(P< 0.05).Conclusions1.The high expression of KLK6 in gastric cancer tissues and cells is related to the expression of VEGF and MMP-9.2.KLK6 was significantly correlated with proliferation,migration and invasion of gastric cancer cells.3.High expression of KLK6 is an independent prognostic factor in patients with gastric cancer. |
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