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Background Y‐box binding protein 1 (YB1 or YBX1) plays a critical role in tumorigenesis and cancer progression. However, whether YB1 affects malignant transformation by modulating non‐coding RNAs remains largely unknown. This study aimed to investigate the relationship between YB1 and microRNAs and reveal the underlying mechanism by which YB1 impacts on tumor malignancy via miRNAs‐mediated regulatory network. Methods The biological functions of YB1 in hepatocellular carcinoma (HCC) cells were investigated by cell proliferation, wound healing, and transwell invasion assays. The miRNAs dysregulated by YB1 were screened by microarray analysis in HCC cell lines. The regulation of YB1 on miR‐205 and miR‐200b was determined by quantitative real‐time PCR, dual‐luciferase reporter assay, RNA immunoprecipitation, and pull‐down assay. The relationships of YB1, DGCR8, Dicer, TUT4, and TUT1 were identified by pull‐down and coimmunoprecipitation experiments. The cellular co‐localization of YB1, DGCR8, and Dicer were detected by immunofluorescent staining. The in vivo effect of YB1 on tumor metastasis was determined by injecting MHCC97H cells transduced with YB1 shRNA or shControl via the tail vein in nude BALB/c mice. The expression levels of epithelial to mesenchymal transition markers were detected by immunoblotting and immunohistochemistry assays. Results YB1 promoted HCC cell migration and tumor metastasis by regulating miR‐205/200b‒ZEB1 axis partially in a Snail‐independent manner. YB1 suppressed miR‐205 and miR‐200b maturation by interacting with the microprocessors DGCR8 and Dicer as well as TUT4 and TUT1 via the conserved cold shock domain. Subsequently, the downregulation of miR‐205 and miR‐200b enhanced ZEB1 expression, thus leading to increased cell migration and invasion. Furthermore, statistical analyses on gene expression data from HCC and normal liver tissues showed that YB1 expression was positively associated with ZEB1 expression and remarkably correlated with clinical prognosis. Conclusion This study reveals a previously undescribed mechanism by which YB1 promotes cancer progression by regulating the miR‐205/200b‒ZEB1 axis in HCC cells. Furthermore, these results highlight that YB1 may play biological functions via miRNAs‐mediated gene regulation, and it can serve as a potential therapeutic target in human cancers., YB1 regulates miR‐205/200b‐ZEB1 axis by inhibiting microRNA maturation.

Previous evidence has highlighted M2 macrophage regulation of cancer cells via exosome shuttling of microRNAs (miRNAs or miRs). The current study set out to explore the possible role of M2 macrophage-derived exosomal miR-155-5p in regard to immune escape of colon cancer cells. Experimental data from quantitative reverse-transcriptase PCR (qRT-PCR) and western blot analysis revealed highly expressed miR-155-5p and interleukin (IL)-6 and poorly expressed ZC3H12B in M2 macrophage-derived exosomes. Additionally, miR-155-5p could be transferred by M2 macrophage-isolated exosomes to colon cancer cells, which targeted ZC3H12B by binding to the 3¢ UTR, as identified by dual luciferase reporter gene. Meanwhile, gain- and loss-of function experimentation on miR-155-5p and ZC3H12B in SW48 and HT29 cells cocultured with M2 macrophage-secreted exosomes demonstrated that miR-155-5p overexpression or ZC3H12B silencing promoted the proliferation and antiapoptosis ability of SW48 and HT29 cells, as well as augmenting the CD3+ T cell proliferation and the proportion of interferon (IFN)-γ+ T cells. Xenograft models confirmed that M2 macrophage-derived exosomal miR-155-5p reduced the ZC3H12B expression to upregulate IL-6, which consequently induced immune escape and tumor formation. Collectively, our findings indicated that M2 macrophage-derived exosomal miR-155-5p can potentially promote the immune escape of colon cancer by impairing ZC3H12B-mediated IL-6 stability reduction, thereby promoting the occurrence and development of colon cancer., Graphical Abstract, miR-155-5p was transferred by M2 macrophage-derived exosomes into colon cancer cells, where miR-155-5p targeted ZC3H12B, which in turn negatively regulated the stability of IL-6 mRNA. By this mechanism, miR-155-5p inhibited T cell immune response and thus promoted immune escape in colon cancer.

