Study On The Diagnositic Value Of Plasma MiRNA And The Mechanism Of Growth-promoting Role Of MiR-95 In Colorectal Cancer

PART I The value of plasma miRNA for detection of CRCObjectiveMicroRNA (miRNA) opens up a new field for molecular diagnosis of cancer. However, the role of circulating miRNAs in plasma/serum in cancer diagnosis is not clear. The aim of this study was to investigate whether plasma miRNAs can be used as biomarkers for the detection of colorectal carcinoma (CRC), and try to find a new method for non-invasive early diagnosis of CRC.MethodsmiRNA expression profiling was performed on 24 plasma samples from 14 CRC patients and 10 normal controls using Agilent Human miRNA Microarray. These miRNAs up-regulated in CRC plasma were selected based on microarray results, and were further selected and validated in an independent cohort using quantitative reverse transcription polymerase chain reaction (qRT-PCR) method. Receiver-operating characteristics (ROC) curves were established to evaluate the diagnostic value of plasma miRNAs for differentiating between CRC, advanced adenomas and controls. Plasma samples from 20 CRC patients were collected before and after the tumor resection, and these samples were used to determine whether those up-regulated miRNA markers in cancer plasma were reduced after the tumor resection.ResultsThere are 54 plasma miRNAs were found to be differentially expressed between CRC and normal control with more than 1.5 fold change according to miRNA microarray analysis. Validation on 32 potential differently expressed miRNAs using qRT-PCR, showed that plasma miR-29a and miR-92a have significant diagnostic value for CRC and advanced adenomas. miR-29a yielded an AUC (the areas under the ROC curve) of 0.844 and miR-92a yielded an AUC of 0.838 in discriminating CRC from controls. ROC curve analyses also showed that both of the two miRNAs could differentiate advanced adenomas from normal controls with an AUC of 0.769 for miR-29a and 0.749 for miR-92a, respectively. Combined ROC analyses using these two miRNAs revealed an elevated AUC of 0.883 with 83.0% sensitivity and 84.7% specificity in discriminating CRC, and an AUC of 0.773 with 73.0% sensitivity and 79.7% specificity in discriminating advanced adenomas. In addition, it was found that the levels of both miR-29a and miR-92a were significantly reduced in the post-operative plasma samples when compared to the pre-operative samples (P<0.05).ConclusionCollectively, these data suggest that plasma miR-29a and miR-92a have strong potential as novel biomarkers for early detection of CRC.Plasma miRNA analysis appears to be a new noninvasive screening tool for colorectal neoplasia. PART II The role of miR-95 on colorectal carcinogenesisObjectiveIn this study, we investigated the influence of miR-95 on malignant phenotype of colorectal cancer (CRC), and disscussed the association of miR-95 expression in CRC tissues with the clinical characteristics of CRC so as to elucidate the application value of miR-95 for CRC diagnosis and prognositcs.MethodsAgilent Human miRNA Microarray was used to profile the miRNA expression profiling on 10 CRC tissues and paired non-cancerous tissues. Of the 26 miRNAs up-regulated in CRC, miR-95 was selected for elucidating it’s potential machanisms in CRC carcinogenesis. Using the CRC cell lines ectopically expressing miR-95 conducted by lentivirus transduction as experimental material, cell culture, cell growth analysis, clone formation, cell transduction, and in vivo tumor xenograft model were used to analyze the potential influence of miR-95 on CRC tumorgenesis. In addition, the miR-95 expression levels in CRC tissues were deteted using quantitative reverse transcription polymerase chain reaction (qRT-PCR) to investigate it’s relationship with clinical characteristics or survival of CRC patients.ResultsAltered miRNA expression profile was found between CRC tissues and adjacent normal tissues, and 49 miRNAs were found differentially expressed with more than 2 fold change according to the results of miRNA microarray. Of the 26 miRNAs up-regulated in tumor tissues, miR-95 was selected for further analysis.Proliferation experiments using cell growth analysis, clone formation and in vivo tumor xenograft model showed that foreced expression of miR-95 in CRC cells could promote proliferation and inhibite cell apoptosis, and the inhibition of miR-95 by siRNA resulted in a significant decrease of cell growth rate of HCT-8 cell.The miR-95 expresssion was further validated in 87 paired CRC tissues using qRT-PCR method and showed significant up-regulation in CRC compared with adjacent normal tissues. However, no significant association was found between miR-95 expression and tumor size, stage, differentiation or overall survival.ConclusionCollectively, there are systemtical variation of miRNA expression profile between CRC tissues and adjacent normal tissues;miR-95 can function as a proliferation-promoting factor in CRC cells, and appears to be a new oncogenic miRNA. PARTⅢThe screening and validation of the target genes of miR-95ObjectiveTo pridict and verify the target genes of miR-95, and to evaluate the potential mechanism of miR-95 in CRC pathogenesis.MethodsGene chip was used to compare the gene expression profile of CRC cell lines stably expressing miR-95 with that of control cells; the potential target genes of miR-95 were also predicted by TargetScan and miRanda software. Potential target genes of miR-95 were selected according to microarray analysis and software predicting. The 3’-UTR of these selected genes were cloned into luciferase reporter vector and were further analyzed using Luciferase assays. Subsequently, qRT-PCR and Western blot assays were used to further verify these candidate targets of miR-95 in CRC cell lines.ResultsNine potential targets of miR-95 were selected for further verification based on microarray analysis and software predicting. Luciferase assays showed that SNX1, PTPN3 and UBE4B are candidate taregets of miR-95;and the protein levels of these genes were significant down-expressed in miR-95 stable cell lines according to Western blot analysis. The expression of SNX1 was significant down-regulated in tumor tissues when compared to the paired normal tissues, and was inversed related with the miR-95 expression(P<0.05).ConclusionThese data verify the predicted SNX1,PTPN3 and UBE4B gene as human targets of miR-95, suggest its contribution to CRC pathogenesis.