| Background:Gastric cancer is one of the most common cancers in the worldwide The numbers of deaths from gastric cancer are the third higest among all tumors,and higer in developing countries especially in China.There are approximately 300,000 new cases deaths from gastric cancer every year in China.Metastasis is an important characteristic of most malignant tumors including gastric cancer,and is also the mainly cause of gastric cancer patients deaths.Therefore,it is necessary to study the mechanism of GC metastasis as well as to find a new potential therapeutic targetE2F3 is a member of E2F transcription factor family.E2F3 plays a vital role in regulation of cell cycle progression,and its dysregulation contributes to oncogenesis Studies have shown that E2F3 is highly expressed and promotes cell proliferation in various cancers including breast cancer,lung cancer.Dysfunction of E2F3 is corresponded with poor prognosis in various cancers.However,the role of E2F3 in gastric cancer has not been fully elucidated.MicroRNAs are small,evolutionarily conserved,non-coding RNAs of 19-24 nucleotides in length,functioning in target gene regulation at the post-transcriptional level.Studies have shown that miRNAs can function as oncogenes or tumor suppressors by regulating different target genes in various human cancers.We thus speculate that dysregulation of E2F3’ regulatory microRNAs might be a main source that leads to its up-regulation in cancersAim:To detect the expression of E2F3 in a large number of gastric cancer tissues and the corresponding non-tumor tissues,and to explore the possible association between E2F3 expression and clinical features.To study the effect of E2F3 on the malignant behavior of gastric cancer.To search possible microRNAs targeting E2F3,and further explore probable mechanism of E2F3 in the malignant behavior of gastric cancerMethods:We investigated the expression of E2F3 in gastric cancer tissuses and the corresponding non-tumor tissues,as well as their relationship with clinical features RT-PCR and western blot were conducted to measure the expression of E2F3 in gastric cancer cell lines,to provide cell model for next work.Transwell and in vivo metastasis assays were performed to study the metastsis in gastric cancer cell lines that E2F3 was overexpressed and knockdown respectively.Possible microRNAs targeting E2F3 was predicted using bioinformatics software and miR-152 was selected.Reporter gene assay,western blot and RT-PCR were performed to verify the prediction.To explore the association between E2F3 and miR-152 expression,RT-PCR was performed to detect expression of E2F3 and miR-152 in 20 paired gastric cancer and non-tumor tissues.Transwell and in vivo metastasis assays were conducted to study the function of miR-152 in invasion and metastasis of gastric cacncer.Chromatin Immunoprecipitation was peformed to find out the possible gene which E2F3 was targeting,and furthermore western blot was performed to confirm that mir-152-E2F3 could regulate the expression of PLK1,which involved in the metastasis of gastric cancerResults:We found that E2F3 was more frequently present in gastric cancer tissues than in the adjacent nontumor tissues.The expression was closely related to clinicopathological features of gastric cancer and correlated with reduced patient survival.The expression of E2F3 was markedly higher in the highly metastasis gastric cells.The migration and invasion abilities of gastric cancer cells were positively correlated with the expression levels of E2F3,overexpression of E2F3 in gastric cells increased cell migration and invasion.In vivo experimental metastasis assays also showed that the downregulation of E2F3 suppressed metastasis.The expression of E2F3 was negative correlated with the expression of miR-152.E2F3 was upregulated in GC tissues while miR-152 downregulated compared to adjacent normal tissues.Luciferase reporter assays showed that miR-152 caused a significant reduction in the relative luciferase activity when the E2F3 plasmid containing wild-type 3’-UTR was present,In vitro tantanswell assays revealed that upregulation of miR-152 significantly suppressed migration and invasion,while downregulation of miR-152 increased migration and invasion.In vivo experimental metastasis assays also showed that upregulation of miR-152 suppressed gastric cell metastasis.Both qRT-PCR and western blott showed that overexpression of miR-152 significantly suppressed E2F3 expression in gastric cells.Chromatin immunoprecipitation showed that E2F3 could bind to PLKland regulate its expression,and further western blot confirmed that miR-152-E2F3 could regulate the expression of PLK1 protein,which involved in the metastasis of gastric cancerConclusion:Taken together,the results demenstrated that overexpression of E2F3 in gastric cancer was correlated with later stages,poorly differentiated and shorter overall survival.Further investigate demonstrated that E2F3 regulated by miR-152 could promoted invasion and metastasis of gastric cancer cells in vitro and vivo miR-152-E2F3 could regulate the expression of PLK1,which involved in the metastasis of gastric cancer. |
