| Objective: Gastric cancer(GC)is the fourth most common malignancy and the second leading cause of death by cancer in the world.According to statistics,there were over 1,000,000 new cases and 783,000 deaths of gastric cancer in 2018.Surgery combined with chemo-radiotherapy remains the preferred choice for gastric cancer,and D2 lymph node dissection plus gastrectomy has become the standard treatment.In recent years,the popularity of gastroscopy has effectively improved the rate of early-stage diagnosis and long-term survival rates of gastric cancer,but the prognosis of gastric cancer is still not ideal,especially for patients with peritoneal metastasis,whose median overall survival after intensive treatment is only 15.6 months.In addition,in clinical practice,although the GC patients without distant metastasis(M0 stage)have a better prognosis,survival heterogeneity is great,even in the same TNM stage patients.And because of the high invasiveness of gastrointestinal tumors,most patients are diagnosed at advanced stage and lose the opportunity for therapeutic resection.Therefore,we urgently need to better understand the pathogenesis of gastric cancer and explore effective therapeutic targets.Methods: 1.GSE79973 was selected from GEO database to explore the different expression genes(DEGs)of gastric cancer.GSE79973 included gene expression data of 10 pairs of gastric cancer tissues and adjacent non-tumor mucosal tissues.DAVID was used for GO/KEGG clustering analysis to screen the enrichment pathways of differential genes.Protein-protein interaction(PPI)was analyzed by STRING to construct important gene expression clusters.In addition,GEPIA database was used to verify the expression of some DEGs in TCGA,and Kaplan-Meier Plotter was used to investigate the effect of genes on the prognosis of gastric cancer.2.Total of 48 cases of gastric cancer who were received gastrectomy at the First Affiliated Hospital of China Medical University were collected and analyzed by immunohistochemistry.Cellular function assays in vitro,such as Transwell,CCK-8 and EdU were performed to investigate the effects of LAMC1 on proliferation,invasion and metastasis of HGC27 and MGC803.Western blot was used to investigate the downstream pathway proteins of LAMC1.The effect of LAMC1 on proliferation and tumorigenesis of gastric cancer cells in vivo was investigated by nude mouse tumorigenicity assay.3.MiR-195-5p mimic was used to detect LAMC1 protein expression.Dual luciferase reporter gene assay confirmed whether miR-195-5p target the 3' UTR of LAMC1 mRNA.Real-time PCR confirmed the facilitation regulatory effect of KLF4 on miR-195-5p expression.In the rescue experiment,mi R-195-5p inhibitor was used after overexpression of KLF4,and then LAMC1 expression level was detected.Finally,the binding sites of KLF4 and miR-195-5p promoter regions were identified by Chromatin Immunoprecipitation(ChIP)and dual luciferase reporter gene assay.Results: 1.A total of 1598 genes were identified as DEGs in GSE79973(|log Fold change| > 1,p<0.01),of which 1298 were up-regulated genes and the other 330 were down-regulated genes.The GO/KEGG analysis revealed that 126 of the 1598 differentially expressed genes were involved in “Cell adhesion”.2.After STING analysis,ITGA2,LAMC1,THBS2,NID2 and FN1 were definited as important gene clusters in “Cell adhesion”,and all of these 5 genes were highly expressed in gastric cancer.Results of Kaplan-Meier Plotter suggested that ITGA2(HR=1.35,log rank p=0.037),LAMC1(HR=1.38,log rank p=0.018)and THBS2(HR=0.77,log rank p=0.014)were associated with prognosis of patients.3.Both immunohistochemistry and Western blotting of tissue indicated that LAMC1 was highly expressed in gastric cancer tissues and was closely related to lymph node metastasis(p=0.003)and the TNM staging(p=0.006).Kaplan-meier survival analysis suggested that patients with high LAMC1 expression had a poor prognosis(Log rank p=0.036).4.After LAMC1 was knocked down,CCK-8 and EdU assays showed a significant decrease of proliferation capacity of gastric cancer cells.Meanwhile,Transwell assay indicated that the invasion and metastasis capacity of gastric cancer was significantly reduced.Through nude mouse tumorigenicity assay,we confirmed that knockdown of LAMC1 can effectively reduce tumorigenesis of HGC-27.5.After inhibiting LAMC1 protein expression,the expression of EMT(N-cadherin,E-cadherin),Erk1/2 pathway(Erk1/2,p-Erk1/2)and cell stemness index(Nanog,Sox2)were significantly changed.6.Western blotting and dual luciferase assay confirmed that mi R-195-5p could target the 3 'UTR region of LAMC1 and inhibit LAMC1 protein expression.7.Real-time PCR confirmed that KLF4 promoted miR-195-5p transcription.8.The rescue assay confirmed that the existence of "KLF4/ mi R-195-5p /LAMC1" regulatory axis in gastric cancer.Both ChIP and dual luciferase assays confirmed that KLF4 could target miR-195-5p promoter region and promote its expression.And the target binding sequence was “GCCCACACCCAG”.Conclusion: 1.ITGA2,LAMC1,THBS2,NID2 and FN1 are important DEGs in gastric cancer,and may be closely related to cell adhesion,invasion and metastasis of gastric cancer.In addition,ITGA2,LAMC1 and THBS2 were associated with the prognosis of gastric cancer patients.2.LAMC1 was highly expressed in gastric cancer,and was associated with lymph node metastasis,TNM staging and prognosis.3.LAMC1 could promote the proliferation,invasion and metastasis of gastric cancer,and affect the protein expression of EMT,Erk1/2 and stemness related pathways.4.MiR-195-5p can target the 3'UTR of LAMC1 mRNA and inhibit LAMC1 expression.As a transcription factor in gastric cancer,KLF4 can target the mi R-195-5p promoter and promote its transcription,then indirectly regulating LAMC1 protein expression.Therefore,there is a "KLF4/ miR-195-5p/ LAMC1" regulatory axis in gastric cancer,which plays an important tumor suppressor role. |
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