The Basic And Clinical Study Of Serum MiRNA As Biomarkers In Colorectal Cancer

Micro RNAs(mi RNAs) are a class of short(18-22 nt in length), evolutionarily conserved, non-protein-coding RNAs that play critical roles in diverse biological processes though negative post-transcriptional regulation. Recent evidence has indicated that mi RNAs can function as oncogenes or tumor suppressors by repressing cancer-related genes. Alterations of mi RNA expression have been observed in a variety of human tumors, and we previously discovered that mi RNAs are stably present in circulating blood at sufficient levels for use as blood-based biomarkers. Colorectal cancer(CRC) is currently the third most common malignancy and the second leading cause of cancer mortality worldwide, with more than 1.2 million new cases and 600,000 deaths annually. Blood-based biomarkers for clinical use caused great interest as easily available and minimally invasive. Unfortunately, studies determining how to monitor dynamic changes, assess prognosis and predict therapeutic outcomes in these patients are lacking. Based on our preliminary studies, in this Ph D thesis, we carried out a series of basic and clinical research to study the potential of serum mi RNAs using as disease novel biomarkers for colorectal cancer, and the research was divided in four parts.In the first section, we investigated the use of a serum mi RNA profile as a prospective indicator to monitor disease dynamics and to predict disease-free survival by comparing the differential expression of serum mi RNAs before and after curative resection for stage II/III CRC. Using the Taq Man Low-Density Array(TLDA), we performed a microarray survey to analyze 749 mi RNAs in the pooled serum of 20 paired pre- and post-operative CRC patients and 20 matched normal subjects. Using individual RT-q PCR verification in 175 stage II/III CRC patients, we identified that mi R-145, mi R-106 a and mi R-17-3p were significantly differentially expressed between pre- and post-operative CRC patients and between pre-operative CRC patients and normal controls(P<0.0001). The area under the ROC curve(AUC) for the three-mi RNA panel was 0.886(95% CI 0.850–0.921) for discriminating between pre-operative CRC patients and normal subjects and 0.850(95% CI 0.809–0.891) for discriminating between pre- and post-operative CRC patients. Furthermore, the prognostic value of the mi RNAs was evaluated using the Kaplan-Meier method and Cox proportional hazards analysis. The survival analysis showed that mi R-17-3p and mi R-106 a were powerful and independent prognostic indicators and that high levels of these mi RNAs were associated with shorter disease-free survival(DFS)(P<0.0001 for mi R-17-3p and P=0.001 for mi R-106a). The present study reveals novel biomarkers for monitoring tumor dynamics as well as for predicting disease recurrence in patients with stage II/III CRC.In the second section, we investigated biomarkers that predict response to oxaliplatin/irinotecan-based therapy in patients with metastatic CRC(m CRC) in this study. Firstly, aiming to identifying potential biomarkers to predict response to oxaliplatin-based chemotherapy as well as monitor therapy response in m CRC patients, we screened global mi RNA expression profiles by using TLDA followed by a large-scale validated study by individual RT-q PCR assay. Furthermore, we evaluated the expression of selected mi RNAs before onset and after four cycles of chemotherapy. Prognostic value of the mi RNAs was evaluated by the Kaplan-Meier method. We identified that mi R-135 b and mi R-208 b were expressed with higher mean expression in non-responder group compared to responder group before treatment(P<0.0001 and <0.0001 respectively). The areas under the ROC curve(AUCs) were 0.877(95% CI 0.811–0.942) for mi R-135 b and 0.771(95% CI 0.688–0.855) for mi R-208 b. Furthermore, both mi R-135 b and mi R-208 b were significantly reduced in responders and elevated in non-responders after four cycles of FOLFOX regimen. After survival analysis, high levels of mi R-135 b and mi R-208 b were associated with shorter progression-free survival(PFS)(Hazard ratios(HR) of 2.08(95% CI, 