Colorectal cancer (CRC) has been one of the most commonly diagnosed cancers in global. The differential expression profiles of microRNAs (miRNAs) in CRC plasma of patients have the potential to serve as a diagnostic biomarker. We conducted a four-stage study to identify the potential plasma miRNAs for CRC detection. In the initial screening phase, Exiqon panel (miRCURY-Ready-to-Use-PCR-Human-panel-I + II-V1.M) including 3 CRC pools and 1 normal controls (NCs) pool were applied to acquire miRNA profiles. In the training stage (30 CRC VS. 30 NCs) and testing stage (79 CRC VS. 76 NCs), quantitative real-time polymerase chain reaction (qRT-PCR) was utilized to conduct candidate miRNA profiles. Then the identified miRNAs were verified in external validation stage (30 CRC VS. 26 NCs). Expression levels of identified miRNAs were assessed in tissue samples (24 pairs) and plasma exosomes (18 CRC VS. 18 NCs). Receiver operating characteristic (ROC) curves were constructed to evaluate the diagnostic accuracy. Seven miRNAs (miR-103a-3p, miR-127-3p, miR-151a-5p, miR-17-5p, miR-181a-5p, miR-18a-5p and miR-18b-5p) were significantly overexpressed in CRC compared with NCs. Area under the ROC curve of the seven-miRNA signature was 0.762, 0.824 and 0.895 for the training, testing and the external validation stages, respectively. Additionally, miR-103a-3p, miR-127-3p, miR-17-5p and miR-18a-5p were discovered significantly up-regulated in CRC tissues; while miR-17-5p, miR-181a-5p, miR-18a-5p and miR-18b-5p were significantly elevated in CRC plasma exosomes. In conclusion, we established a seven-miRNA signature in the peripheral plasma for CRC detection.

This work aimed to investigate the inter-regulatory functions of hsa-mir-127 and replication initiator 1 (REPIN1) on the proliferation and metastasis of glioma cells. The in-silico data on the implication of hsa-mir-127 and REPIN1 in glioma were retrieved from The Cancer Genome Atlas (TCGA). The expression levels of hsa-mir-127 and REPIN1 mRNA were determined by qRT-PCR, whereas Western blot was used to detect REPIN1 protein expression in glioma cell lines. The proliferation of glioma cells was determined by means of the MTT assay, whereas the transwell assay was employed for assessing the extent of cell migration and invasion. The interaction among REPIN1 and hsa-mir-127 was checked using the luciferase reporter assay. The expression of hsa-mir-127 was markedly increased in clinical data obtained from TCGA and in glioma cells compared with normal tissues and control cells, respectively. Increased expression of hsa-mir-127 and decreased expression of REPIN1 were both associated with poor overall survival. Moreover, hsa-mir-127 overexpression noticeably promoted proliferation, inhibited apoptosis and increased the invasive and migratory capacities of glioma cells. Inverse effects were found with hsa-mir-127 antisense inhibitor. Interestingly, overexpression of hsa-mir-127 downregulated REPIN1 expression, and luciferase reporter assay showed that the tumorigenesis effect of hsa-mir-127 requires, in part, its direct targeting of REPIN1. In conclusion, the hsa-mir-127/REPIN1 pathway is involved in gliomas and could be a potential therapeutic target.

By routinely and systematically being able to perform quantitative stem-loop reverse transcriptase (RT) followed by TaqMan® minor-groove binding (MGB) probe, real-time quantitative PCR analysis on exfoliated enriched colonocytes in stool, using human (Homo sapiens, hsa) micro(mi)RNAs to monitor changes of their expression at various stages of colorectal (CRC) progression, this method allows for the reliable and quantitative diagnostic screening of colon cancer (CC). Although the expression of some miRNA genes tested in tissue shows less variability in normal or cancerous patients than in stool, the noninvasive stool by itself is well suited for CC screening. An miRNA approach using stool promises to offer more sensitivity and specificity than currently used genomic, methylomic, or proteomic methods for CC screening.To present an application of employing miRNAs as diagnostic markers for CC screening, we carried out global microarray expression studies on stool colonocytes isolated by paramagnetic beads, using Affymetrix GeneChip miRNA 3.0 Array, to select a panel of miRNAs for subsequent focused semiquantitative PCR analysis studies. We then conducted a stem-loop RT-TaqMan® MGB probes, followed by a modified real-time qPCR expression study on 20 selected miRNAs for subsequent validation of the extracted immunocaptured total small RNA isolated from stool colonocytes. Results showed 12 miRNAs (miR-7, miR-17, miR-20a, miR-21, miR-92a, miR-96, miR-106a, miR-134, miR-183, miR-196a, miR-199a-3p, and miR214) to have an increased expression in stool of CC patients, and that later TNM stages exhibited more increased expressions than adenomas, while 8 miRNAs (miR-9, miR-29b, miR-127-5p, miR-138, miR-143, miR-146a, miR-222, and miR-938) showed decreased expressions in stool of CC patients, which becomes more pronounced as the cancer progresses from early to late TNM stages (0-IV).

