The Role And Molecular Mechanism Of MiR-214 In The Pathogenesis And Progression Of Gastric Carcinoma

The high incidence of gastric cancer is a common and serious threat to the safety of human life of malignant tumor of digestive tract,its lack of specificity and sensitivity for early gastric cancer screening index is the most important factor in the diagnosis of early gastric cancer,most of the patients with gastric cancer clinical diagnosis is already in advanced gastric cancer,resulting in poor clinical prognosis and clinical therapeutic effect.Therefore,it is particularly important to strengthen the study of the pathogenesis,development,pathology and molecular mechanism of gastric cancer.The development and progression of gastric cancer is a multi factor and multistage process,and transcription factors play an important role.The gene encoding RNA(miRNA)is a hot topic in clinical research,which can specifically bind to mRNA and inhibit the expression of regulatory genes of degradation or translation post transcriptional levels,thereby affecting tumor cell development,growth,development,differentiation and apoptosis and other biological functions.Studies have confirmed that miR-214 is involved in the occurrence and development of liver cancer,ovarian cancer,colon cancer,The results of this research show that the role of miR-214 in tumor cells is specific,showing high expression or low expression in the tissues of patients with different cancers,the molecular mechanism of gene targeting or inhibiting cancer gene is also different.However,the expression of miR-214 in cancer tissues of gastric cancer is rarely reported,and the mechanism of miR-214 in the development and progression of gastric cancer is unclear.In view of this,this study is divided into two parts to study the role and molecular mechanism of miR-214 in the pathogenesis and development of gastric cancer.First of all,the expression of miR-214 in human gastric cancer tissues and non tumor gastric mucosa tissues was analyzed,and the clinical significance of miR-214 expression was analyzed.The miR-214 gastric cancer cell function experiments were carried out to identify the target genes of miR-214.Secondly,using the CREB1 target chromatin immunoprecipitation to link miR-214 gene,and from the perspective of molecular mechanism of miR-214 link targeting CREB1 gene in gastric cancer cell function and molecular mechanism,providing a theoretical basis for the clinical diagnosis and treatment of gastric cancer.Part 1: expression and biological behavior of miR-214 in gastric cancer cellsResearch purpose:(1)to study the expression of miR-214 in tumor tissues and gastric cancer cells of gastric cancer patients;(2)to analyze the clinical significance of miR-214 expression in the occurrence and development of gastric cancer patients;(3)to analyze the effects of miR-214 expression on the apoptosis biological of behaviors gastric cancer cells invasion,proliferation,metastasis and;(4)to determine the direct target gene of miR-214 expression.Research methods:(1)using the PCR theorem of real-time detection method,detection of gastric cancer tissues(97 cases)and non tumor gastric mucosa specimens(47 cases),four human gastric cancer cell lines(MKN-28,MKN-45,SGC-7901,BGC-823)and the expression of human gastric epithelial cell line GES-1 miR-214.(2)to analyze the relationship between the expression of miR-214 and the clinical pathological indexes and clinical prognosis of gastric cancer,and to examine the value of miR-214 expression in the diagnosis of gastric cancer by ROC curve.(3)miR-214 Lentivirus Expression Vector and miR-214 inhibitor transfection by real-time quantitative PCR detection method to detect the expression of miR-214 after transfection,and the EdU cell growth experiment,cell apoptosis assay and cell migration invasion experiment,verify the effect of miR-214 expression on the biological behavior of gastric cancer cell proliferation,invasion,metastasis,apoptosis etc.(4)the potential target genes using TargetScan prediction method to identify miR-214,constructed by the target gene 3 '-UTR al fluorescence experiment and Western blot experiment carrier,determine miR-214 expression directly to the target gene.Experiment result:(1)the expression of miR-214 in gastric cancer tissue specimens about expression of miR-214 in the samples of non tumor gastric mucosa 1/6,human gastric cancer cell lines miR-214 expression was lower than that of human gastric epithelial cell line GES-1miR-214.(2)the expression of miR-214 can distinguish gastric cancer tumor diameter,metastasis in patients with lymph node gastric cancer,there is a correlation between the expression of miR-214.(3)high expression of miR-214 could inhibit the proliferation,invasion and metastasis of gastric cancer cells,but had no significant effect on the apoptosis of gastric cancer cells.(4)FGFR1,CREB1,NOTCH2,AGAP2 and GSF1 are potential target genes for low expression of human gastric cancer cell line miR-214,while CREB1 is a direct target gene of low expression in human gastric cancer cell line miR-214.Experimental conclusions and significance:MiR-214 is low expression in tumor cells of gastric cancer patients,and has a certain value in the diagnosis of gastric cancer tissues and non tumor tissues,and for the identification of gastric cancer cells with or without lymph node metastasis.MiR-214 has the proliferation of gastric cancer and tumor cell migration,invasion,excessive expression of miR-214 can significantly inhibit the metastasis and invasion of gastric cancer patients with tumor cells,expression can promote the proliferation,migration and invasion of gastric cancer cells by cytology specific manner,but the expression of apoptosis on tumor cells had no significant effect in patients with gastric cancer.MiR-214 in gastric cancer cell proliferation,migration,invasion and the expression of CREB1 is closely related to CREB1 is the direct effect of miR-214 targeted genes,but its mechanism needs further verification.The second part: the function and molecular mechanism of miR-214 linked CREB1 targeting gene in gastric cancerResearch purpose:(1)to observe the cellular function of miR-214 linked CREB1 targeting gene in gastric cancer(2)to analyze the gene function of promoter regions in miR-214 linked CREB1 targeting gene model and analyze its molecular mechanismResearch methods:(1)in gastric cancer cell line CREB1 gene targeted low expression plasmid(siRNA,si-CREB1)and targeting CREB1 gene expression plasmid(pcDNA3.1(+)CREB1),EdUcell proliferation assay,apoptosis assay and cell migration invasion experiments verify the miR-214 expression,influence on the biological behavior of gastric cancer cell proliferation invasion,metastasis,apoptosis,etc.(2)using chromatin immunoprecipitation sequencing to determine the gastric cancer cell lines miR-214 link targeting CREB1 gene promoter region and the use of Gene peak concentration,Ontology analysis method to analysis the function,then uses Pathway analysis method to determine the biological pathways of gene function.Experiment result:(1)miR-214 link CREB1 targeting gene can promote tissue growth,gastric cancer cell migration and invasion ability.(2)molecular mechanisms such as transcription dysregulation,ubiquitin protein hydrolysis,autophagy,ErbB signaling pathway activation,and PI3K-Akt pathway activation are miR-214 linked CREB1 targeting genes.Experimental conclusions and significance:MiR-214 link CREB1 targeting gene can promote tissue growth,gastric cancer cell migration and invasion ability.For the first time using chromatin immunoprecipitation identified miR-214 links CREB1 gene targeted after the whole genome of gastric cancer cell line with co precipitation spectrum sequencing technology,and confirmed that transcription dysregulation,ubiquitin protein hydrolysis,autophagy,ErbB signaling pathway,PI3K-Akt pathway activation is the molecular mechanism of CREB1 gene targeted miR-214 links,and on patients with to promote the development of gastric cancer molecular mechanism,which provides a theoretical basis for the study of molecular mechanism of miR-214 in the development of gastric cancer.