| Background and Objectives:Gastric cancer is a common malignant tumor of digestive system,doing serious damage to human health.MicroRNA(miRNA)is a kind of small RNA with a length of about 20-24 nucleotides.It is found that miRNA regulates about two-thirds of the human gene.miRNA plays a huge role in cell differentiation,biological progression and disease development.More and more attentions have been paid to miRNAs.With the further study of miRNA effects,it has been found that regulations of miRNA play an important role in the development of a series of tumors including gastric cancer.In this paper,we studied the expression profile of miRNAs in gastric cancer tissues and its adjacent tissues from the population of high incidence by microarray,and screened the specific miRNAs related to gastric cancer.Then,qRT-PCR was.performed to deeply analyze the expression of miRNAs.At the same time,the clinical data correlation analysis was carried out.On the basis of the previous study,we further investigated the effect of the related miRNA on cell biological function.The study could clarify the role of miRNAs in the development and progression of gastric cancer and provide potential biomarkers for the diagnosis and treatment of gastric cancer.Methods and Results:1.Screening of gastric cancer-associated miRNAIn this study,we collected the cancer tissues and adjacent tissues of 10 patients with gastric cancer in Wuwei city,Gansu province,and the patients were diagnosed by pathology,without radiotherapy and chemotherapy.The clinical data were collected as well.The total RNA was extracted from Trizol lysate and the purity of RNA was determined by OD260/280.The results of OD260/280 were between 1.8 and 2.0 and the total RNA purity was qualified.The miRNA/mRNA expression profiles of these 10 patients were detected by high-throughput microarray.61 differentially expressed miRNAs were recognized,of which 38 up-regulated miRNAs and 23 down-regulated miRNAs.GO functional enrichment and KEGG pathway analysis of miRNA/mRNA were performed by DIANA-mirPath V.3 software.MiRNA-mRNA co-expression network was constructed and the potential functions of differentially expressed miRNAs were analyzed.KEGG results showed that differentially expressed miRNAs in gastric cancer are mainly involved in cancer-related signaling pathways such as ErbB,PI3K-Akt,cell cycle and apoptosis.2 Analysis of differentially expressed miRNA in gastric cancerA total of 82 pairs of cancer tissues and adjacent tissues were collected in the area of high gastric cancer incidence.Three differentially expressed miRNAs randomly selected from the chip results were quantitatively detected by qRT-PCR.The results were calculated with the application of paired T test for statistical analysis.Compared with paired adjacent tissues,miR-9-5p was significantly up-regulated in gastric cancer tissues(p<0.05),and miR-3123 and miR-133a-3p were significantly down-regulated in gastric cancer tissues(p<0.05),which were consistent with the results of the microarray.MiRNAs were analyzed in early and late stages of gastric cancer.The results showed that miR-9-5p and miR-3123 in the early stage of gastric cancer showed significant changes in the level of expression,and the expression of miR-133a-3p in early stage of gastric cancer was lower than that in the middle and late stages,suggesting that miR-9-5p and miR-3123 can be used as potential biomarkers for the early diagnosis of gastric cancer,and miR-133a-3p can be used as a potential biomarker associated with progression of gastric cancer.An independent sample T test was conducted to analyze the relationships between the differential expression miRNA and the clinical data of gastric cancer patients,including gender,age,tumor size,differentiation,lymphatic metastasis,TNM staging,etc.The results showed that miR-133a-3p downregulation was significantly associated with lymph node metastasis(p<0.05).3.Study on the biological function of differentially expressed miRNA in gastric cancerQRT-PCR was performed in human gastric mucosal epithelial cells GES-1 and different gastric cancer cells MKN-45 and MGC-803,founding that miR-9-5p was significantly higher in gastric cancer cells than in normal gastric mucosal epithelial cells,which was consistent with the results of chip and population studies.GES-1 was selected as the research object,and the expression of miR-9-5p in GES-1 cells was up-regulated by miR-9-5p mimics.The effects of miR-9-5p expression on cell proliferation,cell apoptosis and cell cycle were detected by MTT assay and flow cytometry.The results showed that the proliferation and apoptosis of GES-1 cells did not change significantly(p>0.05)after miR-9-5p upregulation.A single miR-9-5p expression did not affect the biological functions of gastric epithelial cells.The effect of single miRNA on cell function was limited,and the effect of multiple miRNA expressions on cell's function should be further studied in the future.Conclusion:In this study,61 differentially expressed miRNAs were screened out from miRNA/mRNA microarray expression profiles.KEGG results showed that differentially expressed miRNAs in gastric cancer were mainly involved in cancer-related signaling pathways such as ErbB,PI3K-Akt,cell cycle and apoptosis.MiR-9-5p were highly expressed in gastric cancer tissues.The expressions of miR-3123 and miR-133a-5p were significantly lower in gastric cancer tissues.Further analysis showed that miR-9-5p and miR-3123 presented significant changes in the early stage of gastric cancer,suggesting that miR-9-5p and miR-3123 could be used as early biomedical markers for the diagnosis of gastric cancer.The expression of miR-133a-3p increased in the early and late stages of gastric cancer,and its expression was significantly correlated with lymphatic metastasis,suggesting that it can be used as a potential biomarker associated with progression of gastric cancer.The biological function studies showed that miR-9-5p was highly expressed in gastric cancer cells while miR-9-5p had no significant effect on cell proliferation,apoptosis and cell cycle of normal gastric mucosa GES-1.This study would provide a theoretical basis for further understanding of the pathogenesis of gastric cancer and the diagnosis and treatments of gastric cancer. |
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