MicroRNA‐30b functions as a tumour suppressor in human colorectal cancer by targeting KRAS,PIK3CD and BCL2

Colorectal cancer (CRC) is the third most common cancer in the USA. MicroRNAs play important roles in the pathogenesis of CRC. In this study, we investigated the role of miR‐30b in CRC and found that its expression was significantly lower in CRC tissues than that in normal tissues. We showed that a low expression level of miR‐30b was closely related to poor differentiation, advanced TNM stage and poor prognosis of CRC. Further experiments showed that over‐expression of miR‐30b suppressed CRC cell proliferation in vitro and tumour growth in vivo. Specifically, miR‐30b promoted G₁ arrest and induced apoptosis. Moreover, KRAS, PIK3CD and BCL2 were identified as direct and functional targets of miR‐30b. MiR‐30b directly targeted the 3′‐untranslated regions of their mRNAs and repressed their expression. This study revealed functional and mechanistic links between miRNA‐30b and oncogene KRAS, PIK3CD and BCL2 in the pathogenesis of CRC. MiR‐30b not only plays important roles in the regulation of cell proliferation and tumour growth in CRC, but is also a potential prognostic marker or therapeutic target for CRC. Restoration of miR‐30b expression may represent a promising therapeutic approach for targeting malignant CRC. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Loss of miR‐101 expression promotes Wnt/β‐catenin signalling pathway activation and malignancy in colon cancer cells

Colorectal cancer (CRC) is the second leading cause of cancer‐related mortality in Western countries. Although the aberrant expression of several microRNAs (oncomiRs) is associated with CRC progression, the molecular mechanisms of this phenomenon are still under investigation. Here we show that miR‐101 expression is differentially impaired in CRC specimens, depending on tumour grade. miR‐101 re‐expression suppresses cell growth in 3D, hypoxic survival and invasive potential in CRC cells showing low levels of miR‐101. Additionally, we provide molecular evidence of a bidirectional regulatory mechanism between miR‐101 expression and important CRC pro‐malignant features, such as inflammation, activation of the Wnt/β‐catenin signalling pathway and epithelial–mesenchymal transition (EMT). We then propose that up‐regulated miR‐101 may function as a tumour suppressor in CRC and that its pharmacological restoration might hamper the aggressive behaviour of CRC in vivo. MiR‐101 expression may also represent a cancer biomarker for CRC diagnosis and prognosis. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Expression of chemokine receptor CXCR7 in colorectal carcinoma and its prognostic significance

Previous studies have shown that chemokine receptor CXCR7 plays critical roles in tumor development. However, the clinicopathological and prognositic significance of CXCR7 in colorectal carcinoma (CRC) has not been fully understood. The aim of our study is to investigate the expression of CXCR7 and its clinical significance in CRC. First, quantitative RT-PCR and Western blot assays were performed to determine the expression of CXCR7 mRNA and protein in 20 paired of CRC tissues and corresponding adjacent non-tumor tissues. Next, immunohistochemistry was performed to detect the expression of CXCR7 protein in another 96 cases of CRC tissues, and analyze its correlation with clinicopathological factors of patients. Finally, the correlation of CXCR7 with 5-year overall survival (OS) and progression free survival (PFS) was statistically analyzed by the Kaplan-Meier method and Cox proportional hazards model. Results showed that the expression levels of CXCR7 mRNA and protein were significantly higher in CRC tissues than in normal tissues. Positive CXCR7 expression was observed to be significantly correlated with lymph nodal metastasis (P < 0.001), distant metastasis (P = 0.017), and advanced TNM stage (P < 0.001). Patients with positive expression of CXCR7 were demonstrated to be associated with worse OS and PFS (P < 0.001, P < 0.001, respectively). Moreover, multivariate survival analysis revealed that CXCR7 expression level might be an independent predictive factor for OS and PFS of CRC patients. Collectively, positive CXCR7 expression in CRC was correlated with tumor development and poor prognosis of patients.     

