miR-200c Levels Research Articles

Prognostic significance of microRNA-200c in various types of cancer: An updated meta-analysis of 34 studies

Previous studies have indicated that miR-200c is a promising cancer biomarker. However, different studies have presented conflicting results. Therefore, the aim of the present study was to perform a meta-analysis of miR-200c based on 34 relevant studies. The Materials and methods sections of papers were carefully identified using the databases PubMed, Web of Science and Embase for publications up to December 4, 2015. Pooled hazard ratios (HRs) and 95% confidence intervals (95% CIs) were systematically calculated to investigate the association between the expression of miR-200c and cancer prognosis. The results demonstrated that elevated expression levels of miR-200c indicated significantly worse overall survival rates (HR=1.37, 95% CI: 1.01, 1.85), and a high level of miR-200c was considered an indicator of an unfavorable prognosis in patients from Europe and America (HR=1.85, 95% CI: 1.27, 2.69). Furthermore, overexpression of miR-200c was significantly associated with progression of the disease in the subgroups of tissue and blood samples (HR=0.68 and 2.45, respectively), and inferior overall survival rates for the blood subgroup were revealed (HR=2.21, 95% CI: 1.04, 4.72). In addition, miR-200c was of prognostic value in several disease subgroups. Taken together, high expression levels of miR-200c are of significant prognostic value in various human malignancies.

Diagnostic and prognostic relevance of circulating exosomal miR-373, miR-200a, miR-200b and miR-200c in patients with epithelial ovarian cancer.

Exosomes are membrane vesicles that mediate intercellular communication by transporting their molecular cargo from cell to cell. We investigated whether serum levels of exosomal miR-373, miR-200a, miR-200b and miR-200c and circulating exosomes have diagnostic and prognostic relevance in a cohort of 163 epithelial ovarian cancer (EOC) patients using TaqMan MicroRNA assays and ELISA. The serum concentrations of exosomal miR-373 (p = 0.0001), miR-200a (p = 0.0001), miR-200b (p = 0.0001) and miR-200c (p = 0.028) were significantly higher in EOC patients than healthy women. The levels of miR-200a (p = 0.0001), miR-200b (p = 0.0001) and miR-200c (p = 0.019) could distinguish between malignant and benign ovarian tumors. While the levels of miR-373 and miR-200a were increased in all FIGO/lymph node stages (p = 0.0001), the levels of miR-200b and miR-200c were higher in patients with FIGO stage III–IV (p = 0.0001, p = 0.008, respectively) including lymph node metastasis (p = 0.0001, p = 0.004, respectively) than FIGO stages I–II. The increased levels of miR-200b and miR-200c were also associated with CA125 values (p = 0.0001, p = 0.0001, respectively) and a shorter overall survival (p = 0.007, p = 0.017, respectively). The levels of exosomes were excessively elevated in EOC patients (p = 0.0001). In all three cohorts, they were positively associated with the serum levels of exosomal miR-373 (p = 0.004), miR-200a (p = 0.0001), miR-200b (p = 0.0001) and miR-200c (p = 0.008). In conclusion, the increased levels of exosomal miR-200b and miR-200c mainly observed in advanced EOC suggest that these microRNAs may be involved in tumor progression. The high concentrations of exosomes in EOC patients imply an excessive, active exosomal secretion in EOC.

Dynamics and plasticity of the epithelial to mesenchymal transition induced by miR-200 family inhibition.

Whereas miR-200 family is known to be involved in the epithelial-to-mesenchymal transition (EMT), a crucial biological process observed in normal and pathological contexts, it has been largely unclear how far the functional levels of these tiny RNAs alone can propagate the molecular events to accomplish this process within several days. By developing a potent inhibitor of miR-200 family members (TuD-141/200c), the expression of which is strictly regulatable by the Tet (tetracycline)-On system, we found using a human colorectal cell line, HCT116, that several direct gene target mRNAs (Zeb1/Zeb2, ESRP1, FN1and FHOD1) of miR-200 family were elevated with distinct kinetics. Prompt induction of the transcriptional suppressors, Zeb1/Zeb2 in turn reduced the expression levels of miR-200c/-141 locus, EpCAM, ESRP1 and E-Cad. The loss of ESRP1 subsequently switched the splicing isoforms of CD44 and p120 catenin mRNAs to mesenchymal type. Importantly, within 9 days after the release from the inhibition of miR-200 family, all of the expression changes in the 14 genes observed in this study returned to their original levels in the epithelial cells. This suggests that the inherent epithelial plasticity is supported by a weak retention of key regulatory gene expression in either the epithelial or mesenchymal states through epigenetic regulation.