Osteosarcoma (OS) is one of the most universal malignant bone tumors that occur mostly in children and adolescents. This study aimed to investigate the roles of miR-127-3p and integrin subunit-α 6 (ITGA6) in OS proliferation, apoptosis, invasion and migration, and to explore the possible molecular mechanism and target relationship. By conducting quantitative real-time polymerase chain reaction (qRT-PCR) and western blot, the microRNA (miRNA) and protein expressions of miR-127-3p and ITGA6 in both tissues and cells were determined. The expression of apoptosis and migration related were also detected by western blot. The target relationship between miR-127-3p and ITGA6 was predicted by TargetScan and verified by dual-luciferase reporter assay. The biological functions of miR-127-3p and ITGA6 in OS were investigated by following experiments: cell counting kit 8 (CCK-8) and colony formation assays to inspect cell proliferation, flow cytometry, and caspase 3 activity assay to examine apoptosis, and transwell and wound healing assays to analyze invasion and migration. Significant down-regulation of miR-127-3p and up-regulation of ITGA6 was found out in OS tissues and cells. ITGA6 was proved to be the downstream target gene of miR-127-3p and functioned as a tumor promotor in OS, while miR-127-3p restrained deterioration of OS by suppressing cell viability, reducing migration and invasion, and promoting apoptosis. MiR-127-3p suppressed proliferation, invasion, and migration while stimulated apoptosis of OS cells through knocking down ITGA6. © 2018 IUBMB Life, 70(5):411-419, 2018.

BACKGROUND: microRNAs (miRNAs) are considered promising cancer biomarkers, showing high reliability, sensitivity and stability. Our study aimed to identify associations between whole blood miRNA profiles, presence of circulating tumor cells (CTCs) and clinical outcome in post-operative early breast cancer patients (EBC) to assess the utility of miRNAs as prognostic markers in this setting.
METHOD: A total of 48 post-operative patients, recruited in frame of the SUCCESS A trial, were included in this retrospective study and tested with a panel of 8 miRNAs (miR-10b, -19a, - 21, - 22, -20a, - 127, - 155, -200b). Additional 17 female healthy donors with no previous history of cancer were included in the study as negative controls. Blood samples were collected at different time points (pre-adjuvant therapy, post-adjuvant therapy, 2 years follow up), total RNA was extracted and the relative concentration of each miRNA was measured by quantitative PCR and compared in patients stratified on blood collection time or CTC detection. Furthermore, we compared miRNA profiles of patients, for each time point separately, and healthy donors. CTCs were visualized and quantified with immunocytochemistry analysis. Data were analyzed using non-parametric statistical tests.
RESULTS: In our experimental system, miR-19a, miR-22 and miR-127 showed the most promising results, differentiating patients at different time points and from healthy controls, while miR-20a, miR-21 and miR-200b did not show any difference among the different groups. miR-10b and miR-155 were never detectable in our experimental system. With respect to patients' clinical characteristics, we found a significant correlation between miR-200b and lymph node status and between miR-20a and tumor type. Furthermore, miR-127 correlated with the presence of CTCs. Finally, we found a borderline significance between Progression Free Survival and miR-19a levels.
CONCLUSIONS: This pilot study suggests that profiling whole blood miRNAs could help to better stratify post-operative EBC patients without any sign of metastasis to prevent later relapse or metastatic events.

Ten-Eleven-Translocation 1 (TET1) plays a role in the DNA methylation process and gene activation. Recent reports suggest TET1 acts as an oncogene in leukemia development. However, the clinical relevance and biological insight of TET1 expression in cytogenetically normal acute myeloid leukemia (CN-AML) is unknown. In this study, quantification of TET1 transcript by real-time quantitative PCR in bone marrow blasts was performed in 360 CN-AML patients. As a result, high TET1 expression was more common in M0/M1 morphology and genes of NPM1 mutations, and underrepresented in CEBPA double allele mutations in our AML patients. In addition, we found overexpression of TET1 was associated with an inferior overall survival and event free survival in the two independent cohorts. Notably, mRNA and miRNA integrative analyses showed aberrant expression of several hub oncogenes appear to be regulated by some miRNAs like miR-127-5p, miR-494, miR-21 and miR-616 in high TET1 expressers. In conclusion, the TET1 gene expression might serve as a reliable predictor for patients survival in AML.

Giant cell tumors of bone (GCTB) are generally benign bone tumors associated with expansive osteolytic defects, a high rate of recurrence and potential malignant transformation. We recently observed silencing of miR-127-3p and miR-376a-3p in GCTB and identified COA1 and PDIA6 as their target genes. Here, we investigate the impact of these microRNAs and their target genes on tumor engraftment and progression of giant cell tumor stromal cells (GCTSC) in vivo by xenotransplantation on the chorioallantoic membrane of chicken eggs. Prior to transplantation, the neoplastic GCTSCs were transfected with miRNA mimics or siRNAs directed against their target genes. Restoration of miR-127-3p and miR-376a-3p reduced the tumor take rate to 17% and 47% compared to 95% in the controls. The tumor volumes were significantly reduced to 29% by both miRNAs. Silencing of COA1 and PDIA6 significantly decreased the tumor volumes to 37.7% and 42.7%, while the tumor take rates remained stable. Our results indicate that re-expression of miR-127-3p and miR-376a-3p induces a strong tumor suppressor effect in GCTSC, which is partially mediated via COA1 and PDIA6.

AIM: To investigate the microRNA expression profile in esophageal neosquamous epithelium from patients who had undergone ablation of Barrett's esophagus.
METHODS: High throughput screening using TaqMan
RESULTS: Forty-four microRNAs were differentially expressed between control squamous mucosa
CONCLUSION: Neosquamous mucosa arising after ablation of Barrett's esophagus expresses microRNAs that may contribute to decreased barrier function and microRNAs that may be involved in the regulation of survival signaling pathways.

