| Gastric cancer is one of the most common malignant tumors worldwide and in our country.Gastric cancer patients usually have poor prognosis.Invasion and metastasis are important features of gastric cancer,which often lead to poor prognosis of patients.It is an important issue in the basic and clinical research to reveal the molecular mechanism of gastric cancer invasion and metastasis.In addition to genetic changes,non-coding RNAs,especially microRNAs(miRNAs,miRs),play important roles in tumor development,invasion and metastasis.MiRNAs are a class of small RNAs of about 19-25 nucleotides in length,which could degrade the target mRNA and/or inhibit the target protein translation by binding to the 3’untranslated region(3’-UTR)of the target mRNA.MiRNAs participate in cell proliferation,apoptosis,development,differentiation,and play crutial roles in the development and progression of a variety of human diseases,including tumors.However,the role and mechanism of miR-214 have not been investigated in detail.We propose to detect the expression of miR-214 in human gastric cancer specimens and gastric cancer cell lines,and to perform miR-214 functional experiments to verify its roles and to identify the target genes.The target gene prediction software suggests that cAMP responsive element binding protein 1(CREB1)may be the target gene of miR-214.We intend to explore the expression,function and mechanism of CREB1 in gastric cancer.This study aims to provide new methods and targets for the diagnosis and treatment of gastric cancer.Part I Clinicopathological significance of miR-214 in gastric cancer and its effect on cell biological behaviourPurpose:1.To detect the expression of miR-214 in gastric cancer tissues and cells.2.To analyze the relationship between miR-214 and clinicopathological parameters and prognosis of patients.3.To evaluate the effect of miR-214 on cell migration,invasion,proliferation and apoptosis of gastric cancer.4.To identify the target genes directly regulated by miR-214.Methods:1.Real-time quantitative PCR(RT-qPCR)was used to detect the expression of miR-214 in 80 cases of gastric cancer,18 cases of non-tumorous gastric mucosa,four kinds of gastric cancer cell lines(MKN28,BGC823,MKN45 and SGC7901)and one non-tumorous immortalized cell line GES-1.2.The clinical data and follow-up information were collected to analyze the relationship between miR-214 and clinicopathological parameters such as tumor diameter and lymph node metastasis.Kaplan-Meier method and Log-rank test were used to analyze the prognostic value of miR-214.3.Gastric cancer cells were transfected with miR-214,miR-214 inhibitor or negative control.Cell migration,invasion,proliferation and apoptosis assays were performed.4.TargetScan was used to predict the target genes of miR-214,and the direct target gene of miR-214 was verified by luciferase assay and Western blot.Results:1.The expression level of miR-214 in 80 cases of gastric carcinoma was decreased by sixfold compared with that of 18 cases of non-tumorous gastric mucosa.MiR-214 expression was even lower in gastric cancer tissues with lymph node metastasis.The expression of miR-214 in four kinds of gastric cancer cells was also downregulated compared to non-tumorous gastric mucosa.2.The expression of miR-214 was negatively correlated with tumor size and lymph node metastasis.However,miR-214 had no significant effect on the prognosis of patients.3.MiR-214 can inhibit the proliferation,migration and invasion of gastric cancer cells,with little effect on cell apoptosis.4.Target gene software prediction suggested that FGFR1,CSF1,NOTCH2,CREB1 and AGAP2 were potential target genes of miR-214.It was confirmed by luciferase assay and Western blot that CSF1 was a direct target of miR-214.Conclusions and and significance:MiR-214 was down-regulated in gastric cancer tissues and multiple gastric cancer cell lines,and was even lower in metastatic gastric cancer tissues.MiR-214 inhibited the proliferation,migration and invasion of gastric cancer cells,but had no significant effect on cell apoptosis.CSF1 is the direct target gene of miR-214,and