miRNA-27b Targets Vascular Endothelial Growth Factor C to Inhibit Tumor Progression and Angiogenesis in Colorectal Cancer

Colorectal cancer (CRC) is one of the most prevalent cancers globally and is one of the leading causes of cancer-related deaths due to therapy resistance and metastasis. Understanding the mechanism underlying colorectal carcinogenesis is essential for the diagnosis and treatment of CRC. microRNAs (miRNAs) can act as either oncogenes or tumor suppressors in many cancers. A tumor suppressor role for miR-27b has recently been reported in neuroblastoma, while no information about miR-27b in CRC is available. In this study, we demonstrated that miR-27b expression is decreased in most CRC tissues and determined that overexpression of miR-27b represses CRC cell proliferation, colony formation and tumor growth in vitro and in vivo. We identified vascular endothelial growth factor C (VEGFC) as a novel target gene of miR-27b and determined that miR-27b functioned as an inhibitor of tumor progression and angiogenesis through targeting VEGFC in CRC. We further determined that DNA hypermethylation of miR-27b CpG islands decreases miR-27b expression. In summary, an anti-tumor role for miR-27b and its novel target VEGFC in vivo could lead to tumor necrosis and provide a rationale for developing miR-27b as a therapeutic agent.     

Overexpression of microRNA-378 attenuates ischemia-induced apoptosis by inhibiting caspase-3 expression in cardiac myocytes

MicroRNAs (miRNAs) are a novel class of powerful, endogenous regulators of gene expression. In an intact rat model of myocardial ischemia caused by coronary artery ligation, this study identified 17 miRNAs that changed more than 1.5-fold in the myocardium subjected to 4-h ischemia. Using miRNA microarray analysis, most of these aberrantly expressed miRNAs were confirmed by quantitative RT-PCR. MiR-378, a significantly down-regulated miRNA, was selected for further function study. In serum deprived rat H9c2 cardiomyocytes exposed to hypoxia (1% O₂), miR-378 expression was down-regulated as well. The overexpression of miR-378 resulting from miR-378 mimic transfection significantly enhanced cell viability, reduced lactate dehydrogenase release, and inhibited apoptosis and necrosis. By contrast, miR-378 deficiency resulting from miR-378 inhibitor transfection aggravated the hypoxia-induced apoptosis and cell injury. In accordance, miR-378 inhibitor caused significant apoptosis and cell injury to cardiomyocytes cultured under normoxia. Using bioinformatic algorithms, caspase-3, a key apoptosis executioner, was predicted as a putative target of miR-378. The quantitative RT-PCR showed no effects of miR-378 mimic or inhibitor on caspase-3 mRNA level. However, the amount of caspase-3 proteins was reduced by miR-378 mimic, whereas increased by miR-378 inhibitor. Furthermore, the luciferase reporter assay confirmed caspase-3 to be a target of miR-378, and the apoptosis and cell injury caused by miR-378 inhibitor in both normoxic and hypoxic cells were abolished by a caspase-3 inhibitor. This study first showed that miR-378 inhibited caspase-3 expression and attenuated ischemic injury in cardiomyocytes. It may represent a potential novel treatment for apoptosis and ischemic heart disease.     

MiR-124 Suppresses Growth of Human Colorectal Cancer by Inhibiting STAT3

Emerging evidence indicate that microRNAs (miRNAs) may play important roles in cancer. Aberrant expression of miRNAs has been frequently identified in different human malignancies, including colorectal cancer (CRC). However, the mechanism by which deregulated miRNAs impact the development of CRC remains largely elusive. In this study, we show that miR-124 is significantly down-regulated in CRC compared to adjacent non-tumor colorectal tissues. MiR-124 suppresses the expression of STAT3 by directly binding to its 3′-untranslated region (3′-UTR). Overexpression of miR-124 led to increased apoptosis of CRC cells and reduced tumor growth in vitro and in vivo. Knocking down STAT3 expression by specific siRNA suppressed the growth of CRC cells in vitro and in vivo, resembling that of miR-124 overexpression. Moreover, overexpression of STAT3 in miR-124-transfected CRC cells effectively rescued the inhibition of cell proliferation caused by miR-124. These data suggest that miR-124 serves as a tumor suppressor by targeting STAT3, and call for the use of miR-124 as a potential therapeutic tool for CRC, where STAT3 is often hyper-activated.     

