Mirna-mRNA Regulatory Networks By Integrating MiRNA And MRNA Expression Profiling Of Esophageal Squamous Cell Carcinoma

PART I Screening and analyzing miRNA and genome wide expression profiling of ESCCObjectiveIn order to further understand the association between miRNA expression and their target mRNA in ESCC, integrated miRNA and mRNA expression profiling in ESCC was performed.Materials and MethodsEight esophageal squamous carcinomas of Stage T2N1M0matched with their corresponding normal tissues were included in the study. Macrodissection was performed to assure that only tissues with more than70%tumor components were used for RNA extraction. Microarray technology (Agilent Human miRNA microarray and Agilent whole Human gene expression microarray) was used for miRNA and genome wide mRNA expression profiling. Specific target genes of miRNA were demonstratd by using the target mRNAs multistep bioinformatics, target gene predicting and integrated analysis of miRNAs methods. The relationship of the miRNA and gene expression was calculated by setting their differentiate expression values according to the interactions of miRNA and genes in Gene Ontology and KEGG database to create the miRNA-Gene-Network and miRNA-GO-Network by the bioinformatic tools.ResultsCompared to esophageal normal epithelia,67differentiately expressed miRNAs and2992mRNAs in ESCC were found, serving as esophageal carcinoma miRNA and gene profile; GO and Pathway analysis showed that the up-regulated target genes could be grouped to206GO and31pathway classifications, while the down-regulated target genes to88GO and16pathway classifications.The up-regulated target genes were associated to many biological processes such as angiogenesis, cell adhesion and mitotic cell cycle, while the down-regulated target genes were associated with blood coagulation, signal transduction and negative regulation of cell migration. These up-and down-regulation could directly or indirectly give rise to tumor development. The up-regulated target genes were involved in the significant pathway such as focal adhesion, pathways in cancer, TGF-beta signaling pathway, Wnt signaling pathway and p53signaling pathway. The down-regulated target genes were involed in MAPK signaling pathway, tight junction, arachidonic acid metabolism, cell adhesion molecules (CAMs). These pathways play vital role in a series of important biological processes such as cell movement, cell proliferation, cell differentiation, gene expression regulation, cell survival, etc.In the miRNA-Gene-Network and miRNA-GO-Network interaction, miR-3Oa-5p, miR-29c-3p, let-7c, miR-126-5p, miR-195-5p, miR-125b-5p, miR-196b-5p and miR-1palyed core regulation role, in which RORA, CDK6, KCNMA1, TRAF3, IGF1, TGFβ2and VEGFA were the main target genes regulated. Signal transduction, positive regulation of transcription from RNA polymerase II promoter, angiogenesis and cell proliferation were major function regulated. These indicate that the miRNAs and their target genes as well as its function paly key role in the development of ESCC.ConclusionIt is the first large-scale study to identify miRNA-mRNA regulatory networks in ESCC by combining expression profiles and bioinformatics analysis, demonstrating that miRNA expression is highly associated with the proliferation, prognosis and metastasis of ESCC and providing a valuable source for future study.PART II Validate the results of miRNA and gene chip in esophageal squamous cell carcinomaObjectiveTo confirm the reliability of miRNA and gene microarray results by validating some of the differentially expressed miRNA and genes in the microarray screeningMaterials and MethodsWe validated7candidate miRNAs (miR-21, miR-196b, miR-185, miR-126,miR-195, miR-125b, miR-1) and7candidate mRNA (Sixl, Six4, IGF1, TGFβ2, CDK6, FLNC, VEGFA) in an independent set of70paired tumors and their adjacent normal tissues using Real-time quantitative PCR (RT-qPCR) and analyzed miRNA and gene expression using2-ΔΔCT method. Relative expression of miRNA was determined in reference to an internal U6snRNA control, and relative mRNA expression was determined in reference to β-actin. In the statistical analysis, we presented results as mean±SD, and assessed differences between the groups was displayed by using T-Test in SPSS18.0.ResultsThe expression level of7miRNA