Osteosarcoma (OS) is one of the most common bone malignancies and occurs almost exclusively in children and adolescents. This study aimed to explore the role of lncRNA maternally expressed gene 3 (MEG3) in OS cells growth and metastasis, and to uncover the possible underlying mechanism. In this study, the expressions of MEG3 in five OS cell lines (MG63, OS-732, SaOS, G292, and 143B) and in a human osteoblast cell line hFOB1.19 were measured by qRT-PCR analysis. The expressions of MEG3, miR-127, and ZEB1 in OS-732 cells were overexpressed or suppressed by transfection. Cell viability, migration, invasion, and apoptosis were then assessed. The results showed that MEG3 was highly expressed in OS cell lines when compared to hFOB1.19 cell. MEG3 silence significantly suppressed OS-732 cells growth and metastasis, as evidenced by the decreases in cell viability, migration, invasion, and increase in apoptotic cell rate. MEG3 acted as an endogenous sponge by binding to miR-127. More interestingly, MEG3 silence could not suppress OS-732 cells growth and metastasis when miR-127 was knocked down. ZEB1 was a target gene of miR-127, and miR-127 overexpression-induced impairments in cell growth and metastasis were attenuated when ZEB1 was overexpressed. Moreover,miR-127 suppression activated JNK and Wnt signaling pathways, while these activations were recovered by ZEB1 silence. To conclude, our findings suggest that lncRNA MEG3 promoted OS cells growth and metastasis in vitro through sponging miR-127. This study provides the evidence that MEG3 may be a potential therapeutic target for OS.

Prostate cancer is the most common non-cutaneous cancer among men; yet, current diagnostic methods are insufficient, and more reliable diagnostic markers need to be developed. One answer that can bridge this gap may lie in microRNAs. These small RNA molecules impact protein expression at the translational level, regulating important cellular pathways, the dysregulation of which can exert tumorigenic effects contributing to cancer. In this study, high throughput sequencing of small RNAs extracted from blood from 28 prostate cancer patients at initial stages of diagnosis and prior to treatment was used to identify microRNAs that could be utilized as diagnostic biomarkers for prostate cancer compared to 12 healthy controls. In addition, a group of four microRNAs (miR-1468-3p, miR-146a-5p, miR-1538 and miR-197-3p) was identified as normalization standards for subsequent qRT-PCR confirmation. qRT-PCR analysis corroborated microRNA sequencing results for the seven top dysregulated microRNAs. The abundance of four microRNAs (miR-127-3p, miR-204-5p, miR-329-3p and miR-487b-3p) was upregulated in blood, whereas the levels of three microRNAs (miR-32-5p, miR-20a-5p and miR-454-3p) were downregulated. Data analysis of the receiver operating curves for these selected microRNAs exhibited a better correlation with prostate cancer than PSA (prostate-specific antigen), the current gold standard for prostate cancer detection. In summary, a panel of seven microRNAs is proposed, many of which have prostate-specific targets, which may represent a significant improvement over current testing methods.

Medullary thyroid carcinoma (MTC) represents 3-5% of thyroid cancers. 75% is sporadic and 25% is the dominant component of the hereditary multiple endocrine neoplasia (MEN) type 2 syndromes. Three different subtypes of MEN2, such as MEN2A, MEN2B, and Familial MTC (FMTC) have been defined, based on presence or absence of hyperparathyroidism, pheocromocytoma and characteristic clinical features. Mutations of the RET proto-oncogene are implicated in the pathogenesis of MTC, but there are many other mutational patterns involved. In MEN2A, Codon 634 in exon 11 (Cys634Arg), corresponding to a cysteine in the extracellular cysteine-rich domain, is the most commonly altered codon. Many other mutations include codons 611, 618, 620. In the genetical testing of RET mutations in MTCs, Next-Generation Sequencing (NGS) is taking an increasingly important role. One of the most important benefit is the comprehensive analysis of molecular alterations in MTC, which allows rapidly to select patients with different risk levels. There is a difference in miRNA expression pathway between sporadic and hereditary MTCs. Among sporadic cases, expression of miR-127 was significantly lower in those who harbor somatic RET mutations than those with wild-type RET. CDKN1B mutations are associated with many clinical pictures of cancers, such as MEN4. V109G polymorphism is associated with sporadic MTCs negative for RET mutations, and might influence the clinical course of the patients affected by MTC. Although surgery (i.e. total thyroidectomy with neck lymph node dissection) is the elective treatment for MTCs, about 80% of patients have distant metastases at diagnosis and in this cases surgery is not enough and an additional treatment is needed. Interesting results come from two large phase III clinical trials with two targeted tyrosine kinase inhibitors (TKIs), vandetanib and cabozantinib.
CONCLUSIONS: New genetical testings and therapeutical approaches open new perspectives in MTC management.

The methylation of promoter CpG islands and the interaction between microRNAs (miRNAs) and messenger RNAs (mRNAs) of target genes are considered two crucial mechanisms for gene and pathway deregulation in malignant tumors. The aim of this study was to analyze the role of promoter methylation in altering the expression of 13 miRNAs that are associated with breast cancer (BC): miR-124, -125b, -127, -132, -137, -148a, -191, -193a, -203, -212, -34b, -375, -9. The role of methylation in the deregulation of these miRNAs has not been previously assessed in the representative set of BC samples. We used a set of 58 paired (tumor/normal) breast tissue samples and methylation-specific PCR to demonstrate significant aberrations in the methylation patterns of 9 miRNA genes. In particular, we observed hypermethylation of MIR-127, -132, and -193a, and hypomethylation of MIR-191 for the first time. Using quantitative PCR, we established a strong correlation between promoter methylation and expression levels for 12 miRNA genes (all except MIR-212); this finding demonstrates the functional importance of altered methylation patterns. We also performed a correlation analysis between expression levels of the 13 miRNAs and 5 cancer-associated genes, namely RASSF1(A), CHL1, APAF1, DAPK1, and BCL2, which were predicted as targets for these miRNAs, to investigate the impact of these miRNAs on these genes with key cellular functions in BC. Significant negative correlation was revealed for the following miRNA-mRNA pairs: miR-127-5p and DAPK1, miR-375 and RASSF1(A), and miR-124-3p and BCL2. Additionally, we also found a strong association between hypermethylation of MIR-127 and MIR-125b-1 and BC progression, particularly metastasis. Thus, our findings provide evidence for the significant role of methylation in the deregulation of 12 miRNA genes in BC, identify putative novel functional miRNA-mRNA pairs, and suggest MIR-127 and MIR-125b-1 hypermethylation to be potential biomarkers of BC metastasis.