Polycystic ovary syndrome (PCOS) is a complex reproductive endocrine disease characterized by polycystic ovary. The aim of the study was to assess microRNA-21 regulates granulosa cell apoptosis and proliferation in polycystic ovary syndrome through target toll-like receptor 8. Granulosa cells were collected from 30 PCOS patients and 30 normal patients with tubal or male factor infertility (control) during in vitro fertilization-Embryo Transfer (IVF-ET) and were flash frozen with liquid nitrogen for storage for subsequent use. PCOS diagnosis was based on the revised standards of the American Society of Reproductive Medicine (ASRM) and the Rotterdam criteria PCOS granulosa cells and control granulosa cells were cultured in DMEM/F12 medium containing 10% fetal bovine serum and 1% antibiotic. After this RT-PCR, Western blot assessment and Detection of apoptosis by flow cytometry were conducted. The results of qPCR showed that the mRNA and protein expression of TLR8 in PCOS granulosa cells were significantly increased compared with the normal group. The results of Western blot also showed that the expression of TLR8, IFN-γ, TNF-α and IL-12 gene protein in the transfected cells was significantly higher than that in the control cells. Here, we show that miR-21 and TLR8 significantly increased in PCOS granulosa cell as compared with normal granulosa cells, and miR-21 enhances the TLR8 mRNA translation and then promotes the IFN-γ, TNF-α, and IL-12 secretion. Our study demonstrates that miR-21/ TLR8 involved in the PCOS inflammation, it provides profound insights into pathogenesis of PCOS.

The phenotypic switch in tumor-associated macrophages (TAMs) mediates immunity escape of cancer. However, the underlying mechanisms in the TAM phenotypic switch have not been systematically elucidated. In this study, long noncoding RNA (lncRNA)-Xist, CCAAT/enhancer-binding protein (C/EBP)α, and Kruppel-like factor 6 (KLF6) were upregulated, whereas microRNA (miR)-101 was downregulated in M1 macrophages-type (M1). Knockdown of Xist or overexpression of miR-101 in M1 could induce M1-to-M2 macrophage-type (M2) conversion to promote cell proliferation and migration of breast and ovarian cancer by inhibiting C/EBPα and KLF6 expression. Furthermore, miR-101 could combine with both Xist and C/EBPα and KLF6 through the same microRNA response element (MRE) predicted by bioinformatics and verified by luciferase reporter assays. Moreover, we found that miR-101 knockdown restored the decreased M1 marker and the increased M2 marker expression and also reversed the promotion of proliferation and migration of human breast cancer cells (MCF-7) and human ovarian cancer (OV) cells caused by silencing Xist. Generally, the present study indicates that Xist could mediate macrophage polarization to affect cell proliferation and migration of breast and ovarian cancer by competing with miR-101 to regulate C/EBPα and KLF6 expression. The promotion of Xist expression in M1 macrophages and inhibition of miR-101 expression in M2 macrophages might play an important role in inhibiting breast and ovarian tumor proliferation and migration abilities., Graphical Abstract, The phenotypic switch in tumor-associated macrophages (TAMs) mediates immunity escape of cancer. The present study indicates that Xist mediates macrophage polarization to affect cell proliferation and migration of breast and ovarian cancer by competing with miR-101 to regulate C/EBPα and KLF6 expression.