Pleiotropic actions of miR-21 highlight the critical role of deregulated stromal microRNAs during colorectal cancer progression

The oncogene microRNA-21 (miRNA; miR-21) is overexpressed in most solid organ tumours; however, a recent examination of stage II colorectal cancer (CRC) specimens suggests this may be a stromal phenomenon and not only a feature of cancer cells. In vitro and in vivo studies show that miR-21 has potent pro-metastatic effects in various malignant carcinoma cell lines. The tumour microenvironment has also been identified as a key actor during the metastatic cascade; however to date the significance of deregulated miR-21 expression within the cancer-associated stroma has not been examined. In the present study, a quantitative RT-PCR-based analysis of laser microdissected tissue confirmed that miR-21 expression is associated with a four-fold mean increase in CRC stroma compared with normal tissue. In situ hybridisation using locked nucleic acid probes localised miR-21 expression predominantly to fibroblasts within tumour-associated stroma. To study the molecular and biological impact of deregulated stromal miR-21 in CRC, stable ectopic expression was induced in immortalised fibroblasts. This resulted in upregulated α-smooth muscle actin expression implying miR-21 overexpression is driving the fibroblast-to-myofibroblast transdifferentiation. Conditioned medium from miR-21-overexpressing fibroblasts protected CRC cells from oxaliplatin-induced apoptosis and increased their proliferative capacity. 3D organotypic co-cultures containing fibroblasts and CRC cells revealed that ectopic stromal miR-21 expression was associated with increased epithelial invasiveness. Reversion-inducing cysteine-rich protein with kazal motifs, an inhibitor of matrix-remodelling enzyme MMP2, was significantly downregulated by ectopic miR-21 in established and primary colorectal fibroblasts with a reciprocal rise in MMP2 activity. Inhibition of MMP2 abrogated the invasion-promoting effects of ectopic miR-21. This data, which characterises a novel pro-metastatic mechanism mediated by miR-21 in the CRC stroma, highlights the importance of miRNA deregulation within the tumour microenvironment and identifies a potential application for stromal miRNAs as biomarkers in cancer.     

Serum microRNA-29a is a promising novel marker for early detection of colorectal liver metastasis

Background: Colorectal cancer (CRC) metastasis occurs in various organs, most frequently in liver. Serological examination including tumor and biochemical markers for liver function evaluation is routinely performed, though its accuracy is not high. MicroRNAs (miRNAs) have been implicated in a variety of human diseases including cancer, and have many characteristics of an ideal biomarker most notably their inherent stability and resilience. Recently, several studies have indicated that circulating miRNAs hold much potential as novel noninvasive biomarkers for cancer and other disease processes. The objective of this study was to investigate the potential of serum miRNAs as novel biomarkers for CRC with liver metastasis. Methods: This study was divided into three phases: (I) 3 candidate serum miRNAs were detected by using real-time RT-PCR, corresponding 38 CRC patients with liver metastasis and 36 CRC patients without metastasis. (II) Marker validation by real-time RT-PCR on a similar cohort of age- and sex-matched CRC patients without (n = 20) and with liver metastasis (n = 20). (III) We examined the correlation between the expressions of candidate serum miRNAs with clinical parameters of CRC patients. Results: Serum miR-29a was significantly higher in colorectal liver metastasis (CRLM) patients than in CRC patients. This marker yielded a receiver operating characteristic curve area of 80.3%. At a cutoff value of 0.155, the sensitivity was 75% and the specificity was 75% in discriminating metastatic from non-metastatic patients. In addition, increased levels of miR-29a expression were also observed in colorectal tumors from CRLM patients compared with CRC patients. No significant difference was observed in the levels of serum miR-92a between metastatic and non-metastatic patients. Conclusions: These findings suggest that serum miR-29a has strong potential as a novel noninvasive biomarker for early detection of CRC with liver metastasis.     