OBJECTIVE: To explore the diagnostic value of combined detection of plasma miR-127-3p and HE4 for breast cancer (BC).
METHODS: Included in this study were 102 patients with pathologically confirmed BC who received treatment in the affiliated hospital of Nantong University between March 2015 and April 2016, 87 patients with benign breast tumors, and 90 healthy volunteers as control. Plasma miR-127-3p was detected by SYBR Green RT-qPCR, and plasma HE4 was detected by chemiluminescent immunoassay. The diagnostic efficacy of miR-127-3p alone, HE4 alone and combined detection of miR-127-3p and HE4 in BC women patients was evaluated by ROC curve analysis.
RESULTS: The relative expression quantity (RQ) of plasma miR-127-3p and HE4 in BC patients was 13.561 (3.345∼18.281) pmol/L and 105.42 (40.28∼156.31) pmol/L. The RQ of plasma miR-127-3p in BC patients was significantly higher than that in benign breast tumor patients and healthy individuals (both P< 0.001), and there was no significant difference between benign breast tumor patients and healthy individuals (P> 0.05). There was no significant correlation between plasma miR-127-3p and HE4 levels (r2= 0.086, P= 0.471). ROC curve analysis on the diagnostic efficacy of plasma miR-127-3p and HE4 in BC diagnosis showed that the cut-off value of miR-127-3p and HE4 in BC diagnosis was 3.471 and 63.21 pmol/L; AUC was 0.767 and 0.670; sensitivity was 78.2% and 64.6%; specificity was 79.1% and 69.3%; accuracy was 73.2% and 65.1%, respectively. Prediction probability (P) obtained from the miR-127-3p and HE4 model established by logistic regression was P= 1/ [1 + exp (-0.142miR-127-3p-0.024HE4 + 2.875)]. AUC calculated from ROC was 0.825 and the sensitivity was increased to 87.4%.
CONCLUSION: Combined detection of plasma miR-127-3p and HE4 greatly improved the sensitivity of BC diagnosis and may prove to be a candidate biomarker for early detection and diagnosis of BC.

The coordination between cellular differentiation and the mesenchymal/stem transition is essential for both embryo development and neoplasia, suggesting a mechanistic link between these two major processes. In this work we show that miR-127, an embryo-expressing lung miRNA, was prominently induced in lung adenocarcinoma and correlated with poor prognosis. Elevated miR-127 level drove a pronounced shift from the epithelial to the mesenchymal phenotype in cancer cells, and this shift was associated with their acquisition of stem-like traits, increased resistance to the epidermal growth factor receptor inhibitor and tumor-propagating potential. In contrast, antagonizing miR-127 markedly reversed this malignant transition, compromised the stem-like properties and the in vivo tumorigenic capability of cancer cells. Importantly, a regulatory loop involving the inflammatory signals NF-κB, miR-127 and tumor necrosis factor alpha-induced protein 3 was uncovered as a self-reinforcing circuitry that ensured an aggressive transition in lung cancer. Thus, this work identifies a novel molecular mechanism linking stemness, malignancy and inflammation, opening a new avenue for cancer treatment.

In this study, we investigated the expression patterns and functional roles of microRNA 127 (miR-127) and its target gene Formin-Like 3 (FMNL3) in human esophageal squamous cell carcinoma (ESCC). Quantitative RT-PCR (qRT-PCR) was used to compare miR-127 expression between ESCC cell lines and normal esophageal epithelium cell line, as well as paired ESCC tumors and adjacent normal esophageal tissues in 33 patients. We found miR-127 was aberrantly downregulated in both ESCC cell lines and human ESCC tumors. In ESCC cell lines TE-1 and ECA109 cells, lentiviral-induced miR-127 upregulation markedly inhibited cancer proliferation and migration in vitro, and tumorigenicity in vivo. Through dual-luciferase assay and qRT-PCR, FMNL3 was confirmed to be the downstream target gene of miR-127 in ESCC. Finally, FMNL3 was downregulated by siRNA in TE-1 and ECA109 cells. And we discovered that SiRNA-induced FMNL3 downregulation had tumor suppressive effect in ESCC, inhibiting cancer proliferation, migration in vitro, and tumorigenicity in vivo. These results suggest that miR-127 is downregulated and acting as tumor suppressor in ESCC. Inversely, FMNL3, the target gene of miR-127, is upregulated and acting as an oncogene in ESCC.