and7mRNA showed significant difference in an extended cohort of70ESCC tumor tissues and the matched normal epithelia in RT-qPCR test (P<0.05), indicating coincidence to the results derived from the miRNA and gene chip study.ConclusionThe results from RT-qPCR by validating7miRNA and7mRNA were comparable to the ones of up-and down-regulated trend detected in miRNA and gene chip. The results from two methods mirrored each other. The miRNA panel and mRNA panel were demonstrated to be of critical in ESCC tumorogenesis, providing the basis for further study.PARTIII Expression of candidate miRNA, Sixl and Six4in292cases of ESCC and the correlation to clinical prognosisObjectiveTo investigate the expression of7candidate miRNAs(miR-21, miR-196b, miR-185, miR-126, miR-195, miR-125b, miR-1) and two protein Sixl and Six4in ESCC, and to explore their correlation to clinicopathological factors and prognosis of the disease.Materials and MethodsIn situ hybridization, immunohistochemical method and tissue microarray technology were used to detect the expression of7miRNA studied in part Ⅱ, Sixl and Six4protein expression in the tumor tissues and adjacent normal epithelium of esophagus from292ESCC patients with comprehensive clinicopathological and prognostic data, whose tumor tissues were built in a set of11tissue microarrays.ResultsIn292ESCC patients, expression of miR-21, miR-185and miR-196b in ESCC tissues were significantly higher than that in adjacent normal tissues (p<0.05), indicating that these miRNA might work as oncogenes promoteing the tumorigenesis and development of ESCC. Meanwhile, the expression of miR-1, miR-125b, miR-126and miR-195in ESCC tissues were significantly lower than that in adjacent normal tissues (p<0.05), seeming to be anti-oncogenes with inhibit function in the tumorigenesis and development of ESCC. Chi square test showed that the expression of miR-21was related to distant metastasis and tumor local invasion. The deeper the tumor invaded, the higher miR-21expression was. The expression of miR-185was negatively related to the degree of tumor differentiation. The poorer the tumor was differentiated, the higher of the positive rate of miR-185showed. The expression of miR-196b was positively related to the patient’s age and lymph node metastasis. The expression of miR-125b was negatively related to tumor local invasion.The deeper the tumor was invaded, the higher of the positive rate of miR-125b expressed. The expression of miR-1was negatively related to lymph node metastasis, vascular invasion, distant metastasis and tumor local invasion. Also, the expression of miR-1was bound up with the survival state of ESCC. All of the above results suggest that these miRNAs may play an important role in the carcinogenesis as well as in the process of invasion and metastasis of ESCC.In292ESCC patients, the positive rate of Sixl and Six4protein expression in tumor tissues were72.9%and56.3%, respectively, much higher than33.2%and32.2% in adjacent normal epithelium of esophagus, there was a statistical difference (p<0.05). Chi square test showed that the expression of Six1protein was related to tumor size, tumor infiltration depth and survival status; Six4protein expression level was related to tumor differentiation and tumor infiltration depth. The poorer the tumor was differentiated, the deeper the tumor was invaded, the higher of the positive rate.Single factor Lok-rank analysis revealed that gender, TNM stage, miR-land Sixl protein expression level were bounded up with the overall survival of ESCC patients (P<0.05), while the five years survival rate was significantly higher in the Sixl negative group than that in the Sixl positive group (51.9%.vs.43.7%). Multi-factor COX proportional risk model analysis shows that TNM stage, down regulation of miR-1and positive expression of Sixl was independent prognostic factor of ESCC patients (P<0.05).ConclusionThe expression levels of7miRNA are consistent with the results of the microarray chip analysis and the RT-qPCR. Six1and Six4are highly expressed in ESCC. Their expression levels are closely related to the progress and prognosis of ESCC. Down regulation of miR-1and over expression of Sixl is related to poorer prognosis of ESCC patients. Thus, miR-1and Sixl could be used as an important prognostic indicator of ESCC patients.