MicroRNA let‐7b is a potent tumor suppressor and targets crucial oncogenes. Previous studies have shown that let‐7b expression is suppressed in ovarian cancer; however, the regulatory mechanisms of let‐7b in ovarian cancer are still not well defined. The cellular role and targets of let‐7b in ovarian cancer remain elusive. In the present study, we showed that histone demethylase, KDM2B, directly suppressed let‐7b expression by H3K36me2 demethylation. Moreover, let‐7b inhibited EZH2 expression in ovarian cancer cells. Based on these results we know that let‐7b antagonizes the enhancement of EZH2 expression caused by KDM2B overexpression, and its expression is negatively correlated with KDM2B and EZH2 expression. More importantly, proliferation, migration, and wound healing assays showed that let‐7b inhibited ovarian cancer cell proliferation and migration in vitro. Additionally, let‐7b overexpression neutralized KDM2B‐promoted cell proliferation and migration. Furthermore, downregulation of let‐7b increased the xenografted tumor volumes in nude mice that were transplanted with KDM2B‐silenced cells. EZH2 silencing reversed the tumor growth enhancement mediated by inhibition of let‐7b. Last, we show that let‐7b expression is suppressed in ovarian carcinomas and its expression is negatively associated with the clinicopathological features of ovarian cancer, including histological type, histological grade, International Federation of Gynecology and Obstetrics (FIGO) stage, and lymph node metastatic status. In conclusion, in ovarian cancer, let‐7b expression is epigenetically suppressed by high expression of KDM2B. The loss of let‐7b upregulates the expression of EZH2, which promotes ovarian cancer growth in vitro and in vivo., In ovarian cancer, let‐7b expression is epigenetically suppressed by the high expression of KDM2B. The loss of let‐7b upregulates the expression of EZH2, which promotes ovarian cancer growth in vitro and in vivo.

Endometriosis is one of the most common gynecological diseases that adversely effects the lives of women. Our previous studies showed that LINC01541 plays a key role in 17β-estradiol (17β-E2)-stimulated endometrial stromal cells (ESCs); however, the mechanism by which LINC01541 exerts if effects requires further elaboration. Here, we report that LINC01541 serves to reduce the bioavailability of miR-506-5p by acting as a molecular sponge. Samples of control endometrial tissue and ectopic endometrial tissue were obtained from 10 healthy volunteers and 18 patients with endometriosis, respectively, and the levels of LINC01541 and miR-506-5p expressions in those tissues were measured. The relationship between LINC01541 and miR-506-5p was verified in 17β-E2-stimulated ESCs. Overexpression or silencing of miR-506-5p in ESCs was performed explore its role in endometriosis, and we also investigated whether WNT inhibitory factor 1 (WIF1) might be a target gene of miR-506-5p. Our results showed that LINC01541 was expressed at low levels and miR-506-5p was expressed at high levels in ectopic tissues. LINC01541 expression was negatively correlated with miR-506-5p expression. We also found that miR-506-5p activated the Wnt/β-catenin pathway by inhibiting WIF1 expression, and thereby induced the proliferation, migration, and invasion of ESCs. Furthermore, silencing of miR-506-5p promoted apoptosis and suppressed the proliferation of 17β-E2-treated ESCs. Overexpression of miR-506-5p could reverse the inhibitory effect of LINC01541 in endometriosis. In summary, this study found that in endometriosis, LINC01541 functions as a ceRNA that modulates the Wnt/β-catenin pathway by decoying miR-506-5p.

Background Coronary artery disease (CAD) leads to the highest mortality worldwide, seriously threatening human health. Tanshinone IIA (Tan IIA), which could be extracted from Danshen, is applied in the treatment of cardiovascular and cerebrovascular diseases. MicroRNAs (miRNAs, miRs) play pivotal roles in cell proliferation and cell apoptosis of the cardiovascular system. The aim of the present study was to explore the role of Tan IIA in CAD in vitro and the underlying molecular mechanism. Methods Real-time polymerase chain reaction (RT-PCR) and Western blot were used for the detection of miRNA/mRNA and protein, respectively. Target genes of miR-133a-3p were searched in TargetScan, and the targeting relationship was verified by dual-luciferase reporter assay. Cell proliferation was determined using a Cell Counting Kit-8 (CCK-8) and EdU labeling. Cell apoptosis was detected by flow cytometry and TUNEL staining. Results In the present study, lower miR-133a-3p level and higher epidermal growth factor receptor (EGFR; the target of miR-133a-3p) level were found in H2O2-induced H9c2 cells. In addition, Tan IIA upregulated miR-133a-3p and downregulated EGFR expression. Moreover, Tan IIA promoted cell proliferation and suppressed apoptosis and enhanced G0/G1, which was reversed by miR-133a-3p inhibitor, while siRNA-EGFR abolished the effects induced by miR-133a-3p in H2O2-induced H9c2 cells. Conclusion Tan IIA reversed H2O2-induced cell proliferation reduction, cell apoptosis induction, and G0/G1 arrest reduction in H9c2 cells by miR-133a-3p/EGFR axis. The findings suggested a potential molecular basis of Tan IIA in treating patients with CAD.