In the present study, the tumor-suppressive role of microRNA-127-3p (miR-127-3p) in epithelial ovarian cancer (EOC) was elucidated. Expression of miR-127-3p was examined by quantitative RT-PCR (qRT-PCR) in 9 EOC cell lines and clinical samples from 13 EOC patients. EOC cell lines, OVCAR-3 and Caov-3, were transduced with a lentivirus to overexpress endogenous miR-127-3p. The tumor-suppressive effects of miR-127-3p on EOC proliferation, bufalin sensitivity, invasion and in vivo growth were investigated through proliferation, bufalin sensitivity wound-closure and in vivo tumorigenicity assays, respectively. In addition, luciferase reporter assay and qRT-PCR were conducted to verify whether the Bcl-2-associated athanogene 5 (BAG5) gene was the downstream target of miR-127-3p in EOC. BAG5 was subsequently upregulated in the OVCAR-3 and Caov-3 cells to examine its functional correlation with miR‑127-3p regulation in EOC. The results revealed that in both EOC cell lines and EOC tumor tissues, miR-127-3p was downregulated. Lentiviral-mediated miR-127-3p overexpression exerted tumor-suppressive effects in OVCAR-3 and Caov-3 cells by reducing in vitro proliferation and invasion, increasing bufalin sensitivity, and inhibiting in vivo tumor growth. miR‑127-3p directly regulated the BAG5 gene in EOC. Subsequent BAG5 upregulation ameliorated the tumor-suppressive effects of miR-127-3p overexpression in EOC. In conclusion, miR-127-3p functions as a tumor suppressor in EOC, and its influence on EOC is directly through regulation of BAG5.

MicroRNAs are small non-coding RNAs with a length of 18-25 nucleotides. They can regulate tumor invasion and metastasis by changing the expression and translation of their target mRNAs. Their expression is substantially altered in colorectal cancer cells as well as in the adjacent tumor-associated stroma. Both of these compartments have a mutual influence on tumor progression. In the development of metastases, cancer cells initially interact with the host tissue. Therefore, compartment-specific expression signatures of these three locations-tumor, associated stroma, and host tissue-can provide new insights into the complex tumor biology of colorectal cancer. Frozen tissue samples of colorectal liver (

MicroRNAs are small non-coding RNAs with 18-22 nucleotides in length and have been proposed to function in various biological processes by targeting genes for post-transcriptional degradation via their 3' untranslated region. Moreover, they have been suggested to improve the chemosensitivity in a panel of tumors. However, the biological functions of microRNA-127-3p in esophageal carcinogenesis are still enigmatic. Thus, in the study, we firstly analyzed the roles of microRNA-127-3p in regulating the growth of esophageal cancer cells both in vitro and in vivo. Afterwards, using the microRNA-targeted gene prediction software and the dual-luciferase reporter assays, we confirmed that microRNA-127-3p specifically reduced the expression of X-ray repair complementing defective repair in Chinese hamster cells 3, one of RAD51 recombinase paralogs, at both mRNA and protein levels. Furthermore, using the homologous recombination repair and non-homologous end joining repair reporter systems, we found that microRNA-127-3p specifically compromised the homologous recombination repair and significantly increased DNA double strand breaks in cells. Besides, it statistically increased the chemosensitivity of esophageal cancer cells to a novel phenanthroline-dione derivative in vivo by mechanistically impairing the recruitment of RAD51 to the damage sites. In summary, our findings not only suggest that microRNA-127-3p can be used as a predictor for evaluating the development of esophageal carcinoma, but also show that it can be used to increase the chemosensitivity of esophageal cancer patients to the phenanthroline-dione derivative, which might be a potential anticancer candidate in the future.

Objective To preliminarily observe miRNA gene profiles in benefit serum of advanced non-small cell lung cancer (NSCLC) treated by TCM combined Western medicine (WM) , and to seek for molecular markers for its efficacy monitoring and prediction. Methods Recruited were 5 advanced NSCLC patients who received TCM combined WM treatment and obtained efficacy benefit ( as the treatment group) , 3 advanced NSCLC patients who received early treatment ( as the lung cancer group) , and 3 healthy subjects (as the control group). Serum samples were collected and total RNA was extracted using Trizol method. Using microRNA PCR ARRAY chip technology (product of Exiqon Company) , differentially miRNA expression profiling in serum between the lung cancer group and the control group, and between the treatment group and the lung cancer group were detected. Benefit miRNA expression profiling was ob- tained based on cluster analysis and comparative analysis. Results After tested by miRNA PCR ARRAY and managed by data analysis, a total of 42 miRNAs with more than 2 folds difference were screened in the lung cancer group and the control group, including 29 up-regulated and 12 down-regulated miRNAs. Be- sides, miR-10b-5p, miR-21-5p, miR-182-5p, miR-361-3p, and miR-382-5p were statistically different (P < 0. 05). A total of 45 miRNAs with more than 2 folds difference were screened in the treatment group and the lung cancer group, including 12 up-regulated and 33 down-regulated miRNAs. Fifteen miRNAs were statistically different including miR-137-3p, miR-182-5p, miR-376a-3p, miR-382-5p, miR-409-3p, miR-10a-5p, miR-21-5p, miR-29a-3p, miR-141-3p, miR-150-5p, miR-200c-3p, miR-342-3p, miR-365a-3p, miR-375, miR- 502-3p (P<0.05). Totally 22 miRNAs were screened in the treatment group with more than 2 folds differ- ence as compared with the lung cancer group and with less than or equivalent to 2 folds difference as com- pared with the control group, including 7 up-regulated and 15 down-regulated miRNAs, of which, miR-127- 3p, miR-182-5p, miR-382-5p, miR-409-3p, miR-10a-5p, miR-21-5p, miR-141-3p, miR-342-3p were statistically different (P <0. 05). Conclusion miRNAs including miR-21-5p, miR-182-5p, miR-382-5p are promising to become molecular markers for efficacy monitoring and prediction of advanced NSCLC treated by TCM combined WM, which provides reference for individualized treating advanced NSCLC.