Sulforaphane targets cancer stemness and tumor initiating properties in oral squamous cell carcinomas via miR-200c induction.

Cancer stem cells (CSCs) are deemed as the driving force of tumorigenesis in oral squamous cell carcinomas (OSCCs). In this study, we investigated the chemotherapeutic effect of sulforaphane, a dietary component from broccoli sprouts, on targeting OSCC-CSCs. The effect of sulforaphane on normal oral epithelial cells (SG) and sphere-forming OSCC-CSCs isolated from SAS and GNM cells was examined. ALDH1 activity and CD44 positivity of OSCC-CSCs with sulforaphane treatment was assessed by flow cytometry analysis. Invitro and invivo tumorigenicity assays of OSCC-CSCs with sulforaphane treatment were presented. We observed that the sulforaphane dose-dependently eliminated the proliferation rate of OSCC-CSCs, whereas the inhibition on SG cells proliferation was limited. Cancer stemness properties including self-renewal, CD44 positivity, and ALDH1 activity were also decreased in OSCC-CSCs with different doses of sulforaphane treatment. Moreover, sulforaphane treatment of OSCC-CSCs decreased the migration, invasion, clonogenicity, and invivo tumorigenicity of xenograghts. Sulforaphane treatment resulted in a dose-dependent increase in the levels of tumor suppressive miR200c. These lines of evidence suggest that sulforaphane can suppress the cancer stemness and tumor-initiating properties in OSCC-CSCs both invitro and invivo.

Microarray analysis of microRNA deregulation and angiogenesis-related proteins in endometriosis.

We examined the aberrant microRNA (miRNA) expression profile responsible for the changes in angiogenesis observed in endometriotic lesions. This study revealed characteristic miRNA expression profiles associated with endometriosis in endometrial tissue and endometriotic lesions from the same patient, and their correlation with the most important angiogenic and fibrinolytic factors. miRNA expression was quantified using a microRNA array and reverse-transcription microRNA polymerase chain reaction. Levels of vascular endothelial growth factor A (VEGFA), epidermal growth factor receptor 2 (EGFR2), phosphatase and tensin homolog (PTEN), and C-X-C chemokine receptor type 4 (CXCR4) were quantified using enzyme-linked immunosorbent assay. The endometrial tissue showed significantly lower levels of miR-200b, miR-15a-5p, miR-19b-1-5p, miR-146a-5p, and miR-200c, and higher levels of miR-16-5p, miR-106b-5p, and miR-145-5p. VEGFA was significantly upregulated, whereas EGFR2, PTEN, and CXCR4 were markedly downregulated, in the endometriotic tissues compared to that in the normal endometrial tissues. In conclusion, differences in the miRNA levels could modulate the expression of VEGFA, EGFR2, PTEN, and CXCR4, and may play an important role in the pathogenesis of endometriosis. The higher angiogenic and proteolytic activities observed in the eutopic endometrium might facilitate the implantation of endometrial cells at ectopic sites.

