Study On The Diagnostic Value Of Plasma MiRNA And The Mechanism Of Growth-inhibitory Role Of MiR-202-3p In Colorectal Cancer

PART Ⅰ The value of plasma miRNA for detection of CRCObjectiveColorectal cancer (CRC) is a major cause of death worldwide. Sensitive, non-invasive diagnostic screen methods are urgently needed to improve survival rates. Stable circulating microRNA offers unique opportunities for the early diagnosis of several diseases, including cancers. Our aim has been to find new plasma miRNAs that can be used as biomarkers for the early detection of CRC.MethodsmiRNA expression profiling was done on2pooled plasma samples from10CRCs (including5stage Ⅱ and5stage Ⅲpatients) and10normal controls using microRNA PCR array. A panel of deregulated miRNAs were found and validated in an independent cohort by quantitative reverse transcription polymerase chain reaction (qRT-PCR) method. Receiver-operating characteristic (ROC) curves were established to evaluate the diagnostic value of plasma miRNAs for differentiating CRC or advanced adenomas from nomal controls.ResultsAccording to the results of miRNA profiling, a panel of miRNAs (hsa-let-7e,-10a,-19a,-22*,-24,-92,-125a-5p,-150,-188-3p,-224*,-376a,-425*,-495,-572,-601,-720and hsa-miR-760) were deregulated in CRC plasma. After large scale validation by real-time PCR performed on another191independent individuals (90CRC patients,43advanced adenoma patients and58healthy participants), we found that the levels of plasma miR-601and miR-760were significantly decreased in colorectal neoplasia (carcinomas and advanced adenomas) compared with healthy controls. ROC curve analysis showed that plasma miR-601and miR-760were of significant diagnostic value for advanced neoplasia. These two miRNAs together yield an AUC(area under the ROC curve) of0.792with83.3%sensitivity and69.1%specificity for separating CRC from normal controls, and yield an AUC of0.683with62.1%sensitivity and72.1%specificity in discriminating advanced adenomas from controls. Complementary use of miR-601and miR-760detected30CEA-missed stage Ⅰ and Ⅱ cases, raising diagnostic sensitivity from27.5%to86.3%.ConclusionIn summary, plasma miR-601and miR-760can potentially serve as promising non-invasive biomarkers for the early detection of CRC.PART Ⅱ The function of miR-202-3p on colorectal carcinogenesisObjectiveIn this study, we investigated the influence of miR-202-3p on malignant phenotype of colorectal cancer (CRC), and discussed the association of miR-202-3p expression in CRC tissues with the clinical characteristics so as to elucidate the application value of miR-202-3p for CRC diagnosis and prognostic evaluation.MethodsmiRNA expression profiling was performed on10CRC tissues and paired non-cancerous tissues by Agilent Human miRNA Microarray. A number of81differentially expressed miRNAs was found and of the46down-regulated miRNAs in CRC, miR-202-3p was selected for elucidating potential mechanisms in CRC carcinogenesis. Using the CRC cell lines ectopically expressing miR-202-3p conducted by lentivirus transduction as experimental material, cell culture, cell growth analysis, clone formation, cell transduction were used to analyze the potential influence of miR-202-3p on CRC tumorigenesis. In addition, the miR-202-3p expression levels in CRC tissues were detected using quantitative reverse transcription polymerase chain reaction (qRT-PCR) to investigate the relationship with clinical characteristics.ResultsAltered miRNA expression profile was found between CRC tissues and paired non-cancerous tissues, and81miRNAs were found differentially expressed according to the results of miRNA microarray (P<0.05, fold change≥and flags≥10). Of the46miRNAs down-regulated in tumor tissues, miR-202-3p was selected for further analysis. Proliferation experiments using cell growth analysis, clone formation showed that over expression of miR-202-3p could repress CRC cell proliferation, and the inhibition of miR-202-3p by siRNA resulted in a significant increase of cell growth rate. The miR-202-3p expression was further validated in136paired CRC tissues using qRT-PCR method and showed significant down-regulation in CRC compared with paired non-cancerous tissues. However, no significant association was found between miR-202-3p expression and tumor size, stage, differentiation or overall survival.ConclusionIn summary, there are systematic variation of miRNA expression profile between CRC tissues and paired non-cancerous tissues; miR-202-3p can function as a proliferation-inhibiting factor in CRC cells, and appears to be a new tumor suppress miRNA. PART III The screening and validation of the target genes of miR-miR-202-3pObjectiveThe objective of this part is to predict and verify the target genes of miR-202-3p, and to evaluate the potential mechanism of miR-202-3p in CRC pathogenesis.MethodsGene chip was used to compare the gene expression profile of CRC cell lines transfecting miR-202-3p mimics with that of control cells; the potential target genes and their binding sites of miR-202-3p were also predicted by TargetScan and miRanda software. Potential target genes of miR-202-3p were selected according to microarray analysis and software predicting. The3’-UTR of these selected genes were cloned into luciferase reporter vector and were further analyzed using luciferase assays. Subsequently, si-RNA gene knockdown technology was used for next function analysis. Finally, qRT-PCR was used to verify these candidate targets of miR-202-3p in CRC tissues.ResultsTwelve potential targets of miR-202-3p were selected for further verification based on microarray analysis and software predicting. Luciferase assays showed that ATXN7L2、ARL5A、LRIG3、STARD13、HECTD2and CPEB4are candidate targets of miR-202-3p; knockdown of ATXN7L2、ARL5A. LRIG3and STARD13by siRNA significantly repress CRC cell proliferation; Down regulating of miR-202-3p expression enhance CRC cell growth, but could not promote cell proliferation in ATXN7L2、ARL5A、LRIG3or STARD13-depleted cells. The expression of ARL5A was significant up-regulated in tumor tissues when compared to the paired normal tissues (P<0.05). ConclusionThese data verify the predicted ATXN7L、ARL5A. LRIG3and STARD13gene as human targets of miR-202-3p, suggest its contribution to CRC pathogenesis.