| Purpose:Gastric cancer(GC)is one of the most common malignant tumors of digestive system,with rapid disease progression and high degree of malignancy.Its incidence ranks the fifth in the global cancer incidence and its mortality ranks the third.The onset of gastric cancer lacks early clinical manifestations,and it is often in TNM stage III or IV at the time of diagnosis,and most of these patients are accompanied by regional lymph node metastasis.Surgery is currently the main treatment for gastric cancer,including radiotherapy and chemotherapy,but the overall prognosis of patients with advanced gastric cancer is still poor.Clinical studies have shown that invasion and migration of tumor cells are the key reasons for the high mortality of gastric cancer,but the molecular basis of invasion and metastasis of gastric cancer is still unclear.Therefore,it is urgent to explore the mechanism of gastric cancer metastasis,find effective molecular markers to predict lymph node metastasis of gastric cancer,improve the diagnostic level of gastric cancer and develop more effective biological targeted drugs.The purpose of our experiment is to screen out the key genes involved in the lymph node metastasis mechanism of gastric cancer,to clarify its influence on invasion and metastasis of gastric cancer cells and its specific mechanism of action,to provide new therapeutic targets for the treatment of advanced gastric cancer,and to help improve the prognosis of gastric cancer patients.Methods:In this study,the cancer in situ tissues(CIS)and lymph node metastasis tissues(LNT)of human gastric cancer were taken as samples,and the mRNA expression profiles of in situ gastric cancer tissues and metastatic lymph node tissues of 3patients with gastric cancer complicated with lymph node metastasis were detected by microdissection technology and high-throughput RNA sequencing technology,and some differentially expressed genes were screened out.The differential genes were verified by quantitative PCR(q-PCR),and the phosphoglycerate mutase 1(PGAM1),which was significantly up-regulated in metastatic lymph node tissues,was selected for further study to clarify its influence on invasion and metastasis of gastric cancer cells.In the following study,we verified the expression difference of PGAM1 in gastric cancer tissues,adjacent tissues and metastatic lymph node tissues by immunohistochemistry in 56 clinical samples,and verified the expression difference of PGAM1 in the prognosis of patients.At the cellular level,gastric cancer cell lines SGC7901 and BGC823 with low expression of PGAM1 were constructed by lentiviral vector,and the down-regulation efficiency was verified by Western-blot.After successfully knocking down PGAM1,CCK-8 was used to detect the proliferation changes of SGC7901 and BGC823 cells.The changes of cell cycle and early apoptosis of SGC7901 and BGC823 were detected by flow cytometry.Scratch wound-healing and transwell assay was used to detect the changes of migration and invasion of SGC7901 and BGC823 cells.Then,we established the subcutaneous tumor-bearing model of nude mice,and further tested the growth rate of two gastric cancer cell lines with low PGAM1 expression.At last,in order to further study the specific mechanism of PGAM1 regulating lymph node metastasis of gastric cancer,the regulatory relationship between PGAM1 and Src/FAK/Paxillin signal transduction pathway were identified by Western-blot assay.Results:In this study,the results of high-throughput RNA sequencing showed that PGAM1 was significantly over-expressed in lymph node metastasis tissues compared with in situ carcinoma tissues of gastric cancer patients,which was related to poor prognosis of patients.Similarly,the results of q-PCR and immunohistochemistry confirmed that PGAM1 was overexpressed in lymph node metastasis of gastric cancer patients.To elucidate the role of PGAM1 in gastric cancer metastasis,we knocked down PGAM1 in metastatic gastric cancer cell line SGC7901 and non-metastatic gastric cancer cell line BGC823,and found that PGAM1 knockdown damaged cell proliferation and arrested cell cycle.In addition,PGAM1 knockdown inhibited the migration of gastric cancer cell lines,which was related to the decreased expression of MMP2,MMP9 and ICAM-1.In the mouse xenotransplantation model,the growth ability of gastric cancer cells with PGAM1 knockdown decreased in vivo.The results of Western blot analysis showed that PGAM1 knockdown in SGC7901 and BGC823 decreased the phosphorylation of Src,FAK and Paxillin.These results indicate that PGAM1 may drive cell proliferation,activate cytoskeleton recombination and cell migration through Src/FAK/Paxillin signal transduction pathway,thus playing an important role in promoting lymph node metastasis of gastric cancer.Conclusion:1.PGAM1 was related to the prognosis of gastric cancer: in patients with gastric cancer with lymph node metastasis,the expression of PGAM1 in LNM was significantly higher than that in CIS.The high expression of PGAM1 in LNM was an independent risk factor for poor prognosis of gastric cancer.2.PGAM1 was closely related to the proliferation of gastric cancer cells: after knocking down PGAM1,the proliferation rate of gastric cancer cells slowed down and G1 block occured;3.PGAM1 could regulate invasion and metastasis of gastric cancer cells: after knocking down PGAM1,the migration rate of gastric cancer cells decreased,the expressions of MMP2,MMP9 and ICAM-1 were decreased,and Src/FAK/Paxillin signal transduction was inhibited,indicating that PGAM1 could promote invasion and metastasis of gastric cancer cells by regulating the expression of proteins closely related to invasion and metastasis of gastric cancer cells and activating related pathways. |
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