the regulation of miR-214 on other target genes(e.g.CREB1)needs to be further validated.MiR-214 can down-regulate the expression of CSF1,and we believe that miR-214 may play a role in inhibiting the proliferation,migration and invasion of gastric cancer by regulating CSF1.Part Ⅱ Expression and prognostic value of CREB1 in gastric carcinoma and its regulation by miRNAsPurpose:1.To detect the expression of CREB1 in gastric cancer tissues.2.To analyze the clinical significance and prognostic value of CREB1 in gastric cancer.3.To verify miRNAs’ regulation of CREB1.Methods:1.Immunohistochemistry was used to detect the expression of CREB1 in 185 cases of primary gastric cancer tissues,50 cases of secondary lymph node foci and 50 cases of non-tumorous gastric mucosa.2.The relationship between CREB1 and clinicopathological parameters was analyzed using chi-square test.The relationship between CREB1 and prognosis was analyzed by Kaplan-Meier method and Log-rank test.3.miRWalk and starBase software were used to predict miRNAs that could regulate CREB1.The regulation of miRNAs on CREB1 was verified by luciferase assay and Western blot.Results:1.CREB1 expression was stepwise increased in primary gastric cancer tissues and secondary lymph node metastatic foci,compared with non-tumorous gastric mucosa.CREB1 expression in cancerous tissues with lymph node metastasis(LNM)was significantly higher than that in cancerous tissues without LNM.2.High expression of CREB1 was positively correlated with lymph node metastasis,distant metastasis and tumor stage in gastric cancer.3.Patients with high CREB1 expression had significantly shorter overall survival and disease-free survival than those with low CREB1 expression.The prognostic model composed of CREB1 and tumor stage is an independent prognostic factor.4 CREB1 is a direct target gene of miR-27b and miR-200b,and is negatively regulated by miR-27b and miR-200b.Conclusions and and significance:High expression of CREB1 is associated with metastasis,tumor stage and poor outcome in gastric cancer.MiR-27b and miR-200b could negatively regulate the expression of CREB1.Our findings suggest that CREB1,as a valuable biomarker of gastric cancer prognosis,may be a promising approach to gastric cancer treatment through the miR-27b/miR-200b-CREB1 pathway.Part III Preliminary study on the function and mechanism of CREB1 in gastric cancerPurpose:1.To explore the function of CREB1 in gastric cancer.2.To identify the target genes of CREB1 in gastric cancer and elucidate its molecular mechanism.Methods:1.CREB1 overexpression plasmid or small interfering RNA(siRNA)was transfected into gastric cancer cells.The effects on cell proliferation,migration and invasion were determined.2.Genome-wide binding profiling of CREB1 was analyzed by Chromatin immunoprecipitation sequencing(ChIP-seq)in gastric cancer cell line.The mRNAs,miRNAs and lncRNAs potentially regulated by CREB1 were screened.3.Gastric cancer cells were transfected with CREB1 overexpression plasmid or CREB1 siRNA,and the expression level of candidate downstream IncRNAs were detected by RT-qPCR.4.ChIP-qPCR was used to show direct binding of CREB1 to lncRNA promoter regions.Results:1.Overexpression of CREB1 could promote the proliferation,migration and invasion of gastric cancer cells.In contrast,inhibition of CREB1 expression attenuated the proliferation,migration and invasion of gastric cancer cells.2.As revealed by ChIP-seq,CREB1 could bind to the promoter regions of 831 mRNAs,276 lncRNAs,and 62 miRNAs.3.Combined with our previous study of gastric cancer metastasis-related lncRNA microarray,nine candidate lncRNAs were screened.4.RT-qPCR analysis revealed that CREB1 positively regulated the expression of lncRNA LOC100270746,PIN1P1 and ZEB1-AS1.5.ChIP-qPCR revealed significant enrichment of CREB1 binding to lncRNA promoter regions.Conclusions and and significance:CREB1 could promote the proliferation,migration and invasion of gastric cancer cells.The expression of lncRNA LOC100270746,PIN1P1 and ZEB1-AS1 was positively regulated by CREB1. |
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