miR-181b functions as an oncomiR in colorectal cancer by targeting PDCD4

Programmed cell death 4 (PDCD4) is a RNA-binding protein that acts as a tumor suppressor in many cancer types, including colorectal cancer (CRC). During CRC carcinogenesis, PDCD4 protein levels remarkably decrease, but the underlying molecular mechanism for decreased PDCD4 expression is not fully understood. In this study, we performed bioinformatics analysis to identify miRNAs that potentially target PDCD4. We demonstrated miR-181b as a direct regulator of PDCD4. We further showed that activation of IL6/STAT3 signaling pathway increased miR-181b expression and consequently resulted in downregulation of PDCD4 in CRC cells. In addition, we investigated the biological effects of PDCD4 inhibition by miR-181b both in vitro and in vivo and found that miR-181b could promote cell proliferation and migration and suppress apoptosis in CRC cells and accelerate tumor growth in xenograft mice, potentially through targeting PDCD4. Taken together, this study highlights an oncomiR role for miR-181b in regulating PDCD4 in CRC and suggests that miR-181b may be a novel molecular therapeutic target for CRC.     

Circulating Exosomal miR-17-5p and miR-92a-3p Predict Pathologic Stage and Grade of Colorectal Cancer

Exosomes are extracellular membrane vesicles of 50- to 130-nm diameter secreted by most tumor cells. Exosomes can mediate the intercellular transfer of proteins and RNAs, including microRNAs (miRNAs), and promote both tumorigenesis and premetastatic niche formation. In this study, we performed exosomal RNA sequencing to identify candidate exosomal miRNAs that could be associated with colorectal cancer (CRC) and its distant metastasis. The expression profiles of exosomal miRNA, as secreted by isogenic human primary CRC cell line SW480 and highly metastatic cell line SW620, were analyzed and the potential targets related to tumorigenesis and metastatic progression were investigated. We found that 25 miRNAs had been up-regulated and 5 miRNAs had been down-regulated in exosomes purified from SW620 culture supernatant. Candidate miRNAs were further evaluated for CRC diagnosis using quantitative real-time polymerase chain reaction in CRC patients. Higher expression levels of circulating exosomal miR-17-5p and miR-92a-3p were significantly associated with pathologic stages and grades of the CRC patients. CONCLUSIONS: Circulating exosomal miR-17-5p and miR-92a-3p may provide a promising noninvasive prognostic biomarker for primary and metastatic CRC.     

Elevated MicroRNA-31 Expression Regulates Colorectal Cancer Progression by Repressing Its Target Gene SATB2

Several studies have brought about increasing evidence to support the hypothesis that miRNAs play a pivotal role in multiple processes of carcinogenesis, including cell growth, apoptosis, differentiation, and metastasis. In this study, we investigated the potential role of miR-31 in colorectal cancer (CRC) aggressiveness and its underlying mechanisms. We found that miR-31 increased in CRC cells originated from metastatic foci and human primary CRC tissues with lymph node metastases. Furthermore, the high-level expression of miR-31 was significantly associated with a more aggressive and poor prognostic phenotype of patients with CRC (p < 0.05). The stable over-expression of miR-31 in CRC cells was sufficient to promote cell proliferation, invasion, and migration in vitro. It facilitated tumor growth and metastasis in vivo too. Further studies showed that miR-31 can directly bind to the 3’untranslated region (3’UTR) of SATB2 mRNA and subsequently repress both the mRNA and protein expressions of SATB2. Ectopic expression of SATB2 by transiently transfected with pCAG-SATB2 vector encoding the entire SATB2 coding sequence could reverse the effects of miR-31 on CRC tumorigenesis and progression. In addition, ectopic over-expression of miR-31 in CRC cells induced epithelial-mesenchymal transition (EMT). Our results illustrated that the up-regulation of miR-31 played an important role in CRC cell proliferation, invasion, and metastasis in vitro and in vivo through direct repressing SATB2, suggesting a potential application of miR-31 in prognosis prediction and therapeutic application in CRC.     