To identify potential therapeutic targets for pulmonary fibrosis induced by silica, we studied the effects of this disease on the expression of microRNAs (miRNAs) in the lung. Rattus norvegicus pulmonary silicosis models were used in conjunction with high-throughput screening of lung specimens to compare the expression of miRNAs in control and pulmonary silicosis tissues. A total of 70 miRNAs were found to be differentially expressed between control and pulmonary silicosis tissues. This included 41 miRNAs that were upregulated and 29 that were downregulated relative to controls. Among them, miR-292-5p, miR-155-3p, miR-1193-3p, miR-411-3p, miR-370-3p, and miR-409a-5p were found to be similarly altered in rat lung and transforming growth factor (TGF)-β1-induced cultured fibroblasts. Using miRNA mimics and inhibitors, we found that miR-1193-3p, miR-411-3p, and miR-370-3p exhibited potent anti-fibrotic effects, while miR-292-5p demonstrated pro-fibrotic effects in TGF-β1-stimulated lung fibroblasts. Moreover, we also found that miR-411-3p effectively reduced pulmonary silicosis in the mouse lung by regulating Mrtfa expression, as demonstrated using biochemical and histological assays. In conclusion, our findings indicate that miRNA expression is perturbed in pulmonary silicosis and suggest that therapeutic interventions targeting specific miRNAs might be effective in the treatment of this occupational disease., Graphical Abstract, Using high-throughput screening, Gao et al. found that 70 miRNAs were differentially expressed after chronic exposure to silica dust in rats. Moreover, they also showed that miR-411-3p could significantly attenuate pulmonary silicosis by inhibiting the Mrtfa gene. These results suggest that therapeutic interventions targeting specific miRNAs might be effective in the treatment of this occupational disease.

Evidence indicates that macrophages play an important role in the immune system. Therefore, research involving inflammatory and oxidative stress responses in macrophages is of great significance. Many factors contribute to inflammation and oxidative stress, including Salmonella. We investigated the effect of the miR-139-5p/TRAF6 axis on the inflammatory and oxidative stress responses of Salmonella -infected macrophages. Our findings revealed that miR-139-5p decreased IL-1β and TNF-α levels to inhibit Salmonella-induced inflammatory responses in the RAW264.7 macrophage cell line. Furthermore, miR-139-5p inhibited Salmonella-induced oxidative stress by strengthening SOD, CAT and GSH-PX activity, as well as lowering the malondialdehyde level in the RAW264.7 macrophages cell line. Subsequently, it was verified that TRAF6 was a downstream target of miR-139-5p in RAW264.7 cells. Rescue assays indicated that the over-expression of miR-139-5p inhibits the effects of TRAF6 on inflammatory and oxidative stress responses including Salmonella infection in RAW264.7 cells. To our knowledge, this study is the first to verify that miR-139-5p inhibits inflammatory and oxidative stress responses of Salmonella-infected macrophages through regulating TRAF6. This discovery may offer new insights on inflammatory and oxidative stress responses in macrophages.

Sperm motility is one of the most important indicators for evaluating roosters' fecundity. However, molecular regulation underlying chickens' sperm motility remains to be understood. MicroRNA (miRNA) plays an epigenetic role in reproduction. In this study, we compared the testicular miRNAs of Beijing-you roosters with high (HS) and low (LS) sperm motility using Illumina sequencing to try to understand the associated molecular regulation. Length distribution analysis showed that the dominant size of small RNAs detected in the testes of the chicken samples was 24-28 nt. In total, 518 known and 106 novel miRNAs were identified. A total of 23 miRNAs were found differentially expressed (P 0.05, |log