Immunomodulatory Effect of MUC1-C in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is characterized by an immunosuppressive milieu in the bone marrow microenvironment that fosters tolerance, immune escape, and disease growth. Upregulation of PD-L1 expression by leukemia cells induces an exhausted phenotype in tumor reactive lymphocytes that blocks the activation and expansion of leukemia specific lymphocytes. Identifying pathways that regulate PD-L1 expression is vital for the development of novel therapeutic targets for immune based therapies. MUC1 is an oncoprotein that is aberrantly expressed on a majority of primary AML. MUC1 contains a transmembrane subunit (MUC1-C) that forms dimers and translocates to the nucleus where it mediates downstream signaling. MUC1-C expression on malignant cells promotes cell proliferation and resistance to apoptosis.We have demonstrated that MUC1-C is a critical regulator of PD-L1 expression on AML cells. Silencing of MUC1 expression was documented following lentiviral transduction with MUC1-C specific shRNA using western blotting. MUC1 silencing results in the near abrogation of MUC1-C expression by the human AML cell lines, MOLM-14 and THP1. Silencing of MUC1-C on MOLM-14 and THP1 AML cells resulted in a two-fold increase in susceptibility to T cell mediated lysis as determined by a flourochrome based CTL assay.To assess the effect of MUC1-C silencing on PD-L1 expression in-vivo, C57BL/6J mice were challenged with 100,000 GFP transfected TIB-49 murine AML cells in which MUC1-C was silenced using a lentiviral shRNA hairpin against MUC1-C. Following leukemia establishment, TIB-49 GFP+ cells were isolated from the bone marrows and spleens and PD-L1 expression on leukemic cells was measured. TIB-49 GFP+ cells of mice engrafted with MUC1 silenced AML cells demonstrated significantly lower PD-L1 expression compared to mice inoculated with TIB-49 cells transduced with a control vector (18% versus 3%; n=4). T cells isolated from bone marrow of mice inoculated with AML with silenced MUC1-C demonstrated a three-fold increase in INF-γ production when stimulated ex-vivo with autologous tumor lysate as compared to T cells from mice inoculated with control AML cells (n=4). Our group has developed a peptide inhibitor drug (G0-203), a cell-penetrating peptide that binds to the MUC1-C CQC motif, disrupting MUC1-C interaction with downstream effectors. As was observed following MUC1 silencing, bone marrow derived CD4+ and CD8+ T cells isolated from mice treated with G0-203 were shown to have two-fold increased intracellular IFN-γ production compared to control mice following ex-vivo exposure to AML lysate (n=4).To investigate the mechanism by which MUC1-C regulates PD-L1 expression we examined the role of noncoding RNAs that have been shown to be effectors of oncogenic modulation. MicroRNAs (miRNAs) are a conserved class of small RNAs that post-transcriptionally regulate gene expression by interacting with the 3′ untranslated region (3′ UTR) of target mRNAs. The 3'UTR of the PD-L1 gene contains putative binding sites for miR-200 family of micro-RNAs, supporting the hypothesis that miR-200 plays a role in regulating the expression of PD-L1.In the present study, we evaluated the effect of targeting MUC1-C mediated signaling, on miR200c levels and PD-L1 expression. MUC1-C silenced MOLM-14 cells demonstrated a two-fold increase in miR-200c expression, as demonstrated by qPCR and was associated with a reduction of PD-L1 expression from 77% to 13%. These data were confirmed in THP1 cells with PDL-1 expression decreasing from 95% to 40% following MUC1-C silencing. In support of the observation that miR200c is involved in the regulation of PD-L1 expression, lentiviral overexpression of miR200c in MOLM-14 cells led to a decrease in PD-L1 expression from 90% to 2% as demonstrated by flow cytometric analysis.In conclusion, these results demonstrate that MUC1-C mediated signaling regulates the expression of PD-L1 on AML cells. Silencing MUC1-C results in reduced expression of PD-L1, interfering with immune escape and enhancing the expansion of leukemia reactive T cells. We define a novel mechanism by which MUC1-C governs PD-L1 expression through its modulation of miR200c. As such, MUC1-C functions as a critical regulator of immune escape and represents a novel target for immunotherapy. DisclosuresKufe:Genus Oncology: Consultancy, Equity Ownership.

The ZEB1/miR-200c feedback loop regulates invasion via actin interacting proteins MYLK and TKS5

Epithelial to mesenchymal transition (EMT) is a developmental process which is aberrantly activated during cancer invasion and metastasis. Elevated expression of EMT-inducers like ZEB1 enables tumor cells to detach from the primary tumor and invade into the surrounding tissue. The main antagonist of ZEB1 in controlling EMT is the microRNA-200 family that is reciprocally linked to ZEB1 in a double negative feedback loop. Here, we further elucidate how the ZEB1/miR-200 feedback loop controls invasion of tumor cells. The process of EMT is attended by major changes in the actin cytoskeleton. Via in silico screening of genes encoding for actin interacting proteins, we identified two novel targets of miR-200c - TKS5 and MYLK (MLCK). Co-expression of both genes with ZEB1 was observed in several cancer cell lines as well as in breast cancer patients and correlated with low miR-200c levels. Depletion of TKS5 or MYLK in breast cancer cells reduced their invasive potential and their ability to form invadopodia. Whereas TKS5 is known to be a major component, we could identify MYLK as a novel player in invadopodia formation. In summary, TKS5 and MYLK represent two mediators of invasive behavior of cancer cells that are regulated by the ZEB1/miR-200 feedback loop.