MiR-1188 at the imprinted Dlk1-Dio3 domain acts as a tumor suppressor in hepatoma cells

The aberrant expression of microRNAs (miRNAs) has frequently been reported in cancer studies; miRNAs play roles in development, progression, metastasis, and prognosis. Recent studies indicate that the miRNAs within the Dlk1-Dio3 genomic region are involved in the development of liver cancer, but the role of miR-1188 in hepatocellular carcinoma (HCC) and the pathway by which it exerts its function remain largely unknown. Here we demonstrate that miR-1188 is significantly down-regulated in mouse hepatoma cells compared with normal liver tissues. Enhanced miR-1188 suppresses cell proliferation, migration, and invasion in vitro and inhibits the tumor growth of HCC cells in vivo. Moreover, overexpressed miR-1188 promotes apoptosis, enhances caspase-3 activity, and also up-regulates the expression of Bax and p53. MiR-1188 directly targets and negatively regulates Bcl-2 and Sp1. Silencing of Bcl-2 and Sp1 exactly copies the proapoptotic and anti-invasive effects of miR-1188, respectively. The expression of apoptosis- and invasion-related genes, such as Vegfa, Fgfr1, and Rprd1b, decreases after enhancement of miR-1188, as determined by gene expression profiling analysis. Taken together, our results highlight an important role for miR-1188 as a tumor suppressor in hepatoma cells and imply its potential role in cancer therapy.     

Circulating plasma microRNAs in colorectal neoplasia: A pilot study in assessing response to therapy

IntroductionCurrent serological surveillance markers to monitor colorectal cancer (CRC) or colorectal advanced adenomas (CAA) are hampered by poor sensitivity and specificity. The aim of this study is to identify and validate a panel of plasma microRNAs which change in expression after resection of such lesions.MethodsA prospectively maintained colorectal surgery database was queried for patients in whom both pre- and post-procedural serum samples had been obtained. An initial screening analysis of CRC and CAA patients (5 each) was conducted using screening cards for 380 miRNAs. Four identified miRNAs were combined with a previously described panel of 7 miRNAs that were diagnostically predictive of CRC and CAA. Differential miRNA expression was assessed using quantitative real-time polymerase chain reaction(qRT-PCR).ResultsFifty patients were included (n = 27 CRC, n = 23 CAA). There was no difference in age, gender, or race profile of CRC patients compared to CAA patients. Six miRNA were significantly increased after CRC resection (miR-324, let7b, miR-454, miR-374a, miR-122, miR-19b, all p<0.05), while three miRNAs were significantly increased following CAA resection (miR-454, miR-374a, miR-122, all p<0.05). Three miRNA were increased in common for both (miR-454, miR-374a, miR-122).DiscussionThe expression of miRNAs associated with neoplasia (either CRC or CAA) was significantly increased following surgical resection or endoscopic removal of CRC or CAA. Future studies should focus on the evaluation of these miRNAs in CRC and CAA prognosis.     

Targeting the lipid metabolic axis ACSL/SCD in colorectal cancer progression by therapeutic miRNAs: miR-19b-1 role

An abnormal acyl-CoA synthetase/stearoyl-CoA desaturase (ACSL/SCD) lipid network fuels colon cancer progression, endowing cells with invasive and migratory properties. Therapies against this metabolic network may be useful to improve clinical outcomes. Because micro-RNAs (miRNAs/miRs) are important epigenetic regulators, we investigated novel miRNAs targeting this pro-tumorigenic axis; hence to be used as therapeutic or prognostic miRNAs. Thirty-one putative common miRNAs were predicted to simultaneously target the three enzymes comprising the ACSL/SCD network. Target validation by quantitative RT-PCR, Western blotting, and luciferase assays showed miR-544a, miR-142, and miR-19b-1 as major regulators of the metabolic axis, ACSL/SCD. Importantly, lower miR-19b-1 expression was associated with a decreased survival rate in colorectal cancer (CRC) patients, accordingly with ACSL/SCD involvement in patient relapse. Finally, miR-19b-1 regulated the pro-tumorigenic axis, ACSL/SCD, being able to inhibit invasion in colon cancer cells. Because its expression correlated with an increased survival rate in CRC patients, we propose miR-19b-1 as a potential noninvasive biomarker of disease-free survival and a promising therapeutic miRNA in CRC.     