The human genome contains millions of putative regulatory elements, which regulate gene expression. We are just beginning to understand the functional consequences of genetic variation within these regulatory elements. Since the bulk of common genetic variation impacting polygenic disease phenotypes localizes to these non-coding regions of the genome, understanding the consequences will improve our understanding of the mechanisms mediating genetic risk in human disease. Here, we define the systemic lupus erythematosus (SLE) risk variant rs2431369 as likely causal for SLE and show that it is located in a functional regulatory element that modulates miR-146a expression. We use epigenomic analysis and genome-editing to show that the rs2431697-containing region is a distal enhancer that specifically regulates miR-146a expression in a cell-type dependent manner. 3D chromatin structure analysis demonstrates physical interaction between the rs2431697-containing region and the miR-146a promoter. Further, our data show that NF-kB binds the disease protective allele in a sequence-specific manner, leading to increased expression of this immunoregulatory microRNA. Our work provides a strategy for using disease-associated variants to define the functional regulatory elements of non-coding RNA molecules such as miR-146a and provides mechanistic links between autoimmune disease risk genetic variation and disease etiology.

IntroductionRecent studies have proved the diverse roles of miRs in different cancer-related processes. This study was undertaken to determine the therapeutic implications of miR-325-3p in breast cancer.Material and methodsExpression analysis was carried out by qRT-PCR. Transfections were performed by Lipofectamine 2000 reagent. MTT assay was used for cell viability. Transwell assays were used for cell migration and invasion. Western blot analysis was used for protein expression analysis.ResultsGene expression analysis revealed miR-325 to be significantly suppressed in breast cancer tissues and cell lines. Nonetheless, ectopic expression of miR-325 resulted in suppression of the growth and colony development potential of the SK-BR-3 and CAMA-1 cells. Transwell assays showed that miR-325 overexpression also resulted in the decline of the migration and invasion of the SK-BR-3 and CAMA-1 cells. Bioinformatic analysis showed that miR-325 targets lipocalin 15 (LNC15) in breast cancer cells. LNC15 was also overexpressed in the breast cancer tissues and cell lines. However, overexpression of miR-325 caused a significant decline in the LNC15 expression in SK-BR-3 cells. Additionally, silencing of LNC15 resulted in inhibition of the growth, migration and invasion of the SK-BR-3 cells. Rescue assay showed that overexpression of LNC15 could promote the growth, migration and invasion of the miR-325 overexpressing effects.ConclusionsTaken together, the evidence shows that miR-325 acts as a tumor suppressor in breast cancer and may be used in the treatment of breast cancer.

Occupational exposure to silica dust particles was the major cause of pulmonary fibrosis, and many miRNAs have been demonstrated to regulate target mRNAs in silicosis. In the present study, we found that a decreasing level of miR-411-3p in silicosis rats and lung fibroblasts induced by TGF-β1. Enlargement of miR-411-3p could inhibit the cell proliferation and migration in lung fibroblasts with TGF-β1 treatment and attenuate lung fibrosis in silicotic mice. In addition, a mechanistic study showed that miR-411-3p exert its inhibitory effect on Smad ubiquitination regulatory factor 2 (Smurf2) expression and decrease ubiquitination degradation of Smad7 regulated by smurf2, result in blocking of TGF-β/Smad signaling. We proposed that increased expression of miR-411-3p abrogates silicosis by blocking activation of TGF-β/Smad signaling through decreasing ubiquitination degradation effect of smurf2 on Smad7.

Several microRNAs (miRNAs) have been closely correlated with the development of hepatocellular carcinoma (HCC). However, the involvement of miR-300 in the development of HCC remains unknown. This study elucidated the potential molecular mechanisms of miR-300 in the modulation of the epithelial-mesenchymal transition (EMT) and invasion of HCC. The expression levels of miR-300 in HCC cells and clinical samples were detected by quantitative real-time PCR and in situ hybridization. The in vitro function of miR-300 in HCC was evaluated using a migration/invasion assay. Quantitative real-time PCR, western blotting, immunofluorescence and immunohistochemistry were used to validate the roles of miR-300 and FAK/PI3K/AKT in EMT progression. A dual-luciferase reporter assay was performed to confirm the target gene. miR-300 was down-regulated in HCC and significantly correlated with a poor prognosis in HCC patients. The down-regulation of miR-300 increased the invasiveness of the HCC cells, and promoted the EMT in both HCC tissues and HCC cells. In contrast, up-regulation of miR-300 led to the opposite results. Ectopic overexpression of miR-300 reversed TGF-β1-induced EMT in SMMC-7721 cells, and according to a dual-luciferase reporter assay and rescue assay, miR-300 inhibits the EMT-mediated migration and invasion of HCC cells via the targeted modulation of FAK and the downstream PI3K/AKT signaling pathway. miR-300 targeting modulates FAK, and the PI3K/AKT signaling pathway inhibits the EMT and suppresses the migration and invasion of HCC cells. Thus, miR-300 represents a promising therapeutic target for HCC.