Overexpression of MAGE-D4 in colorectal cancer is a potentially prognostic biomarker and immunotherapy target

Melanoma-associated antigen D4 (MAGE-D4) is a novel member of MAGE family. This study aimed to examine the expression and immunogenicity of MAGE-D4 in colorectal cancer (CRC) to determine its potential as a prognosis and immunotherapeutic target. The expression of MAGE-D4 mRNA and protein was determined by RT-PCR and immunohistochemistry (IHC) in CRCs with paired adjacent non-tumor tissues, colorectal adenomas and normal colorectal tissues, respectively. Sera from 64 CRC patients were tested for MAGE-D4 antibody by ELISA. MAGE-D4 mRNA was more frequently expressed in CRCs (76.7%, 46/60) than in adjacent non-tumor tissues (15.0%, 9/60). MAGE-D4 protein was detected in all the CRC tissues tested, 70.0% of which showed high expression. There was no MAGE-D4 protein detected in any paired adjacent non-tumor tissue. No MAGE-D4 expression was found in colorectal adenomas and normal colorectal tissues by either RT-PCR or immunohistochemistry. Patients with high MAGE-D4 protein expression had significantly shorter overall survival than those with low MAGE-D4 protein expression (median, 68.6 vs 122.2 months; P=0.030). Furthermore, multivariate analysis exhibited high MAGE-D4 protein expression had a trend toward an independent prognostic factor (hazard ratio: 6.124; P=0.050). Humoral immunity to MAGE-D4 was detected in 12 of 64 (18.8%) CRC patients’ sera but not in 77 healthy donors. There was no correlation between MAGE-D4 expression, serum antibody and clinicopathological parameters. These findings suggest MAGE-D4 may serve as a potentially prognostic biomarker and an attractive target of immunotherapy in CRC.     

Expression of miR‐100 and miR‐138 as prognostic biomarkers in non‐muscle‐invasive bladder cancer

microRNA alterations are involved in bladder cancer tumorigenesis. The aim of the current study was to evaluate the potential role of miR‐100 and miR‐138 as prognostic biomarkers in Ta/T1 non‐muscle‐invasive bladder cancer (NMIBC). We assessed a quantitative RT‐PCR analysis of miR‐100 and miR‐138 in 50 bladder tumor samples (stage Ta/T1) and four healthy adjacent tissues. Western blot analysis was used to measure protein expression of FGFR3 and cyclin D3 in order to know whether these targets can be regulated by miR‐100 and miR‐138, respectively. The statistical analysis included non‐parametric tests (Mann–Whitney U and Kruskal–Wallis) and univariate survival analysis by Kaplan–Meier method and the log‐rank test. Low expression of miR‐138 characterized recurrent tumors (p = 0.043), and higher expression levels were associated with longer recurrence‐free survival (p = 0.012). However, low miR‐100 expression correlated with longer progression‐free survival (marginal significance; p = 0.053) and cancer‐specific overall survival (p = 0.006). Additionally, higher levels of miR‐100 were associated with negative FGFR3 protein expression (p = 0.032) and higher levels of miR‐138 were associated with positive cyclin D3 protein expression (p = 0.037). Our results support miR‐138 and miR‐100 as prognostic biomarkers in patients with NMIBC.     

Prognostic relevance of miR‐124‐3p and its target TP53INP1 in pediatric ependymoma

Ependymoma is a malignant pediatric brain tumor, often incurable under the current treatment regimen. We aimed to evaluate the expression of microRNAs (miRs) in pediatric ependymoma tumors in an attempt to identify prognostic molecular markers which would lead to potential therapeutic targets. Following miR‐array expression analysis, we focused on 9 miRs that correlated with relapse which were further validated by quantitative real‐time PCR (qRT‐PCR) in a cohort of 67 patients. Western blotting and immunohistochemistry were used to measure target protein expression in 20 and 34 tumor samples, respectively. High expression of miR‐124‐3p significantly correlated with the lower progression‐free survival (PFS) of 16% compared to 67% in those expressing low levels (P = .002). Interestingly, in the group of patients with local disease (n = 56) expression levels of this miR distinguished 2 subgroups with a significantly different outcome (P = .001). miR‐124‐3p was identified as an independent prognostic factor of relapse in the multivariate analysis performed in the whole cohort and in the group with localized disease. In the localized group, a patient expressing high levels of miR‐124‐3p had a 4.1‐fold increased risk for relapse (P = .005). We demonstrated the direct binding of miR‐124‐3p to its target TP53INP1. Negative TP53INP1 protein levels correlated with a poor outcome (P = .034). We propose miR‐124‐3p and TP53INP1 as new biomarkers for prognostic stratification that may be possible therapeutic targets for ependymoma.     

