| Part 1: Screening of differentially expressed circRNA in plasma of gastric cancer patientsObjective: To determine the circRNA expression profile in gastric cancer plasma and screening of differentially expressed circRNA genes.Methods: The differences of circRNA expression profiles between gastric cancer plasma and healthy controls were analyzed by circRNA microarray.Then,we selected the circRNAs whose expression was up-regulated or down-regulated by more than two folds,and the gene ontology enrichment analysis and kyoto encyclopedia of genes and genomes pathway enrichment analysis was used to further screening out the candidate for gastric cancer markers circRNA.Results: We identified 566 circRNAs that were differentially expressing in plasma of gastric cancer patients compared with healthy persons by analyzing circRNA expression profiles?P<0.05?,including 68 circRNAs,whose differential expression level was more than two fold?28 up-regulated and 40 down-regulated?.Further bioinformatics analysis,the final screening of 20 candidate circRNA genes?8 up-regulated,12 down-regulated?.Conclusion: The circRNA expression profiles of gastric cancer plasma were successfully constructed,and 20 circRNA genes expressed in the gastric cancer plasma were screened.Part 2: Expression of primary screening circRNA in gastric cancer plasmaObjective: To verify the expression of primary screening circRNAs in gastric cancer plasma,and to explore its correlation with the clinicopathological features of gastric cancer.Methods: Eighty plasma samples were collected from patients with gastric cancer and healthy persons,and the screening results were verified by qRT-PCR.Combined with patients' age,gender,tumor size,TNM stage,histological grade,lymph node metastasis and other clinical data or pathological features,then clarify the clinicopathological significance of gastric cancer-related circRNA.Results: The expression of 4 circRNAs was verified from 20 circRNAs.The expression of hsacirc0010697,hsacirc0025603 and hsacirc0051494 in plasma was not significantly correlated with gastric cancer?P>0.05?.The plasma level of hsacirc0033634 was significantly increased in patients with gastric cancer?P<0.05?.The area under the ROC curve was 0.739?95%CI: 0.661-0.818?,the sensitivity and specificity were 78.8% and 68.7%,respectively,and hsacirc0033634 was associated with lymph node metastasis of gastric cancer?P<0.05?.Conclusion: Hsacirc0033634 may be involved in the invasion and metastasis of gastric cancer.It has some clinical value for the diagnosis and prognosis of gastric cancer,and may serve as a potential biomarker for gastric cancer.Part 3: Regulation of hsacirc0033634 on biological behavior of gastric cancerObjective: To detect the effect of hsacirc0033634 on the biological behavior of gastric cancer cells,and to understand the relationship between hsacirc0033634 and gastric cancer.Methods: Hsacirc0033634 overexpressing gastric cancer cell SGC-7901 model was established by lentiviral infection technique.The effects of hsacirc0033634 overexpressing on the proliferation,apoptosis,migration and invasion of gastric cancer cells were analyzed by qRT-PCR,CCK-8,flow cytometry,Transwell cell migration and invasion test.Results: The expression level of hsacirc0033634 in overexpressing gastric cancer cells was 3.47±0.13,which was significantly higher than that in negative control group?0.91±0.05??P<0.05?.After transfection with hsacirc0033634 mimics,the proliferation of gastric cancer cells SGC-7901 gradually increased,and the migration ability and invasion ability were significantly increased?P<0.05?,but the apoptosis rate did not change significantly?P>0.05?.Conclusion: Hsacirc0033634 can promote the proliferation of gastric cancer cells,enhance the migration and invasion of gastric cancer cells,and play a positive regulatory role in the process of tumor development.Part 4: Mechanism of hsacirc0033634 in the development of gastric cancerObjective: To investigate the effect of hsacirc0033634 on the biological function of downstream microRNAs,and to explore the molecular mechanism of hsacirc0033634 in the development of gastric cancer.Methods: The bioinformatics method was used to predict the downstream miRNAs and their target genes of hsacirc0033634,and the selected miRNAs were verified by qRT-PCR.Then,the effects of hsacirc0033634 on the biological functions of miRNA were investigated by flow cytometry and tranwell assay.Finally,Western blot was used to verify the effects of hsacirc0033634 and the selected miRNA on target gene expression.Results: The combined analysis of bioinformatics and literature search screened hsa-miR-331-3p and RAS-ERK as possible target genes and signaling pathways of hsacirc0033634.Hsa-miR-331-3p inhibited the proliferation,migration and invasion of gastric cancer cells and promoted apoptosis?P<0.05?,while hsacirc0033634 significantly decreased the expression of hsa-miR-331-3p in gastric cancer cells,and negative regulate its anti-cancer effect.Western blot analysis showed that hsa-miR-331-3p significantly decreased the expression levels of CDK4 protein,Cyclin D1 protein,E2F1 protein,p-ERK1/2 protein and RAS protein in gastric cancer cells?P<0.05?,while hsacirc0033634 significantly decreased the inhibitory effect of hsa-miR-331-3p on the expression of RAS-ERK1/2 signaling pathway and increased the expression level of related target protein?P<0.05?.Conclusion: Hsacirc0033634 participates in the development of gastric cancer through the circ-0033634/miR-331-3p/RAS-ERK regulatory network.Summary:In this study,the expression of circRNA in gastric cancer plasma was screened and verified by gene chip and qRT-PCR technology.The relationship between circRNA and clinicopathological features of gastric cancer was analyzed.The role and molecular mechanism of hsacirc0033634 in the development of gastric cancer was explored by CCK-8,flow cytometry,tranwell and Western blot.Based on the findings,we conclude that: Hsacirc0033634 promotes the proliferation of gastric cancer cells through circ-0033634/miR-331-3p/RAS-ERK regulatory network,and enhances the migration and invasion of gastric cancer cells,and plays a positive regulatory role in the development of tumors. |
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