Hong Xu,1–3,* Yuting Wu,2–4,* Li Li,1–3 Weifeng Yuan,1–3 Deming Zhang,2–4 Qitao Yan,2,3 Zhenhui Guo,2,3 Wenjie Huang1–3 1Department of RespiratoryMedicine, 2Guangdong Provincial Key Laboratory of Geriatric Infection andOrgan Function Support, 3Guangzhou Key Laboratory of Geriatric Infection andOrgan Function Support, 4Department of Geriatric RespiratoryMedicine, General Hospital of Guangzhou Command of PLA, Guangzhou, Guangdong, People’sRepublic of China *These authors contributed equally tothis work Objectives: COPD is an abnormal inflammatory response characterized by decreased expression of TLR2 in patients, which is suggested to induce invasive pulmonary aspergillosis (IPA). MicroRNAs (miRNAs) have been shown to play important roles in the pathogenesis of human respiratory system disorders. Therefore, the aim of this study was to identify the miRNAs involved in the regulation of TLR2 signaling in COPD. Materials and methods: miRNA microarray analysis was performed to screen for the dysregulated miRNAs in alveolar macrophages (AMs) isolated from COPD rats. The interaction between these miRNAs and TLR2 gene was predicted using miRBase and validated using dual luciferase assay. Based on the analysis, a novel miR-344b-1-3p was identified as a novel modulator of TLR2 gene. Then, the mechanism through which miR-344b-1-3p regulated TLR2 expression was explored using cigarette smoke extract (CSE)-pretreated NR8383 cells. Moreover, by subjecting CSE-pretreated NR8383 cells to Pam3CSK4, the effect of miR-344b-1-3p on NF-κB activity and other important mediators of COPD, including IRAK-1, ERK, TNF-α, IL-1β, and MIP-2, was also assessed. Results: COPD rat model was successfully induced by smoke inhalation. Among the 11 upregulated miRNAs in AMs from COPD rats, miR-344b-1-3p was predicted to be a novel miRNA targeting TLR2 gene. In the CSE pretreated NR8383 cells exposed to Pam3CSK4, miR-344b-1-3p inhibition increased the expression levels of TLR2, TNF-α, and IL-1β and decreased the expression levels of MIP-2. In addition, the phosphorylation of IRAK-1, IκBα, and IRK was augmented by miR-344b-1-3p inhibition. Conclusion: Findings outlined in this study suggest that miR-344b-1-3p was an effective modulator of TLR2 gene, which can be employed as a promising therapeutic and preventive target of IPA in COPD patients. Keywords: COPD, miR-344b-1-3p, TLR2, invasive pulmonary aspergillosis

MicroRNA-24 (miR-24), a member of the miRNA family, functions as an oncogene in various types of human cancer. However, the underlying mechanisms of miR-24 involvement in the development and progression of hepatocellular carcinoma (HCC) remain poorly understood. The present study revealed that miRNA-24 down-regulates p53 through binding to the 3'-UTR of p53 mRNA based on a luciferase reporter assay, and that the expression level of miR-24 could affect the invasion of HCC lines via p53. Down-regulation of p53 significantly attenuated the inhibitory effects of miR-24 knockdown on the invasion of HCC cells, suggesting that miR-24 could be a potential target for HCC treatment. Moreover, our results revealed that miR-24 expression was significantly increased in HCC metastatic tumor tissues compared with matched non-metastatic tumor tissues, and that the up-regulation of miR-24 was significantly associated with down-regulation of p53 in the HCC tissues. In conclusion, this study demonstrates that miR-24 functions as an oncogene in HCC, at least partly by promoting cell invasion through down-regulation of p53. Therefore, miR-24 may be a potential therapeutic target for treatment of HCC.

