结肠癌组织和癌旁组织miRNA表达谱研究

目的探讨结肠癌癌组织和癌旁组织中miRNA的表达差异,寻找潜在的结肠癌特异表达谱。方法miRNA表达芯片检测手术切除未发生转移的原位结肠癌组织和癌旁组织标本中miRNA表达水平,并采用Real-time PCR对差异表达的miRNA进行验证。结果miRNA表达芯片分析发现肿瘤细胞中24种miRNA表达下调,27种表达上调。miR-145、miR-143在癌组织中表达显著下调,miR-451在癌组织中表达显著上调,并被Real-time PCR方法进一步证实。结论结肠癌癌组织和癌旁组织中miRNA的表达存在差异,miR-145、miR-143在结肠癌组织中表达下调,miR-451表达上调,结肠癌组织具有特异miRNA表达谱,可作为结肠癌潜在诊断芯片进行开发。     

Identification of aberrantly expressed miRNAs in rectal cancer

Disturbance of miRNA expression may play a key role in the initiation and progression of colorectal cancer (CRC). CRC should be viewed as a heterogeneous disease, but previous studies have only screened dysregulated miRNAs in CRC from a panel of 96, 145, 287 and 455 miRNAs, respectively. It is necessary to identify new aberrantly expressed miRNAs in rectal cancer. In this study tissue samples were derived from patients undergoing a surgical procedure to remove a portion of cancers. The expression profile of 904 miRNAs was analyzed using a miRCURY? LNA Array from 6-paired rectal cancers and normal tissues. The expression levels of 4 miRNAs were compared by real-time PCR between colon and rectal cancer, and also the expression levels of metastatic miRNAs in different stages of rectal cancer were analyzed. We found that 67 miRNA precursors are upregulated in rectal cancer (p<0.05) and 21 of those have never been reported in colorectal cancer (CRC); 39 miRNA precursors are downregulated (p<0.05) and 24 novel dysregulated miRNAs were identified in rectal cancer. miR-31, miR-126 and miR-143 are differentially expressed between colon cancer and rectal cancer. Here, we report an miRNA profile of rectal cancer, and we identified differential expression patterns of miRNAs between rectal and colon cancers. This novel information may suggest the potential roles of these miRNAs in the diagnosis of rectal cancer.     

Clinical value of integrated-signature miRNAs in colorectal cancer: miRNA expression profiling analysis and experimental validation

MicroRNA (miRNA) expression profiling of colorectal cancer (CRC) are often inconsistent among different studies. To determine candidate miRNA biomarkers for CRC, we performed an integrative analysis of miRNA expression profiling compared CRC tissues and paired neighboring noncancerous colorectal tissues. Using robust rank aggregation method, we identified a miRNA set of 10 integrated-signature miRNAs. In addition, the qRT-PCR validation demonstrated that 9 miRNAs were consistent dysregulated with the integrative analysis in CRC tissues, 4 miRNAs (miR-21-5p, miR-183-5p, miR-17-5p and miR-20a-5p) were up-regulated expression, and 5 miRNAs (miR-145-5p, miR-195-5p, miR-139-5p, miR-378a-5p and miR-143-3p) were down-regulated expression (all p < 0.05). Consistent with the initial analysis, 7 miRNAs were found to be significantly dysregulated in CRC tissues in TCGA data base, 4 miRNAs (miR-21-5p, miR-183-5p, miR-17-5p and miR-20a-5p) were significantly up-regulated expression, and 3 miRNAs (miR-145-5p, miR-139-5p and miR-378a-5p) were significantly down-regulated expression in CRC tissues (all p < 0.001). Furthermore, miR-17-5p (p = 0.011) and miR-20a-5p (p = 0.003) were up-regulated expression in the III/IV tumor stage, miR-145-5p (p = 0.028) and miR-195-5p (p = 0.001) were significantly increased expression with microscopic vascular invasion in CRC tissues, miR-17-5p (p = 0.037) and miR-145-5p (p = 0.023) were significantly increased expression with lymphovascular invasion. Moreover, Cox regression analysis of CRC patients in TCGA data base showed miR-20a-5p was correlated with survival (hazard ratio: 1.875, 95%CI: 1.088–3.232, p = 0.024). Hence, the finding of current study provides a basic implication of these miRNAs for further clinical application in CRC.     