Abstract 2775: Prognostic significance of triple miRNAs, miR-9, miR-221, and miR-200c in breast cancer

Abstract MicroRNAs (miRNA) mediated epithelial-mesenchymal transition have been considered as a major event in cancer aggression and accounted for cancer progression. However, clinical association between synergistic effect of deregulated expression levels of the EMT-related miRNAs, including miR-9, miR-221, and miR-200c, and breast cancer prognoses remains underestimated. Initially, tissue samples were from 184 enrolled female patients, with invasive ductal carcinoma, followed up for more than ten years. miRNA expression levels between paired laser capture microdissected tumor and non-tumor cells from the primary tumor site was analyzed by quantitative real-time PCR (qRT-PCR). Differentially expressed status of the target miRNA in tumors relative to non-tumor parts (T/N ratio) was assessed their relationship with pathological characteristics and clinical outcomes of breast cancer. While miR-9 or miR-221 showed significantly higher expression level in tumor cells compared to the non-tumor ones, miR-200c exhibited the opposite (p<0.05). Based on the ROC curve, T/N ratio of 2.80- and 1.96-fold increase for miR-9 and miR-221, whereas, 1.80-fold decrease for miR-200c as risk genotypes, respectively. According to these cutoffs, high miR-9 expression in tumors was associated with advanced tumor stages (p<0.05); significant risk associations were shown between LNM-positive tumors and miR-9 (OR = 3.12) or miR-221 (OR = 1.88) (p<0.05). A joint effect on risk elevation of tumor invasion and nodal metastasis was seen in tumors having miR-9 overexpression alongside increased miR-221 or reduced miR-200c expression (p-trend<0.05). Considering the combined effect of these three miRNAs, patients harboring greater numbers of deregulated expression levels in tumors were found to have a higher risk of poor prognoses (high-stage (p-trend = 0.003) and LNM (p-trend = 0.028)) and reduced probability of 10-year overall survival, showing hazard ratio of 2.39 (p<0001 for trend test). In conclusion, deregulated levels of miR-9, miR-221, and miR-200c, may serve as prognostic biomarkers to predict disease progression and shorter OS in breast cancer. Citation Format: Chun-Wen Cheng, Jyh-Cherng Yu, Yi-Hsien Hsieh, Po-Ming Chen, Hsiao-Wei Wang, Hui-Chun Wang, Chen-Yang Shen. Prognostic significance of triple miRNAs, miR-9, miR-221, and miR-200c in breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2775. doi:10.1158/1538-7445.AM2015-2775

Abstract 4299: MicroRNA 200c regulates cytochrome P450 1B1-mediated docetaxel resistance in renal cell cancers

Abstract Cytochrome P450 1B1 (CYP1B1) is up-regulated in many types of cancer. It has been suggested that CYP1B1 may be a general tumor biomarker and have an important function in cancers. Despite its high protein expression and enzymatic activity in renal cell cancer (RCC), functional significance of CYP1B1 has not been elucidated. Here we explored the functional role and regulatory mechanism of CYP1B1 in RCC. Reduction of CYP1B1 levels fail to prevent in vitro tumorigenicity such as proliferation, apoptosis, and cell cycle progression of RCC cells. Moreover, the expression levels are not associated with tumor type, stage, Fuhrman grade and 5-year survival probability after surgery. Instead, alteration of CYP1B1 expression regulates the chemosensitivity of RCC cells to docetaxel suggesting its critical contribution to the RCC chemoresistance. Additionally, miR-200c, which is significantly down-regulated in RCC tissues and cell lines regulates CYP1B1 mRNA, protein and enzyme activity. An inverse association was also observed between the expression levels of miR-200c and CYP1B1 protein in RCC tissues. Thus, high expression of CYP1B1 could be a result of decreased miR-200c expression in RCC. Finally, alteration of miR-200c levels affects the chemosensitivity of RCC cells. Restoration of docetaxel resistance by exogenous expression of CYP1B1 in miR-200c-over-expressing cells indicates that CYP1B1 is a functional target of miR-200c. These results suggest that CYP1B1 up-regulation mediated by low miR-200c is one of the mechanisms underlying resistance of RCC cells to docetaxel. Therefore, expression of CYP1B1 and miR-200c in RCC may be useful as a prediction for docetaxel response. Citation Format: Inik Chang, Yozo Mitsui, Shinichiro Fukuhara, Ankurpreet Gill, Darryn K. Wong, Soichiro Yamamura, Varahram Shahryari, Z. Laura Tabatabai, Rajvir Dahiya, Dong Min Shin, Yuichiro Tanaka. MicroRNA 200c regulates cytochrome P450 1B1-mediated docetaxel resistance in renal cell cancers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4299. doi:10.1158/1538-7445.AM2015-4299

Abstract 2235: miR-200c reduces P-gp mediated MDR and metastasis by targeting JNK2/c-Jun signaling pathway in colon cancer