SP1 mediates the link between methylation of the tumour suppressor miR‐149 and outcome in colorectal cancer

Although recent studies indicate that DNA methylation contributes to the down‐regulation of microRNAs (miRNAs) in colorectal cancer (CRC), this field remains largely unexplored. To identify methylation‐silenced miRNAs and clarify their role in CRC, we performed a microarray analysis and screened for miRNAs that were induced in CRC cells by 5‐aza‐2′‐deoxycytidine treatment or by the knockdown of DNA methyltransferases. The DNA methylation status of the candidate miRNA was analysed by bisulphite sequencing PCR and methylation‐specific PCR. We found that miRNA‐149 (miR‐149) was epigenetically silenced in CRC and down‐regulation of miR‐149 was associated with hypermethylation of the neighbouring CpG island (CGI). Quantitative RT‐PCR analysis demonstrated that the miR‐149 level was markedly reduced in 51.6% of the CRC tissues compared with matched non‐cancerous tissues. In addition, low expression of miR‐149 was associated with a greater depth of invasion ($\it{p} = 0.012$ ), lower 5‐year survival rate ($\it{p} = 0.025$ ), and was found to be an independent prognostic factor for overall survival ($\it{p} = 0.016$ ) in a multivariate analysis. Moreover, transfection of miR‐149 inhibited cell growth and invasion of CRC cells in vitro. We also identified mRNA for Specificity Protein 1 (SP1, Sp1), a potential oncogenic protein, as a target of miR‐149. Our data suggest that, as a methylation‐sensitive miRNA, miR‐149 may play an important role as a tumour suppressor in CRC, which has prognostic and therapeutic implications. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

结直肠癌中Keratin 17的表达及其临床预后价值

目的 分析角蛋白17(Keratin 17,KRT17)在结直肠癌(colorectal cancer,CRC)中的表达及其临床预后价值.方法 采用生物信息学方法分析KRT17在CRC数据库中的表达及预后价值;进一步,采用免疫组织化学技术检测KRT17蛋白在95对CRC临床样本组织中的表达水平,并分析其表达与患者临床病...     

Human papillomavirus‐stratified analysis of the prognostic role of miR‐21 in oral cavity and oropharyngeal squamous cell carcinoma

Human papillomavirus (HPV) infection plays a significant role in the development and progression of head and neck squamous cell carcinoma (HNSCC). Expression of miR‐21 has a prognostic role in a wide variety of cancers. The upregulation of miR‐21 suppresses a number of target genes, including phosphatase tensin homologue (PTEN) and programmed cell death 4 (PDCD4). We investigated the association between the expression of miR‐21 and the clinical features of HNSCC using stratified analysis based on HPV infection status. HPV status and miR‐21 expression in HNSCC tissues from 167 patients were evaluated using in situ hybridization. The expression of PDCD4 and PTEN was examined by immunohistochemistry. The up‐regulation of stromal miR‐21 expression occurred in 40.6% of HPV‐negative samples and 28.3% of the HPV‐positive group. In HPV‐stratified multivariate analysis, high miR‐21 expression was associated with poor cancer‐specific survival in HPV‐negative tumors, but not in HPV‐positive tumors. There was a significant association between miR‐21 and cytoplasmic PDCD4 overexpression in HPV‐negative HNSCCs. We suggest that stromal miR‐21 expression is an independent prognostic factor in HPV‐negative tumors and miR‐21 may play different roles depending on HPV infection status.     

High expression of meningioma 1 is correlated with reduced survival rates in colorectal cancer patients

The identification of prognostic markers for colorectal cancer (CRC) has important clinical implications. However, the association between meningioma 1 (MN1) expression and clinical outcomes of CRC has not been fully investigated. The aim of this study was to investigate the expression of MN1 in the clinical context of CRC. We first used immunohistochemistry (IHC) staining to examine and compare MN1 expression between multiple human cancer tissues and normal tissues. Initial screening revealed that the expression of MN1 proteins was significantly higher in tumor tissues of the breast, colon, and liver than in normal tissues. In further testing conducted on 59 paired CRC samples, we observed that the expression of MN1 in CRC tissue samples was significantly higher than in adjacent normal tissues. Moreover, high MN1 expression was not significantly associated with clinicopathological characteristics. Kaplan-Meier survival analysis revealed that high expression of MN1 mRNA or MN1 protein was significantly associated with poor CRC prognosis. Furthermore, univariate Cox analysis revealed that a high MN1 score was significantly associated with prognostic factors. Multivariate Cox analysis further indicated that gender, histologic grade, tumor-node-metastasis (TNM) stage, and a high MN1 score were independent factors of overall CRC survival rates. Finally, MN1 and PCNA protein levels were positively correlated, which suggests that MN1 may be involved in the cell proliferation process during CRC formation. Our results, which confirm those of other studies, indicate that (1) high levels of MN1 expression contribute to poor CRC prognosis and (2) MN1 can serve as a novel potential biomarker in predicting the prognosis of CRC patients.     

