| Background:Gastric carcinoma is one of the most aggressive malignancies and the fifth most common malignancy in the world.Stomach cancer is still the second leading cause of cancer-related death worldwide,although its incidence in western countries has declined significantly in the past few years.Ki67 is a nuclear antigen closely related to tumor cell proliferation.Because of its high fertility,Ki67 is thought to be associated with the protein marker index.Although Ki67 is recognized as a key marker associated with cancer cell proliferation and poor prognosis,its full potential for proliferation has not been assessed.Because of its important role in cell proliferation,Ki67 is considered a potential therapeutic target for cancer.Therefore,the mechanism of Ki67 in the occurrence and development of gastric cancer is worthy of further study.Objective:To explore the possible molecular mechanism of Ki67 in the development of gastric cancer.Method:1)Tumor tissues removed from gastric cancer patients were collected,and the expression of Ki67 protein in gastric cancer tissues and adjacent tissues were detected by immunohistochemistry.The relationship between Ki67 protein expression in gastric cancer tissues and the clinicopathological characteristics of patients was statistically analyzed,as well as survival analysis.2)By constructing CRISPR/Cas9 lentivirus plasmid targeted at Ki67 to down-regulate the expression of Ki67 in 4 kinds of gastric cancer cells,qRT-PCR,western blotting,cell proliferation activity detection,flow cytometry detection of cell apoptosis and other experiments,the effect of Ki67 on the proliferation and apoptosis of 4 kinds of gastric cancer cells was studied in vitro.3)By constructing tumor-bearing mice with 4 tumor-bearing gastric cancer cells,in vitro study was conducted to determine whether the down-regulation of Ki67 could inhibit the growth of transplanted tumors in tumor-bearing mice with 4 types of gastric cancer cells.4)The mechanism of Ki67,C-Myc and EGFR in the occurrence and development of gastric cancer was studied in vivo and in vitro by qrt-pcr and western blot experiments respectively.Results:The positive expression rate of Ki67 protein in gastric cancer patients was significantly higher than that in adjacent tissues(P<0.05).In gastric cancer,the high expression of Ki67 was significantly correlated with tumor diameter,differentiation degree,infiltration depth,lymphoid infiltration and clinical stage(P<0.05).Kaplan-meier prognostic analysis showed that Overall survival(OS)of patients with gastric cancer with high expression of Ki67 protein was significantly lower than that of patients with low expression of Ki67 protein,and the expression level of Ki67 protein could be used as an independent factor to predict the prognosis of patients with gastric cancer(P<0.05).The results of CCK8 cytotoxic activity test showed that,compared with normal gastric cancer cell lines MKN45,N87,SGC and KATOIII,the proliferation efficiency of the above four types of gastric cancer cells using lentivirus to down-regulate Ki67 gene was significantly reduced(p<0.05).Annexinv-APC single-staining flow cytometry assay on apoptosis showed that the apoptosis rates of the above four gastric cancer cells were significantly increased by downregulation of Ki67 gene by lentivirus compared with normal human gastric cancer cell lines MKN45,N87,SGC and KATOIII(P<0.01).By constructing tumor-bearing mice with 4 tumor-bearing gastric cancer cells,we investigated in vitro whether the down-regulation of Ki67 could inhibit the growth of transplanted tumors in four kinds of tumor-bearing mice with gastric cancer cells.The results showed that,after 12 days of inoculation,the growth of transplanted tumor was significantly inhibited in tumor-carrying mice of the above 4 types of gastric cancer cells using lentivirus to down-regulate Ki67 gene compared with normal gastric cancer cell lines MKN45,N87,SGC and KATOIII(P<0.05)In vitro and in vivo studies,the results of qRT-PCR and Western blotting experiments showed that,in terms of protein level,Ki67 and c-myc,as well as Ki67 and EGFR were significantly decreased in the 4 types of gastric cancer cells after the down-regulation of Ki67 compared with the normal group(p<0.05).At mRNA level,C-Myc and EGFR in the 4 gastric cancer cells decreased significantly after the regulation of Ki67 compared with the normal group(p<0.05).Conclusion:1)Compared with the adjacent tissues,Ki67 protein was highly expressed in the tumor tissues of patients with gastric cancer.2)The high expression of Ki67 was significantly correlated with the clinicopathological characteristics of gastric cancer patients.The expression of Ki67 protein was higher in gastric cancer patients with larger tumor,deeper invasion,lower differentiation,lymphatic invasion and later clinical stage.3)The high expression of Ki67 protein is related to the prognosis of gastric cancer patients.The median survival time of gastric cancer patients with high positive Ki67 protein expression group was significantly lower than that of gastric cancer patients with weak positive and negative Ki67 protein expression group.4)Down-regulation of Ki67 can reduce the proliferation of 4 kinds of human gastric cancer cells,promote the apoptosis of 4 kinds of human gastric cancer cells,and inhibit the growth of transplanted tumors in 4 kinds of human gastric cancer tumor-bearing mice.5)Ki67 is associated with C-Myc and EGFR and jointly promotes the development of gastric cancer. |
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