Abstract MicroRNA-200c (miR-200c) recently emerged as an important regulator of tumorigenicity and cancer metastasis, however, its role in regulating multidrug resistance (MDR) remains unknown. In the current study, we found that the expression levels of miR-200c in recurred and metastatic colorectal cancers (CRC) were significantly lower, while the JNK2 expression was higher compared to primary tumors. We showed that in MDR CRC cells, miR-200c targeted the 3′UTR of JNK2 gene. Over-expression of miR-200c attenuated the levels of p-JNK, p-c-Jun, P-gp and MMP-2/-9, the downstream factors of JNK signaling pathway, resulting in increased sensitivity to chemotherapeutic drugs, which was accompanied by heightened apoptosis and decreased cell invasion and migration. Moreover, in an orthotopic MDR CRC mouse model, we demonstrated that over-expression of miR-200c effectively inhibited the tumor growth and metastasis. At last, in the tumor samples from locally advanced CRC patients with routine post-surgical chemotherapy, we observed an inverse correlation between the levels of mRNA expression of miR-200c and JNK2, ABCB1, MMP-9 thus predicting patient therapeutic outcomes. In summary, we found that miR-200c negatively regulated the expression of JNK2 gene and increased the sensitivity of MDR CRC cells to chemotherapeutic drugs, via inhibiting the JNK2/p-JNK/p-c-Jun/ABCB1 signaling. Restoration of miR-200c expression in MDR CRC may serve as a promising therapeutic approach in MDR induced metastasis. Citation Format: Qi Li. miR-200c reduces P-gp mediated MDR and metastasis by targeting JNK2/c-Jun signaling pathway in colon cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2235. doi:10.1158/1538-7445.AM2015-2235

Abstract 143: A long non-coding RNA activated by TGF-β can predict the prognosis of gastric cancer patients

Abstract Background: Long non-coding RNA (lncRNA) is known to play important roles in proliferation or metastasis in several human cancers. A recent study reported that lncRNA activated by TGF-β (lncRNA-ATB) induced Epithelial-Mesenchymal Transition (EMT) through the TGF-β/miR-200s/ZEB axis in hepatocellular carcinoma (HCC), therefore the high lncRNA-ATB patients in HCC had poor prognosis. EMT through the TGF-β/miR-200s/ZEB axis reported to be involved in invasion, metastasis, or dessemination in gastric cancer. Therefore, we focused on the clinical significance of lncRNA-ATB expression in gastric cancer. Materials and Methods: qRT-PCR was performed to examine expression of lncRNA-ATB, miR-200b, and miR-200c in gastric cancer tissues (n = 183). Then we divided patients into high and low lncRNA-ATB expression groups using lncRNA-ATB/GAPDH ≥0.55 or <0.55 to determine the clinicopathological significance of lncRNA-ATB in gastric cancer. Moreover we evaluated expressions of lncRNA-ATB, miR-200s and ZEB1 in gastric cancer cell lines by qRT-PCR. Gastric cancer cell lines with low lncRNA-ATB expression were treated by recombinant TGF-β1 to examine morphologic changes and genetic alterations, such as lncRNA-ATB, miR-200s, and ZEB1 levels, with respect to the EMT phenotype. Results: LncRNA-ATB expression levels in gastric cancer patients weren't associated with the clinicopathological factors, gender, age, tumor size, histological type, depth of tumor invasion, lymph node metastasis, lymphatic invasion, vascular invasion, liver metastasis, peritoneal dissemination, or distant metastasis. However, the high lncRNA-ATB group experienced a lower 5-year survival rate compared to the low lncRNA-ATB group (P = 0.003), and multivariate analysis indicated that lncRNA-ATB was an independent prognostic factor (HR = 3.18; 95%CI: 1.47-7.29; P = 0.0031). The lncRNA-ATB level was inversely associated with miR-200s in gastric cancer cell lines. The inverse association was validated in miR-200c and lncRNA-ATB in gastric cancer patients. Moreover, lncRNA-ATB and ZEB1 levels increased and miR-200c levels reduced in low lncRNA-ATB cells treated with TGF-β1 after 10 d of treatment. Morphologies also changed from epithelial to mesenchymal form. Conclusions: LncRNA-ATB plays an important role in EMT to promote invasion and metastasis through the TGF-β/miR-200s/ZEB axis, resulting in a poor prognosis in gastric cancer. Further investigations will demonstrate the significance of lncRNA-ATB as a novel lncRNA biomarker indicative of a poor prognosis in gastric cancer patients. Citation Format: Tomoko Saito, Junji Kurashige, Sho Nambara, Hisateru Komatsu, Hidenari Hirata, Masami Ueda, Shotaro Sakimura, Ryutaro Uchi, Yuki Takano, Yoshiaki Shinden, Tomohiro Iguchi, Hidetoshi Eguchi, Keishi Sugimachi, Kazunari Murakami, Koshi Mimori. A long non-coding RNA activated by TGF-β can predict the prognosis of gastric cancer patients. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 143. doi:10.1158/1538-7445.AM2015-143

Decreased expression of MicroRNA-200 family in human breast cancer is associated with lymph node metastasis.