Pancreatic carcinoma is one of the most malignant human cancers. In this study, we intended to explore the molecular functional of microRNA-127 (miR-127) in regulating pancreatic cancer development both in vitro and in vivo. Quantitative real-time PCR (qRT-PCR) was performed to evaluate endogenous miR-127 expression in in vitro pancreatic cancer cell lines and in vivo clinical samples of pancreatic carcinoma. Lentiviral technology was applied to overexpress miR-127 in capan-1 and PANC-1 cells. Pancreatic cancer proliferation, cell-cycle progression, and invasion were assessed in vitro, and capan-1-derived tumorigenicity was evaluated in vivo. Dual-luciferase reporter assay and qRT-PCR were performed to assess the downstream target gene of miR-127 in pancreatic cancer, human Bcl-2-associated athanogene 5 (BAG5). BAG5 was subsequently upregulated in miR-127-overexpressed capan-1 and PANC-1 cells to evaluate its effect on pancreatic cancer progression. MiR-127 was preferentially downregulated in both pancreatic carcinoma cell lines and human pancreatic tumors. In lentivirus-infected capan-1 and PANC-1 cells, miR-127 overexpression significantly inhibited cancer progression, cell-cycle transition and invasion in vitro, as well as tumorigenicity in vivo. Human BAG5 was confirmed to be the downstream target of miR-127 in pancreatic cancer. Forced overexpression of BAG5 in capan-1 and PANC-1 cells reversed the tumor-suppressing effect of miR-127 on cancer development. MiR-127 is downregulated and acting as a tumor suppressor in pancreatic carcinoma. The functional regulation of miR-127 in pancreatic carcinoma is very likely through the inverse correlation of its downstream target gene of BAG5.

BACKGROUND: Therapeutic management of Locally Advanced Rectal Cancer (LARC) involves pre-operative chemoradiotherapy (pCRT) followed by surgery. However, after pCRT the complete pathological response is approximately 20%, whereas in 20 to 40% of patients the response is poor or absent.
METHODS: Cancer biopsy specimens (n= 38) and serum samples (n= 34) obtained before pCRT from 38 LARC patients were included in the study. Patients were classified in responders (R, tumor regression grade [TRG] 1-2; n= 16) and non-responders (NR, TRG 3-5; n= 22) according to the pathological response observed upon surgery. We performed miRNA microarrays analysis on biopsy specimens, and validated the selected candidates both by qRT-PCR (tissue and serum) and by in situ hybridization (tissue, miR-125b) analyses.
RESULTS: Eleven miRNAs were significantly different between R and NR (miR-154, miR-409-3p, miR-127-3p, miR-214*, miR-299-5p and miR-125b overexpressed in NR; miR-33a, miR-30e, miR-338-3p, miR-200a and miR-378 decreased). In particular, miR-125b resulted to be the best candidate to discriminate the two groups (AUC of 0.9026; 95% CI, 0.7618-1.043). Additionally, miR-125b serum levels were significantly overexpressed in NR patients compared to R (p-value=0.0087), with an excellent discriminating power (AUC of 0.782; 95% CI, 0.6123-0.9518).
CONCLUSIONS: The obtained results further support the clinical impact of miRNA analysis. High miR-125b expression in tissue and serum were associated with a poor treatment response in LARC patients, therefore miR-125b could be considered as a possible novel non-invasive biomarker of response in LARC treatment.

The levels of expression of O6-methylguanine-DNA methyltransferase (MGMT) are relevant in predicting the response to the alkylating chemotherapy in patients affected by glioblastoma. MGMT promoter methylation and the published MGMT regulating microRNAs (miRNAs) do not completely explain the expression pattern of MGMT in clinical glioblastoma specimens. Here we used a genome-wide microarray-based approach to identify MGMT regulating miRNAs. Our screen unveiled three novel MGMT regulating miRNAs, miR-127-3p, miR-409-3p, and miR-124-3p, in addition to the previously identified miR-181d-5p. Transfection of these three novel miRNAs into the T98G glioblastoma cell line suppressed MGMT mRNA and protein expression. However, their MGMT- suppressive effects are 30-50% relative that seen with miR-181d-5p transfection. In silico analyses of The Cancer Genome Atlas (TCGA) and Chinese Glioma Genome Atlas (CGGA) revealed that miR-181d-5p is the only miRNA that consistently exhibited inverse correlation with MGMT mRNA expression. However, statistical models incorporating both miR-181d-5p and miR-409-3p expression better predict MGMT expression relative to models involving either miRNA alone. Our results confirmed miR-181d-5p as the key MGMT-regulating miRNA. Other MGMT regulating miRNAs, including the miR-409-3p identified in this report, modify the effect of miR-181d-5p on MGMT expression. MGMT expression is, thus, regulated by cooperative interaction between key MGMT-regulating miRNAs.

