Research On Screening Plasma MiRna As Biomarkers In Diagnosis Of Colorectal Cancer By Using S-poly(T)PCR

Objective: Colorectal cancer(Colorectal Cancer, CRC) is one of the most common malignant tumors, and it has high morbidity and mortality, the key way to reduce fatality rate and improve the prognosis is early diagnosis and treatment. It is crucial to find an effective and reliable way for diagnosis of CRC for the lack of method which is with less trauma,lower cost, higher sensitivity and specificity. at present, more and more researchs find the abnormal expression of non-coding RNA(micro RNA, miRNA) which is detected in plasma of CRC patient, and is expected to become a tumor marker in diagnosing CRC, this study will explore the feasibility and value of miRNA as biomarks by using S-poly(T) PCR. The selected miRNAs will be further analyzed by bioinformatics analysis to search the theoretical basis for the diagnosis of colorectal cancerMethods: All the plasma samples of 101 patients with CRC,20 with colorectal non-cancerous polyps are diagnosed with Paraffin pathology,134 healthy controls are also included.In the first step called the first screening phase we made two plasma mixture samples of 53 patients diagnosed with CRC and 87 healthy controls for screening analyses by S-poly(T) PCR. The aberrant exprssed miRNAs in CRC patients' plasma were filtered from 485 human miRNAs. In the second step called the second screening phase we devided the 53 CRC samples into different groups according to the TNM stage.33 CRC of stage I-II, 20 CRC of stage III-IV,87 healthy controls are mixed into three plasma samples individually tested by S-poly(T) PCR for selecting the differently expressed miRNAs from the filtered miRNAs in the first screening phase.The third step called the validation phase we detected the expression level of the selected miRNAs in each plasma samples of 48 patients with CRC,20 with colorectal non-cancerous polyps,47 healthy controls. In order to evaluate the clinical diagnostic efficacy,we analyzed the relationship of the expression level of these aberrant miRNAs with clinicopathological factors,such as TNM stage, tumor size and tumor location.Result: After comparing the aberrant expression level of miRNAs detecting by S-poly(T)PCR in the two mixed plasma samples of 53 patients with CRC and 87 healthy controls,we chose 16 different-expressed miRNAs from the 485 human miRNAs accordding to the following request: FC(fold change)>1 or FC<-1,p value<0.05,Ct value<35. 2 of 16 miR NAs are up-regulated,others are down-regulated. In the second screening stage,we still use S-poly(T) PCR for selecting the differently expressed miRNAs from the 16 filtered miRN As. we chose 9 miRNAs for the validation phase finally. 2 of 9 miRNAs are up-regulate d,others are down-regulated.we detected the 9 miRNAs from each plasma sample of 48 pat ients with CRC,20 with colorectal non-cancerous polyps, 47 healthy controls. we suggest th at miR-27a-3p,miR-143-3p,miR-144-3p,miR-148a-3p,miR-424-5p,miR-425-5p, miR-1260 b hav e the ability to distinguish CRC from the normals,all the 7 miRNAs are down-regulated,the sensitivities and specificities are 75.0% and 85.0%, 72.9% and 78.7%, 93.8% and 78.7%,79.2% and 91.5%,79.2% and 93.6%,83.3% and 91.5%,81.3% and 83.3% respectively.T he consistency check result shows a good agreement between the miRNA and the gold stand ard paraffin pathology diagnosis. By non-conditional Logistic regressionanalysis, we get the optimal combination of miRNAs in CRC diagnosis: miR-144-3p?miR-425-5p?miR-1260 b. t he sensitivity and specificity of the combined determination were 93.8%and 91.3%.No signi fcant miRNAs are found when analyzing the relationship of the expression level of these a berrant miRNAs with clinicopathological factors,such as TNM stage, tumor size and tumor location.By bioinformatics analysis of the first part selected three miRNAs(miR-144-3p, miR-425-5p, miR-1260b),we predict their target gene and then analysis their over-represented path way by using KEGG, reactome database. Analysising the result details of over-represented KEG G pathways associated with miRNA target genes, we found miR-144-3p significantly enriched in axonal guidance pathway and miR-425-5p enriched in Ca signaling pathway. Analysisin g the result details of over-represented KEGG pathways, we found miR-144-3p significantly enric hed in PI3 K, PDGF and other classic pathway, miR-425-5p mainly enriched in the pathwa y ralated to c AMP,c GMP.Conclusion: This research is aimed at screening miRNA as biomarkers in diagnosis of colo rectal cancer by using S-poly(T) PCR,we finally select 7 miRNAs which are potential for diagnosis of colorectal cancer as clinical biomarkers:miR-27a-3p,miR-143-3p,miR-144-3p,miR-148a-3p,miR-424-5p,miR-425-5p,miR-1260 b.The diagnostic value of miRNA and the value of pathological paraffin section have a good consistency; Both the sensitivity and specificity of combined determination: miR-144-3p,miR-425-5p,miR-1260 b are more than 90%, superior to the current clinical common-used methods such as CEA,they have the potential clinical app lication value.Bioinformatics analysis shows miR-144-3p significantly is enriched in axonal guidance pa thway, PI3 K, PDGF and other classic pathway, miR-425-5p enriched in Ca signaling pathw ay and the pathway ralated to c AMP,c GMP. Because of the rare report about the relation ship between miR-1260 b and CRC,we can find the over-represented pathways about miR-1260 b.But it can still prove miRNA could participate in the regulation of signaling pathway s by rugulating its target genes and it plays an important role in the occurrence and devel opment of CRC.It also lays a good theoretical foundation of surpporting plasma miRNA f or the diagnosis of colorectal cancer...