Candidate microRNA biomarkers in human colorectal cancer: systematic review profiling studies and experimental validation

Colorectal cancer (CRC) is a major cause of cancer mortality worldwide. There is an urgent need to search for specific and sensitive biomarkers for early diagnosis of CRC. We carried out a comprehensive systematic review of published studies that compared the miRNA expression profiles between CRC tissue and paired neighboring noncancerous colorectal tissue to determine candidate miRNA biomarkers for CRC. A miRNA ranking system that takes the number of comparisons in agreement, total study sizes and direction of differential expression into the consideration was devised and used. One of the most up-regulated miRNAs, miRNA-106a, was consistently reported to be differentially expressed in six studies and the five most down-regulated miRNAs, miR-30a-3p, miR-139, miR-145, miR-125a and miR-133a, were consistently reported to be differentially expressed in four studies. Moreover, we further validated five miRNAs in a clinical setting using qRT-PCR, which demonstrated that miR-106a expression was increased, whereas the expression of miR-30a-3p, miR-145, miR-125a and miR-133a was decreased in the CRC tissues. Therefore, these miRNAs may be the candidates to develop a panel of biomarkers with sufficient sensitivity and specificity for the diagnosis of CRC in a clinical setting.     

Identification of new aberrantly expressed miRNAs in intestinal-type gastric cancer and its clinical significance

miRNAs are small 19 to 22 nucleotide sequences of RNA that negatively regulate gene expression. miRNA expression profiles may become useful biomarkers for diagnostics, prognosis and prediction of response to treat, and it could be a powerful tool for cancer prevention and therapeutics. Several miRNA expression profiles of miRNAs in gastric cancer have been reported, but these studies screened only few miRNAs and samples used in experiments include several different subtypes of gastric cancers, which decrease the sensitivity to identify new aberrant miRNAs. In this study, a miRNA expression profile was identified by miRCURY LNA Array (v.14.0) between intestinal-type gastric cancers and normal tissues. Forty miRNA precursors were up-regulated and thirty-six miRNA were down-regulated in intestinal-type gastric cancers (p<0.01). Sixteen new miRNAs were found in intestinal-type gastric cancers. Seventeen new miRNAs were found in intestinal-type gastric cancers. miR-145, miR-27a, miR-494 are differently expressed between intestinal-type and diffuse-type gastric cancers. miR-32, miR-182 and miR-143 dysregulated expression levels are related with different pathological stages of intestinal-type gastric cancers (p<0.01). Taken together, aberrantly expressed miRNAs may offer new clues to tumorigenesis of gastric cancers. miR-32, miR-182 and miR-143 may be potential diagnostic biomarkers for intestinal-type gastric cancers.     

Plasma Circulating Mirnas Profiling for Identification of Potential Breast Cancer Early Detection Biomarkers

Objective: This study aimed to characterize the miRNA expression profiles from plasma samples of our local breast cancer patients in comparison to healthy control by using miRNA PCR Array. Methods: In this study, plasma miRNA profiles from eight early-stage breast cancer patients and nine age-matched (± 2 years) healthy controls were characterized by miRNA array-based approach, followed by differential gene expression analysis, Independent T-test and construction of Receiver Operating Characteristic (ROC) curve to determine the capability of the assays to discriminate between breast cancer and the healthy control. Results: Based on the 372-miRNAs microarray profiling, a set of 40 differential miRNAs was extracted regarding to the fold change value at 2 and above. We further sub grouped 40 miRNAs of breast cancer patients that were significantly expressed at 2-fold change and higher. In this set, we discovered that 24 miRNAs were significantly upregulated and 16 miRNAs were significantly downregulated in breast cancer patients, as compared to the miRNA expression of healthy subjects. ROC curve analysis revealed that seven miRNAs (miR-125b-5p, miR-142-3p, miR-145-5p, miR-193a-5p, miR-27b-3p, miR-22-5p and miR-423-5p) had area under curve (AUC) value > 0.7 (AUC p-value < 0.05). Overlapping findings from differential gene expression analysis, ROC analysis, and Independent T-Test resulted in three miRNAs (miR-27b-3p, miR-22-5p, miR-145-5p). Cohen’s effect size for these three miRNAs was large with d value are more than 0.95. Conclusion: miR-27b-3p, miR-22-5p, miR-145-5p could be potential biomarkers to distinguish breast cancer patients from healthy controls. A validation study for these three miRNAs in an external set of samples is ongoing.     