MicroRNA-200 family (miR-200f) has been consistently reported to be deregulated and modulate the metastatic process in multiple cancers. In the present study, we detected the expression of miR-200f in breast cancer (BC) tissue and explored its relationships with clinicopathological characteristics, especially with lymph node metastasis. Expression levels of miR-200a, miR-200b, miR-200c, miR-141, and miR-429 in 99 pairs of BC tissues and adjacent normal tissues were measured by real-time quantitative PCR. The correlation between miR-200f level and multiple clinicopathological factors was then examined by Mann-Whitney test, ANOVA, and operating characteristic (ROC) analysis. All members of the miR-200f were down-regulated in BC tissue compared with that in normal adjacent tissue; miR-200a, miR-200b, and miR-200c were highly decreased (p < 0.05), while the differences of miR-141 and miR-429 between patients and the control group were not statistically significant. Furthermore, all five members were found to be distinctly decreased with the incidence of lymph node metastasis (p < 0.05); When the patients were divided into three groups according to the number of lymph node metastasis (0; 1-3; ≥4), a gradual decrease of miR-200f expression was observed with the increasing number of lymph node metastasis; ROC revealed that miR-200b can differentiate patients with lymph node metastasis from those without lymph node metastasis. These observations imply that the down-regulation of miR-200f in human BC is associated with an invasive phenotype, and miR-200b may be useful to estimate the likelihood of the presence of pathologically positive lymph nodes.

The analysis of microRNAs miR-200C and miR-145 expression in colorectal cancer of different molecular subtypes.

The expression levels of microRNAs miR-200c and miR-145 in two groups of colorectal cancer differing by the presence/absence of epithelial-mesenchymal transition (EMF) were studied. In the EMF-positive cancer, the level of miR-145 is increased, whereas the level of miR-200c is reduced. The reverse situation is observed in the EMI-negative cancer. MiR-145 can serve as a marker of the mesenchymal subtype of cancer. Gene expression profiles and microRNAs allow prognostically unfavorable tumors of the mesenchymal subtype to be distinguished.

Expression of serum miR-200a, miR-200b, and miR-200c as candidate biomarkers in epithelial ovarian cancer and their association with clinicopathological features.

MicroRNAs (miRs) have been implicated in the etiology of various human cancers. The aim of this study was to investigate the association of the expression of three members--miR 200a, miR 200b, and miR 200c belonging to the miR-200 family with clinicopathological characteristics and their impact on the progression of epithelial ovarian cancer (EOC). Total RNA from serum was isolated by Trizol method, polyadenylated, and reverse transcribed into cDNA. Expression levels of miR-200a, miR-200b, and miR-200c were detected by using miRNA qRT-PCR. We measured miR expression in 70 serum samples of EOC patients with matched controls using U6 snRNA as a reference. Levels of miR expression was compared with distinct clinicopathological features. Expression of miR-200a was found to be greater than six-fold (p = 0.01), miR-200b and miR-200c greater than three-fold (p = 0.01) in comparison with matched normal controls. Association of miRNA expression with clinicopathological factors and progression was statistically evaluated. The expression levels of miR-200a and miR-200c were found to be significantly associated with disease progression (p = 0.04 and p < 0.001, respectively). miR-200a overexpression was found be associated with tumor histology and stage. Patients with lymph node metastasis showed significant elevation of miR-200c (p = 0.006). The AUC in ROC curve also indicated that serum levels of miR-200a and miR-200c might be worthwhile as a diagnostic tool in the near future. Our findings suggest that miR-200a, miR-200b, and miR-200c overexpressions are associated with the aggressive tumor progression and be recognized as reliable markers to predict the prognosis and survival in EOC patients.

A Long Non-coding RNA Activated by Transforming Growth Factor-β is an Independent Prognostic Marker of Gastric Cancer.