Deemed a putative tumor suppressor in breast cancer, the tight junction protein claudin 1 has now been shown to be highly expressed in the basal-like molecular subtype. Moreover, recent in vitro studies show that claudin 1 can regulate breast cancer cell motility and proliferation. Herein, we investigated whether microRNA (miRNA) dysregulation is associated with alterations in the level of claudin 1. Using next-generation sequencing (NGS), we identified seven miRNAs (miR-9-5p, miR-9-3p, let-7c, miR-127-3p, miR-99a-5p, miR-129-5p, and miR-146a-5p) that were deregulated as a consequence of claudin 1 overexpression in the MDA-MB231 human breast cancer (HBC) cell line. Most of these miRNAs have been associated with tumor suppression in a variety of cancers, including breast cancer. Moreover, through gene expression profiling analysis, we identified epithelial-mesenchymal transition-related genes, including platelet-derived growth factor receptor-beta (PDGFRB) and cadherin 1 (CDH1, E cadherin), whose downregulation correlated with claudin 1 overexpression. Collectively, we show for the first time that in HBC, claudin 1 can alter the dynamics of a number of miRNAs involved in tumor progression. Our data suggest that the dysregulated expression of these miRNAs, in conjunction with the high claudin 1 levels, could serve as a useful biomarker that identifies a subset of tumors within the poorly characterized basal-like subtype of breast cancer. Further studies are warranted to determine the role of these miRNAs in facilitating the function of claudin 1 in breast cancer.

miRNAs are small RNAs and control the expression of protein-encoding genes. The aim of this study was to determine the association between miRNA profile and clinical variables including age, stage, B symptom, histopathologic subtype, response to treatment, disease-free survival (DFS) and overall survival (OS) in classical Hodgkin lymphoma (cHL). A total of 377 miRNAs were studied by qPCR in 32 cases with cHL, and results were compared with 60 samples taken from cases with reactive lymphadenopathy. Biogazelle qbasePLUS 2.0 software was used to analyze the results. miR-582-3p, miR-525-3p, miR-448, miR-512-3p, miR-642a-5p, miR-876-5p, miR-532-3p, miR-654-5p, miR-128, miR-145-5p, miR-15b-5p, miR-328 and miR-660-5p were found to be decreased in cHL compared with controls. In contrast, miR-34a-5p (2.626-fold), miR-146a-5p (4.32-fold), miR-93-5p (2.347-fold), miR-20a-5p (4.930-fold), miR-339-3p (4.948-fold), miR-324-3p (4.98-fold), miR-372 (7.038-fold), miR-127-3p (8.234-fold), miR-155-5p (4.947-fold), miR-320a (17.502-fold) and miR-370 (21.479-fold) (p < 0.05) were found to be increased in cHL. There was no difference in miRNA profile according to the age, sex, stage, response to treatment, DFS and OS. However, miR-889 was found to be increased in patients with B symptom and miR-127-3p was found to be increased in nodular sclerosing subtype. Some miRNAs increase and some decrease in cHL. However, there was no clinical association between clinical variables and with the majority of the miRNA profile studied in this study. miR-889 and miR-127-3p were related to B symptom and nodular sclerosis subtype, respectively. We need more studies evaluating miRNA profile and clinical outcome in Hodgkin Lymphoma.

Overexpression of IQGAP1 and microRNA (miRNA) dysregulation are frequent in human tumors, but little is known about the role of IQGAP1 and its relationship to miRNA in endometrial carcinogenesis. We demonstrate that IQGAP1 activates the epithelial-mesenchymal transition (EMT) program and that miR-124 directly represses IQGAP1 expression in endometrial cancer (EC) cells. The overexpression of IQGAP1 stimulates EMT features and enhances migration, invasion and proliferation of EC cells, whereas knocking down IQGAP1 expression reverses EMT and inhibits these malignant properties. Using miRNA microarray profiling, we identified 29 miRNAs (let-7b, let-7f, miR-10b, miR-15b, miR-23a, miR-24, miR-25, miR-27a, miR-29b, miR-30a-5p, miR-34a, miR-124, miR-127, miR-130b, miR-148a, miR-155, miR-191*, miR-194, miR-224, miR-362, miR-409-3p, miR-422b, miR-424, miR-453, miR-497, miR-518d, miR-518f*, miR-526a and miR-656) that are significantly down-regulated in an in vitro-selected highly invasive derivative cell line (HEC-50-HI) relative to the parental HEC-50 cells. We further identified miR-124 as a direct regulator of IQGAP1 in EC cells. Enforced expression of miR-124 suppresses EC cell invasion and proliferation. The expression of IQGAP1 mRNA was significantly elevated in EC tissues, while the expression of miR-124 was decreased. The downregulation of miR-124 correlates with a poor survival outcome for patients with EC. Treating EC cells with the demethylating agent 5-aza-2'-deoxycytidine increased miR-124 expression and down-regulated IQGAP1 levels. Our data suggest that IQGAP1 promotes EMT, migration and invasion of EC cells. MiR-124, a novel tumor suppressor miRNA that is epigenetically silenced in EC, can reverse EMT and the invasive properties, by attenuating the expression of the IQGAP1 oncogene.

Nuclear factor-κB (NF-κB) activation is one of the major mediators of inflammation-induced cancer cell growth and progression. In previous studies, we screened a series of microRNAs (miRNAs) that targeted the NF-κB signaling pathway. In this study, we showed that miR-127-5p suppressed NF-κB activity through inhibition of p65 nuclear translocation. In addition, miR-127-5p also inhibited the transcription of downstream targets of the NF-κB signaling pathway. While exploring the mechanism of the inhibition of NF-κB activity by miR-127-5p, we found that miR-127-5p decreased the phosphorylation of p65. MicroRNA-127-5p inhibited the growth and colony formation of hepatocellular carcinoma (HCC) cells and decreased biliverdin reductase B (BLVRB) expression by directly binding to its 3'-UTR. RNA interference of BLVRB suppressed HCC cell growth, whereas the overexpression of BLVRB promoted HCC cell growth. Furthermore, BLVRB blockade inhibited the phosphorylation of p65 protein and the expression of downstream targets of the NF-κB signaling pathway, mimicking the function of miR-127-5p. The restoration of BLVRB in HCC cells overexpressing miR-127-5p impaired the suppression of HCC growth by miR-127-5p. Moreover, miR-127-5p was downregulated in 58% of HCC samples. In summary, we found that miR-127-5p suppressed NF-κB activity by directly targeting BLVRB in HCC cells, and this finding improves our understanding of the molecular mechanism of inflammation-induced HCC growth and proliferation and the successful inhibition of NF-κB activity by cancer treatment.