MicroRNA-143 targets DNA methyltransferases 3A in colorectal cancer

Background:

MicroRNAs (miRNAs) are 19-25-nucleotides regulatory non-protein-coding RNA molecules that regulate the expressions of a wide variety of genes, including some involved in cancer development. In this study, we investigated the possible role of miR-143 in colorectal cancer (CRC).

Methods:

Expression levels of human mature miRNAs were examined using real-time PCR-based expression arrays on paired colorectal carcinomas and adjacent non-cancerous colonic tissues. The downregulation of miR-143 was further evaluated in colon cancer cell lines and in paired CRC and adjacent non-cancerous colonic tissues by qRT–PCR. Potential targets of miR-143 were defined. The functional effect of miR-143 and its targets was investigated in human colon cancer cell lines to confirm miRNA–target association.

Results:

Both real-time PCR-based expression arrays and qRT–PCR showed that miR-143 was frequently downregulated in 87.5% (35 of 40) of colorectal carcinoma tissues compared with their adjacent non-cancerous colonic tissues. Using in silico predictions, DNA methyltranferase 3A (DNMT3A) was defined as a potential target of miR-143. Restoration of the miR-143 expression in colon cell lines decreased tumour cell growth and soft-agar colony formation, and downregulated the DNMT3A expression in both mRNA and protein levels. DNMT3A was shown to be a direct target of miR-143 by luciferase reporter assay. Furthermore, the miR-143 expression was observed to be inversely correlated with DNMT3A mRNA and protein expression in CRC tissues.

Conclusion:

Our findings suggest that miR-143 regulates DNMT3A in CRC. These findings elucidated a tumour-suppressive role of miR-143 in the epigenetic aberration of CRC, providing a potential development of miRNA-based targeted approaches for CRC therapy.     

MicroRNA-143 is a putative predictive factor for the response to fluoropyrimidine-based chemotherapy in patients with metastatic colorectal cancer

Approximately half of the colorectal cancer (CRC) patients develop metastatic disease. Fluoropyrimidine-based chemotherapy forms the backbone of treatment in these patients. However, the response to this therapy varies between individuals. Therefore, an important challenge in CRC research is to identify biomarkers that are predictive of this response. In this study, we explored the potential of miRNAs, and the miRNA producing protein Dicer, as biomarkers that can predict chemo-sensitivity to fluoropyrimidine chemotherapy in patients with metastatic colorectal cancer (mCRC). We analyzed the levels of 22 miRNAs and the Dicer protein in primary tumors from patients with mCRC who were treated with first-line capecitabine monotherapy within the CAIRO trial of the Dutch Colorectal Cancer Group. Correlation between the expression status of miRNAs or Dicer in primary tumors and the progression free survival (PFS) were investigated. Patients with low expression of miR-143 in their primary tumor had increased median PFS compared to those with high expression of miR-143. Furthermore, FXYD3, an ion transport regulator and a putative target of miR-143, also showed an association with PFS. These findings warrant further studies to investigate the relationship between miR-143, FXYD3 and fluoropyrimidines, and the clinical utility of miR-143 as biomarker.     