Hong Chen,1 Hong Xu,1 Yu-gang Meng,1 Yun Zhang,2 Jun-ying Chen,1 Xiao-ning Wei1 1Department of Gynaecology, The First Affiliated Hospital of Guangxi Medical University, Nanning, Guangxi, 2Department of Gynaecology, The People’s Hospital of Suzhou High Tech District, Suzhou, Jiangsu, People’s Republic of China Background: Uterine leiomyoma is one of the most common benign tumors in women. It dramatically decreases the quality of life in the affected women. However, there is a lack of effective treatment paradigms. Micro-RNAs are small noncoding RNA molecules that are extensively expressed in organisms, and they are interrelated with the occurrence and development of the tumor. miR-139-5p was found to be downregulated in various cancers, but its function and mechanism in uterine leiomyoma remain unknown. The aim of this study was to investigate the role of miR-139-5p and its target gene in uterine leiomyoma.Methods: By using a bioinformatic assay, it was found that TPD52 was a potential target gene of miR-139-5p. Then, expressions of miR-139-5p and TPD52 in uterine leiomyoma and adjacent myometrium tissues were evaluated by quantitative real-time polymerase chain reaction and Western blot. Proliferation, apoptosis, and cell cycle of uterine leiomyoma cells transfected by miR-139-5p mimics or TPD52 siRNA were determined.Results: It was observed that the expression of miR-139-5p in uterine leiomyoma tissues was significantly lower (P

Gastric cancer is a common malignant tumor. Studies from our laboratory or others have shown that long non-coding RNA (lncRNA) zinc finger antisense (ZFAS)1 often acts as an oncogene. However, the molecular underpinnings of how ZFAS1 regulates gastric cancer remain to be elucidated. Results showed that ZFAS1 expression was upregulated, and microRNA-200b-3p (miR-200b) expression was downregulated in gastric cancer tissues. MiR-200b overexpression suppressed the proliferation, cell cycle process, and Wnt/β-catenin signaling of gastric cancer cells. Subsequently, we identified miR-200b is a target of ZFAS1 and Wnt1 is a target of miR-200b. Furthermore, promotion of cancer malignant progression and activation of Wnt/β-catenin signaling induced by ZFAS1 was counteracted by increasing miR-200b expression. In vivo, ZFAS1 knockdown suppressed the tumorigenesis with the upregulated miR-200b and the inactive Wnt/β-catenin signaling. Summarily, we demonstrated a critical role of miR-200b in gastric cancer, and ZFAS1 can promote malignant progression through regulating miR-200b mediated Wnt/β-catenin signaling.

There is growing evidence supporting dysregulated microRNAs (miRNAs) as potential prognostic biomarkers in cancer. The present study aimed to identify an miRNA model set with prognostic power for patients with gastric adenocarcinoma. miRNA‑seq data from 155 patients and 37 controls were downloaded from The Cancer Genome Atlas (TCGA) database for a comprehensive analysis of miRNA expression profiles and were used as training data. A total of 5 prognostic miRNAs, which have not been previously reported, were identified using univariate and multivariate Cox regression analyses. A separate 155‑patient TCGA cohort was used as a validation set for evaluation of the risk model. Patients in the training set were assigned into high‑ and low‑risk groups according to the 5‑miRNA signature risk scores. Kaplan‑Meier survival analyses demonstrated that patients with high risk scores had significantly shorter survival times than those with low risk scores. The risk model validation confirmed the prognostic ability of this 5‑miRNA signature in predicting the risk status of patients. Stratification analysis for clinical prognostic variables demonstrated recurrence and age were significant prognostic factors in the low‑ and high‑risk groups, respectively. In conclusion, the present 5‑miRNA signature is a potential independent risk factor for patient outcomes. The risk model based on the 5‑miRNA signature performed well in predicting overall survival time in patients with gastric adenocarcinoma.