1.36-3.18), p<0.0001 and HR of 1.90(95% CI, 1.24-2.90), p=0.001; respectively). The present study demonstrates that serum mi RNAs analyzed before treatment may serve as non-invasive markers predicting chemotherapy sensitivity and clinical outcome and monitoring chemotherapy response in m CRC patients treated with 5-FU and oxaliplatin-based chemotherapy. Secondly, we sought to identify a serum mi RNA expression profile to predict for irinotican-based chemotherapy sensitivity prior to treatment and monitoring disease dynamics during treatment. Using the TLDA, we performed an initial microarray survey of 749 mi RNAs to analyze pooled serum on 12 responders and 12 nonresponders to FOLFIRI regimen. Using individual RT-q PCR from 38 responders and 32 nonrespoders, we validated the selected mi RNAs to identify significantly differentially expressed serum mi RNAs in patients responsive and nonresponsive to FOLFIRI regimen. Furthermore, we evaluated the expression of selected mi RNAs before onset and after four cycles of chemotherapy. For the FOLFIRI regimen, we found that mi R-135 b, mi R-10 a and mi R-627 were overexpressed in non-responder group compared to responder group before treatment(P<0.0001, <0.001 and <0.001 respectively). The areas under the ROC curve(AUCs) were 0.84(95% CI 0.749–0.932) for mi R-135 b, 0.789(95% CI 0.681–0.897) for mi R-10 a and 0.79(95% CI 0.686–0.894). The present study demonstrates that plasma mi RNAs analyzed before treatment may serve as non-invasive markers predicting chemotherapy sensitivity and monitoring chemotherapy response in m CRC patients treated with 5-FU and irinotican-based chemotherapy.In the third section, we explore the role of mi R-135 b in cancer progression by targeting transforming growth factor beta receptor II(TGFBR2) in colorectal cancer. We found that the TGFBR2 protein levels were consistently down-regulated in CRC tissues, whereas its m RNA levels varied in these tissues, suggesting that a post-transcriptional mechanism is involved in the regulation of TGFBR2. Because mi RNAs are powerful post-transcriptional regulators of gene expression, we performed bioinformatic analyses to search for mi RNAs that potentially target TGFBR2. We identified the specific targeting site of mi R-135 b in the 3’-untranslated region(3’-UTR) of TGFBR2. We further identified an inverse correlation between the levels of mi R-135 b and TGFBR2 protein, but not m RNA, in CRC tissue samples. By overexpressing or silencing mi R-135 b in CRC cells, we experimentally validated that mi R-135 b directly binds to the 3’-UTR of the TGFBR2 transcript and regulates TGFBR2 expression. Furthermore, the biological consequences of the targeting of TGFBR2 by mi R-135 b were examined using in vitro cell proliferation and apoptosis assays. We demonstrated that mi R-135 b exerted a tumor-promoting effect by inducing the proliferation and inhibiting the apoptosis of CRC cells via the negative regulation of TGFBR2 expression. Taken together, our findings provide the first evidence supporting the role of mi R-135 b as an oncogene in CRC via the inhibition of TGFBR2 translation.In summary, we studied the mi RNA expression profile in serum evaluate their clinical value for CRC diagnosis, we also investigate the potential role of the selected mi RNA in CRC and found that:(1) Three serum mi RNA including mi R-145, mi R-106 a and mi R-17-3p can be used as a potential diagnostic marker for stage II/III CRC. The survival analysis showed that mi R-17-3p and mi R-106 a were powerful and independent prognostic indicators and that high levels of these mi RNAs were associated with shorter disease-free survival.(2) Serum mi R-135 b and mi R-208 b can be used as markers predicting chemotherapy sensitivity and clinical outcome and monitoring chemotherapy response in m CRC patients treated with FOLFOX regimen.(3) Serum mi R-135 b, mi R-10 a and mi R-627 can be used as markers predicting chemotherapy sensitivity and monitoring chemotherapy response in m CRC patients treated with FOLFIRI regimen.(4) Mi R-135 b promotes cancer progression by targeting transforming growth factor beta receptor II(TGFBR2) in colorectal cancer.