MicroRNA profiling and prediction of recurrence/relapse-free survival in stage I lung cancer

About 30% stage I non-small cell lung cancer (NSCLC) patients undergoing resection will recur. Robust prognostic markers are required to better manage therapy options. MicroRNAs (miRNAs) are a class of small non-coding RNAs of 19-25 nt and play important roles in gene regulation in human cancers. The purpose of this study is to identify miRNA expression profiles that would better predict prognosis of stage I NSCLC. MiRNAs extracted from 527 stage I NSCLC patients were profiled on the human miRNA expression profiling v2 panel (Illumina). The expression profiles were analyzed for their association with cancer subtypes, lung cancer brain metastasis and recurrence/relapse free survival (RFS). MiRNA expression patterns between lung adenocarcinoma and squamous cell carcinoma differed significantly with 171 miRNAs, including Let-7 family members and miR-205. Ten miRNAs associated with brain metastasis were identified including miR-145*, which inhibit cell invasion and metastasis. Two miRNA signatures that are highly predictive of RFS were identified. The first contained 34 miRNAs derived from 357 stage I NSCLC patients independent of cancer subtype, whereas the second containing 27 miRNAs was adenocarcinoma specific. Both signatures were validated using formalin-fixed paraffin embedded and/or fresh frozen tissues in independent data set with 170 stage I patients. Our findings have important prognostic or therapeutic implications for the management of stage I lung cancer patients. The identified miRNAs hold great potential as targets for histology-specific treatment or prevention and treatment of recurrent disease.     

Tumor microRNA-29a expression and the risk of recurrence in stage II colon cancer

There is emerging evidence for the prognostic role of various microRNA (miRNA) molecules in colon cancer. The aim of this study was therefore to compare the miRNA profiles in the primary tumor of patients with recurrent and non-recurrent colon cancer. The study population included 110 patients, 51 (46%) with stage I and 59 (54%) with stage II disease, who underwent curative colectomies between 1995 and 2005 without adjuvant therapy and for whom reliable miRNA expression data were available. RNA was extracted from formalin-fixed paraffin-embedded (FFPE) tumor samples. Initial profiling, using microarrays, was done in order to identify potential biomarkers of recurrence. The miRNA expression was later verified by quantitative real-time polymerase chain reaction (qRT-PCR). Findings were compared between patients who had a recurrence within 36 months of surgery (bad prognosis group, n=23, 21%) and those who did not (good prognosis group, n=87, 79%) in the entire group and within each stage. The results showed that in stage I, none of the 903 miRNAs tested showed differential expression between patients with good prognosis compared with those with poor prognosis. In contrast, in stage II, one miRNA, miR-29a, showed a clear differential expression between the groups (p=0.028). High expression of miR-29a was associated with a longer disease-free survival (DFS), on both univariate and multivariate analyses. Using miR-29a, the positive predictive value for non-recurrence was 94% (2 recurrences among 31 patients). The differential expression of miR-29a was verified by qRT-PCR, showing a similar impact of this miR on DFS. In conclusion, this study demonstrated a significant impact of miR-29a on the risk of recurrence in patients with stage II but not in patients with stage I colon cancer. Based on these results, a validation study is planned.     

microRNA-320a inhibits tumor invasion by targeting neuropilin 1 and is associated with liver metastasis in colorectal cancer

MicroRNAs (miRNAs) have been implicated in regulating diverse cellular pathways. Although there is emerging evidence that various miRNAs function as oncogenes or tumor suppressors in colorectal cancer (CRC), the role of miRNAs in mediating liver metastasis remains unexplored. The expression profile of miRNAs in liver metastasis and primary CRC tissues was analyzed by miRNA microarrays and verified by real-time polymerase chain reaction (PCR). In 62 CRC patients, the expression levels of miR-320a were determined by real-time PCR, and the effects on migration and invasion of miR-320a were determined using a transwell assay. miR-320a target genes were confirmed by luciferase assay, real-time PCR and western blot analysis. A set of miRNAs was found to be dysregulated in the liver metastasis tissues compared to matched primary CRC tissues, and the expression levels of miR-320a were significantly decreased in the liver metastasis tissues examined. miR-320a was correlated with tumor progression in CRC. miR-320a was downregulated in liver metastatic colon cancer cells and inhibited liver metastatic colon cancer cell migration and invasion. miR-320a directly binds to the 3'UTR of neuropilin 1 (NRP-1), a protein that functions as a co-receptor of vascular epithelial growth factor. miR-320a downregulated the expression of NRP-1 at both the mRNA and protein levels. These data demonstrated that miR-320a may be useful for identifying CRC patients that are at an elevated risk for developing liver metastasis. Our findings suggest that miR-320a may be a novel therapeutic candidate for the treatment of colorectal cancer.