MicroRNA-218 Inhibits Cell Cycle Progression and Promotes Apoptosis in Colon Cancer by Downregulating BMI1 Polycomb Ring Finger Oncogene

Deregulated miRNAs participate in colorectal carcinogenesis. In this study, miR-218 was found to be downregulated in human colorectal cancer (CRC) by miRNA profile assay. miR-218 was silenced or downregulated in all five colon cancer cells (Caco2, HT29, SW620, HCT116 and LoVo) relative to normal colon tissues. miR-218 expression was significantly lower in 46 CRC tumor tissues compared with their adjacent normal tissues (P < 0.001). Potential target genes of miR-218 were predicted and BMI1 polycomb ring finger oncogene (BMI-1), a polycomb ring finger oncogene, was identified as one of the potential targets. Upregulation of BMI-1 was detected in CRC tumors compared with adjacent normal tissues (P < 0.001) and in all five colon cancer cell lines. Transfection of miR-218 in colon cancer cell lines (HCT116, HT29) significantly reduced luciferase activity of the wild-type construct of BMI-1 3′ untranslated region (3′UTR) (P < 0.001), whereas this effect was not seen in the construct with mutant BMI-1 3′UTR, indicating a direct and specific interaction of miR-218 with BMI-1. Ectopic expression of miR-218 in HCT116 and HT29 cells suppressed BMI-1 mRNA and protein expression. In addition, miR-218 suppressed protein expression of BMI-1 downstream targets of cyclin-dependent kinase 4, a cell cycle regulator, while upregulating protein expression of p53. We further revealed that miR-218 induced apoptosis (P < 0.01), inhibited cell proliferation (P < 0.05) and promoted cell cycle arrest in the G2 phase (P < 0.01). In conclusion, miR-218 plays a pivotal role in CRC development through inhibiting cell proliferation and cycle progression and promoting apoptosis by downregulating BMI-1.     

MicroRNA-18a Attenuates DNA Damage Repair through Suppressing the Expression of Ataxia Telangiectasia Mutated in Colorectal Cancer

Background

miR-18a is one of the most up-regulated miRNAs in colorectal cancers (CRC) based on miRNA profiling. In this study, we examined the functional significance of miR-18a in CRC.

Methods

Expression of miR-18a was investigated in 45 CRC patients. Potential target genes of miR-18a were predicted by in silico search and confirmed by luciferase activity assay and Western blot. DNA damage was measured by comet assay. Gene function was measured by cell viability, colony formation and apoptosis assays.

Results

The up-regulation of miR-18a was validated and confirmed in 45 primary CRC tumors compared with adjacent normal tissues (p<0.0001). Through in silico search, the 3′UTR of Ataxia telangiectasia mutated (ATM) contains a conserved miR-18a binding site. Expression of ATM was down-regulated in CRC tumors (p<0.0001) and inversely correlated with miR-18a expression (r = -0.4562, p<0.01). Over-expression of miR-18a in colon cancer cells significantly reduced the luciferase activity of the construct with wild-type ATM 3′UTR but not that with mutant ATM 3′UTR, inferring a direct interaction of miR-18a with ATM 3′UTR. This was further confirmed by the down-regulation of ATM protein by miR-18a. As ATM is a key enzyme in DNA damage repair, we evaluated the effect of miR-18a on DNA double-strand breaks. Ectopic expression of miR-18a significantly inhibited the repair of DNA damage induced by etoposide (p<0.001), leading to accumulation of DNA damage, increase in cell apoptosis and poor clonogenic survival.

Conclusion

miR-18a attenuates cellular repair of DNA double-strand breaks by directly suppressing ATM, a key enzyme in DNA damage repair.