A recent study reported that long non-coding RNA activated by TGF-β (lncRNA-ATB) induced epithelial-mesenchymal transition (EMT) through the transforming growth factor-β (TGF-β)/miR-200s/ZEB axis in hepatocellular carcinoma. Herein, we focused on the clinical significance of lncRNA-ATB in gastric cancer (GC) patients. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) was performed to examine expression of lncRNA-ATB, miR-200b, and miR-200c in GC tissues (n = 183). Patients were divided into high and low lncRNA-ATB expression groups using a cutoff of lncRNA-ATB/GAPDH ≥0.60 or <0.60 to determine the clinicopathological significance of lncRNA-ATB in GC. Moreover, we evaluated the expression of TGF-β, lncRNA-ATB, miR-200s, and ZEB1 in GC cell lines by qRT-PCR. GC cell lines were treated by recombinant TGF-β1 or TGF-β receptor inhibitor to examine morphologic changes and genetic alterations, such as lncRNA-ATB, miR-200s, and ZEB1 levels, with respect to the EMT phenotype. The high lncRNA-ATB group experienced a lower overall survival rate compared with the low lncRNA-ATB group, and multivariate analysis indicated that lncRNA-ATB was an independent prognostic factor (hazard ratio 3.50; 95 % CI 1.73-7.44; p = 0.0004). miR-200c levels were lower and ZEB1 levels were higher in the high lncRNA-ATB group than in the low lncRNA-ATB group. Treatment with TGF-β in GC cell lines resulted in morphological EMT changes, upregulation of lncRNA-ATB and ZEB1, and downregulation of miR-200c and CDH1. SB431542 reduced lncRNA-ATB expression. LncRNA-ATB plays an important role in EMT to promote invasion and metastasis through the TGF-β/miR-200s/ZEB axis, resulting in a poor prognosis in GC. LncRNA-ATB is a novel biomarker of lncRNA, indicative of a poor prognosis in GC patients.

Alterations of miR-132 are novel diagnostic biomarkers in peripheral blood of schizophrenia patients.

Alterations in microRNAs (miRNAs) have been considered to have diagnostic implications in most diseases, but few studies have reported dysregulated miRNAs in schizophrenia (SCZ). In order to observe an association between miRNAs and SCZ, this study was designed to investigate expression profiling of miRNAs in peripheral blood mononuclear cells (PBMCs). miRNA microarray technology was employed to compare the expression of miRNAs in PBMCs from SCZ patients (n=105) and normal controls (n=130), and real-time quantitative polymerase chain reaction (QPCR) was used to analyze the results. Several important miRNA levels were examined before and after antipsychotic treatment in first-onset SCZ patients. In addition, an SCZ-like rat model was established using dizocilpine (MK-801), and miR-132 expression in PBMCs and whole-brain tissue from SCZ-like rats was studied using QPCR. In humans, dysregulated miRNAs were observed before treatment and QPCR verified that miR-132, miR-134, miR-1271, miR-664(⁎), miR-200c and miR-432 levels were significantly decreased (P<0.01 for all) in PBMCs of SCZ patients compared with healthy controls. After antipsychotic treatment there was a marked increase in miR-132 (P<0.01), miR-664(⁎) (P<0.05) and miR-1271 (P<0.05) levels in SCZ patients compared with the levels before treatment. In the animal assays, miR-132 levels declined in PBMCs and whole-brain tissues (both P<0.05) of the SCZ-like rats compared to controls. For the first time, our results suggest that miR-132 is a potential and superior biomarker in peripheral blood that will allow discrimination of SCZ patients from healthy controls.

Circulating miR-200c and miR-141 and outcomes in patients with breast cancer.

BackgroundThe deregulation of microRNAs in both tumours and blood has led to the search for microRNAs to indicate the presence of cancer and predict prognosis. We hypothesize the deregulation of miR-200c/miR-141 in the whole blood can identify breast cancer (BC), and could be developed into a prognostic signature.MethodsThe expression of miR-200c and miR-141 were examined in bloods (57 stage I-IV BC patients and 20 age-matched controls) by quantitative reverse-transcription PCR. The associations of circulating microRNAs with clinic and pathological characteristics were analysed. Their effects on survival were analysed by the Kaplan-Meier method and Cox regressions.ResultsMiR-200c was down regulated (P < 0.0001) in the blood of BC patients, yielded an area under the ROC curve of 0.79 (90% sensitivity, 70.2% specificity) in discriminating BC from controls. Circulating miR-141 was not discriminating. MiR-200c and miR-141 in the blood of BC patients were inversely correlated (P = 0.019). The miR-200c levels were numerically higher in stage IV and tumours with lower MIB-1. MiR-141 was significantly higher in the blood of patients with stage I-III, lymph node metastasis, and HER2 negative tumours. High blood expression of miR-200c and/or low expression of miR-141 was associated with unfavourable overall survival (hazard ratio, 3.89; [95% CI: 1.28-11.85]) and progression-free survival (3.79 [1.41–10.16]) independent of age, stage and hormonal receptors.ConclusionsCirculating miR-200c and miR-141 were deregulated in BC comparing with controls. Furthermore, miR-200c and miR-141 were independent prognostic factors and associated with distinct outcomes of BC patients.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1238-5) contains supplementary material, which is available to authorized users.