MicroRNAs (miRNAs) play an essential role in cancer development. Several studies have indicated that miRNAs mediate tumorigenesis processes, such as, inflammation, proliferation, apoptosis and invasion. In the present study, we focused on the influence of the miR-127-3p on the proliferation, migration and invasion of osteosarcoma (OS). MiR-127-3p was found at reduced levels in OS tissues and cell lines. Overexpression of miR-127-3p in the OS cell lines significantly inhibited the cell proliferation, migration and invasion; however, inhibition of miR-127-3p increased the proliferation, migration and invasion of OS in vitro. SETD8 was identified as a direct target of miR-127-3p, and SETD8 expression decreased post miR-127-3p overexpression, while SETD8 overexpression could reverse the potential influence of miR-127-3p on the migration and invasion of OS cells. MiR-127-3p is suggested to act mainly via the suppression of SETD8 expression. Overall, the results revealed that miR-127-3p acts as a tumor suppressor and that its down-regulation in cancer may contribute to OS progression and metastasis, suggesting that miR-127-3p could be a potential therapeutic target in the treatment of OS.

Although generally benign, giant cell tumors of bone (GCTB) display an aggressive behavior associated with significant bone destruction and lung metastasis in rare cases. This and the very high recurrence rate observed after surgical resection ranging from 20 to 55% necessitates the development of more effective treatment strategies. To identify valuable therapeutic targets, we screened a previously identified microRNA signature consisting of 23 microRNAs predominantly down-regulated in GCTB. We preselected eight candidate microRNAs and analyzed the impact of their restored expression on the neoplastic phenotype of GCTB stromal cells (GCTSC). A consistent and significant inhibition of cell proliferation, migration, colony formation and spheroid formation could be induced by transfection of primary GCTSC cell lines with miR-127-3p and miR-376a-3p, respectively. Genome wide expression analysis of miR-127-3p and miR-376a-3p transfected cells revealed four novel target genes for each microRNA. Luciferase reporter assays demonstrated direct interactions of miR-127-3p with COA1 and direct interaction of miR-376a-3p with GLE1 and PDIA6, suggesting a pivotal role of these genes in the molecular etiology of GTCB. Interestingly, both microRNAs are located within a chromosomal region frequently silenced in GCTB and many other types of cancers, indicating that these microRNAs and their target genes are valuable therapeutic targets for the treatment of GCTB and possibly other tumor entities.

Aberrant expression of miRNAs, cytokines and chemokines are involved in pathogenesis of colon cancer. However, the expression of p53 mediated miRNAs, cyto- and chemokines after radiation and SN38 treatment in colon cancer remains elusive. Here, human colon cancer cells, HCT116 with wild-type, heterozygous and a functionally null p53, were treated by radiation and SN38. The expression of 384 miRNAs was determined by using the TaqMan® miRNA array, and the expression of cyto- and chemokines was analyzed by Meso-Scale-Discovery instrument. Up- or down-regulations of miRNAs after radiation and SN38 treatments were largely dependent on p53 status of the cells. Cytokines, IL-6, TNF-α, IL-1β, Il-4, IL-10, VEGF, and chemokines, IL-8, MIP-1α were increased, and IFN-γ expression was decreased after radiation, whereas, IL-6, IFN-γ, TNF-α, IL-1β, Il-4, IL-10, IL-8 were decreased, and VEGF and MIP-1α were increased after SN38 treatment. Bioinformatic analysis pointed out that the highly up-regulated miRNAs, let-7f-5p, miR-455-3p, miR-98, miR-155-5p and the down-regulated miRNAs, miR-1, miR-127-5p, miR-142-5p, miR-202-5p were associated with colon cancer pathways and correlated with cyto- or chemokine expression. These miRNAs have the potential for use in colon cancer therapy as they are related to p53, pro- or anti-inflammatory cyto- or chemokines after the radiation and SN38 treatment.

The aim of this study was to investigate signaling pathways for reversal of microRNA-127-mediated multi-drug resistance (MDR) in gliomas cells. Adriamycin-resistant glioma cell lines U251/adr and U87-MG/adr were established and we found that anti-microRNA-127 markedly reduced microRNA-127 expression levels in a time-dependent manner, leading to distinct inhibition of cell proliferation and increased apoptosis and the content of intracellular Rh123. Silencing of microRNA-127 significantly increased the sensitivity of U251/ADR and U87-MG/adr cells to adriamycin, compared to cells transfected with negative control siRNA. Silencing of microRNA-127 also significantly reduced the mRNA and protein expression levels of MDR1 and MRP1, which are major ATP-binding cassette (ABC) transporter linked to multi-drug resistance in cancer cells. And Runx2, p53, bcl-2 and survivin, which are important role in cell apoptosis, also markedly changed after microRNA-127 silencing. In addition, down-regulating microRNA-127 decreased the level of phosphorylated-Akt. Our data indicate that down-regulation of micorRNA-127 can trigger apoptosis and overcome drug resistance of gliomas cells. Therefore, this resistance of adriamycin in gliomas can be cancelled by silencing expression of microRNA-127.