Catenin‐δ1, negatively regulated by miR‐145, promotes tumour aggressiveness in gastric cancer

Increasing evidence supports the association of catenin‐δ1 (CTNND1, p120ctn) with tumour development and progression. However, the mechanism and clinical significance of CTNND1 deregulation in gastric cancer remain unknown. The expression level and cellular localization of CTNND1 were determined by immunohistochemistry in 126 human gastric cancer and 50 non‐tumourous tissues. The cellular localization of CTNND1 and epithelial cadherin (E‐cadherin) were detected by immunofluorescence. Cell proliferation, apoptosis, migration and invasion assays were performed to assess the effect of CTNND1 cDNA or CTNND1 siRNA transfection on gastric cancer cells. Luciferase assay, western blot analysis and in vivo assays were used to determine whether CTNND1 could be regulated by miR‐145. The results demonstrate that the cytoplasmic localization of CTNND1 protein, rather than expression level, was indicative of higher clinical stage, positive lymph node metastasis and poorer prognosis in gastric cancers. CTNND1 could promote gastric cancer cell migration and invasion with little effect on cellular proliferation and apoptosis. CTNND1 was proved to be a direct target gene for miR‐145. Besides suppressing cytoplasmic CTNND1 expression, miR‐145 could recover the membranous localization of CTNND1 and E‐cadherin. We conclude that cytoplasmic CTNND1 can serve as an independent prognostic factor for patients with gastric cancers. MiR‐145 inhibits invasion of gastric cancer cells not only by down‐regulating cytoplasmic CTNND1 expression but also by inducing the translocation of CTNND1 and E‐cadherin from the cytoplasm to the cell membrane through down‐regulating N‐cadherin. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

MicroRNA9 regulates neural stem cell differentiation by controlling Hes1 expression dynamics in the developing brain

Earlier studies show that Hes1 expression is oscillatory in neural stem cells but sustained and high in the roof plate and the floor plate, and that such different dynamics of Hes1 expression (oscillatory versus sustained) regulate different proliferation and differentiation characteristics of these cells (active in neural stem cells but rather dormant in roof/floor plate cells). The mechanism of how different dynamics of Hes1 expression is controlled remains to be determined. Here, we found that the seed sequence of microRNA‐9 (miR‐9) is complementary to the 3′‐UTR sequence of Hes1 mRNA. MiR‐9 is highly expressed in the ventricular zone of the developing brain, which contains neural stem cells, but it is not expressed in the roof plate or the floor plate. Over‐expression of miR‐9 negatively regulates the Hes1 protein expression by interacting with the 3′‐UTR of Hes1 mRNA, thereby inducing cell cycle exit and neuronal differentiation. Conversely, knockdown of miR‐9 inhibits neuronal differentiation. Furthermore, knockdown of miR‐9 inhibits the oscillatory expression of Hes1 mRNA in neural stem cells. These results indicate that miR‐9 regulates the proliferation and differentiation of neural stem cells by controlling the dynamics of Hes1 expression in the developing brain.     

Differential CXC receptor expression in colorectal carcinomas

In this study, we aimed to assess the expression profile of chemokine receptors CXCR1–4 in inflammatory and malignant colorectal diseases and corresponding hepatic metastases of synchronous and metachronous origin to elucidate their role in colorectal cancer (CRC) progression and metastasis. Chemokine receptor expression was assessed by quantitative real-time PCR, immunohistochemistry (IHC) and Western blot analysis in resection specimens from patients with ulcerative colitis (UC, n  = 25), colorectal adenomas (CRA, n  = 8), different stages of CRC ( n  = 48) as well as colorectal liver metastases (CRLM) along with their corresponding primary colorectal tumours ( n  = 16). While none of the chemokine receptors were significantly upregulated or downregulated in UC or CRA tissues, CXC receptors 1, 2 and 4 demonstrated a significant increase in expression in all tumour stages of CRC specimens with CXCR4 correlating with tumour grading ( P  < 0.05). On the other hand, CXCR3 showed no significant upregulation in either tumour stage, but significant overexpression in CRLM. While CXCR4 demonstrated significant upregulation in both tumour entities, IHC analysis revealed that the predominate cell type expressing CXCR4 in CRC is represented by tumour cells, whereas in CRLM the majority of positive CXCR4 signals is due to hepatocytes along the tumour invasion front. In conclusion, our findings show a very differential expression pattern of the four receptors in colorectal carcinomas and their corresponding liver metastases with prominent expression profiles that indicate a potential role in the pathogenesis of CRC.