MiR-200c-SUMOylated KLF4 feedback loop acts as a switch in transcriptional programs that control VSMC proliferation

The regulation of vascular smooth muscle cell (VSMC) proliferation is an important issue because it has major implications for the prevention of pathological vascular conditions. Using microRNA array screen, we found the expression levels of 200 unique miRNAs in hyperplasic tissues. Among them, miR-200c expression substantially was down-regulated. The objective of this work was to assess the function of miR-200c and SUMOylated Krϋppel-like transcription factor 4 (KLF4) in the regulation of VSMC proliferation in both cultured cells and animal models of balloon injury. Under basal conditions, we found that miR-200c inhibited the expression of KLF4 and the SUMO-conjugating enzyme Ubc9. Upon PDGF-BB treatment, Ubc9 interacted with and promoted the SUMOylation of KLF4, which allowed the recruitment of transcriptional corepressors (e.g., nuclear receptor corepressor (NCoR) and HDAC2) to the miR-200c promoter. The reduction in miR-200c levels led to increased target gene expression (e.g., Ubc9 and KLF4), which further repressed miR-200c levels and accelerated VSMC proliferation. These results demonstrate that induction of a miR-200c-SUMOylated KLF4 feedback loop is a significant aspect of the PDGF-BB proliferative response in VSMCs and that targeting Ubc9 represents a novel approach for the prevention of restenosis.

Loss of miR-200c up-regulates CYP1B1 and confers docetaxel resistance in renal cell carcinoma.

Despite high protein expression and enzymatic activity of cytochrome P450 1B1 (CYP1B1) in renal cell cancer (RCC), its functional significance has not been elucidated. Here we explored the functional role and regulatory mechanism of CYP1B1 in RCC. Reduction of CYP1B1 levels fail to prevent in vitro tumorigenicity such as proliferation, apoptosis, and cell cycle progression of RCC cells. Moreover, the expression levels are not associated with tumor type, stage, Fuhrman grade and 5-year survival probability after surgery. Instead, alteration of CYP1B1 expression regulates the chemosensitivity of RCC cells to docetaxel suggesting its critical contribution to the chemoresistance. Additionally, miR-200c, which is significantly down-regulated in RCC regulates CYP1B1 expression and activity. An inverse association was also observed between the expression levels of miR-200c and CYP1B1 protein in RCC tissues. Finally, alteration of miR-200c levels affects the chemosensitivity of RCC cells. Restoration of docetaxel resistance by exogenous expression of CYP1B1 in miR-200c-over-expressing cells indicates that CYP1B1 is a functional target of miR-200c. These results suggest that CYP1B1 up-regulation mediated by low miR-200c is one of the mechanisms underlying resistance of RCC cells to docetaxel. Therefore, expression of CYP1B1 and miR-200c in RCC may be useful as a prediction for docetaxel response.

MicroRNA-200c contributes to injury from transient focal cerebral ischemia by targeting Reelin.

MicroRNA (miR)-200c increases rapidly in the brain after transient cerebral ischemia but its role in poststroke brain injury is unclear. Reelin, a regulator of neuronal migration and synaptogenesis, is a predicted target of miR-200c. We hypothesized that miR-200c contributes to injury from transient cerebral ischemia by targeting reelin. Brain infarct volume, neurological score and levels of miR-200c, reelin mRNA, and reelin protein were assessed in mice subjected to 1 hour of middle cerebral artery occlusion with or without intracerebroventricular infusion of miR-200c antagomir, mimic, or mismatch control. Direct targeting of reelin by miR-200c was assessed in vitro by dual luciferase assay and immunoblot. Pretreatment with miR-200c antagomir decreased post-middle cerebral artery occlusion brain levels of miR-200c, resulting in a significant reduction in infarct volume and neurological deficit. Changes in brain levels of miR-200c inversely correlated with reelin protein expression. Direct targeting of the Reln 3' untranslated region by miR-200c was verified with dual luciferase assay. Inhibition of miR-200c resulted in an increase in cell survival subsequent to in vitro oxidative injury. This effect was blocked by knockdown of reelin mRNA, whereas application of reelin protein afforded protection. These findings suggest that the poststroke increase in miR-200c contributes to brain cell death by inhibiting reelin expression, and that reducing poststroke miR-200c is a potential target to mitigate stroke-induced brain injury.