Objective: Abnormal proliferation or migration of vascular smooth muscle cells (VSMCs) can lead to vessel lesions, resulting in atherosclerosis and in stent-restenosis (IRS). The purpose of our study was to establish the role of miR-378a-5p and its targets in regulating VSMCs function and IRS. Methods: EdU assays and Cell Counting Kit-8 (CCK-8) assays were applied to evaluate VSMCs proliferation, wound healing assays and transwell assays were applied to assess cells migration. Furthermore, quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was performed to investigate the expression level of miR-378a-5p IRS patients and healthy individuals. Target genes were predicted using Target Scan and miRanda software, and biological functions of candidate genes were explored through bioinformatics analysis. Moreover, RNA-binding protein immunoprecipitation (RIP) was carried out to analyze the miRNAs interactions with proteins. We also used Immunofluorescence (IF) and fluorescence microscopy to determine the binding properties, localization and expression of miR-378a-5p with downstream target CDK1. Results: The expression of miR-378a-5p was increased in the group with stent restenosis compared with healthy people, as well as in the group which VSMCs stimulated by platelet-derived growth factor-BB (PDGF-BB) compared with NCs. MiR-378a-5p over-expression had significantly promoted proliferative and migratory effects, while miR-378a-5p inhibitor suppressed VSMC proliferation and migration. CDK1 was proved to be the functional target of miR-378a-5p in VSMCs. Encouragingly, the expression of miR-378a-5p was increased in patients with stent restenosis compared with healthy people, as well as in PDGF-BB-stimulated VSMCs compared with control cells. Furthermore, co-transfection experiments demonstrated that miR-378a-5p over-expression promoted proliferation and migration of VSMCs specifically by reducing CDK1 gene expression levels. Conclusion: In this investigatory, we concluded that miR-378a-5p is a critical mediator in regulating VSMC proliferation and migration by targeting CDK1/p21 signaling pathway. Thereby, interventions aimed at miR-378a-5p may be of therapeutic application in the prevention and treatment of stent restenosis.

// Jing-Song Chen 1 , Li-Li Liang 2 , Hong-Xu Xu 3 , Fan Chen 4 , Shun-Li Shen 5 , Wei Chen 5 , Lian-Zhou Chen 4 , Qiao Su 6 , Long-Juan Zhang 4 , Jiong Bi 4 , Wen-Tao Zeng 4 , Wen Li 4 , Ning Ma 7 and Xiao-Hui Huang 4 1 Department of Gastrointestinal Surgery, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou, People’s Republic of China 2 Departments of Pediatrics, The First Affiliated Hospital of Sun Yat-Sen University, Guangzhou, China 3 Departments of Laboratory, The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou, China 4 Departments of General Surgical Laboratory, The First Affiliated Hospital of Sun Yat-Sen University, Guangzhou, China 5 Departments of Hepatobiliary Surgery, The First Affiliated Hospital of Sun Yat-Sen University, Guangzhou, China 6 Animal Center, The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou, China 7 Department of Gastrointestinal, Hernia and Abdominal Surgery, The Sixth Affiliated Hospital, Sun Yat-Sen University, Guangzhou, China Correspondence to: Ning Ma, email: maning85322@163.com Xiao-Hui Huang, email: hxiaohui2006@126.com Keywords: miR-338-3p, epithelial-mesenchymal transition, Snail1, N-cadherin, sonic hedgehog Received: November 25, 2015 Accepted: June 04, 2016 Published: June 17, 2016 ABSTRACT Down-regulation of the miRNA miR-338-3p correlates with the invasive ability of hepatocellular carcinoma (HCC) cells. However, it is currently unclear whether down-regulation of miR-338-3p induces epithelial-mesenchymal transition (EMT), which may be the underlying mechanism governing HCC invasion. Here, we demonstrate that restoration of miR-338-3p expression via transfection of a miR-338-3p mimic reversed EMT and inhibited the motility and invasiveness of HCC cells. Conversely, silencing of endogenous miR-338-3p expression with a miR-338-3p-specific inhibitor induced EMT and enhanced HCC cell motility. Additionally, Snail1 (an upstream regulatory protein of EMT) and Gli1 (a key transcription factor in the sonic hedgehog (SHH) signaling pathway) expression was up-regulated in cells treated with the miR-338-3p inhibitor and down-regulated by the miR-338-3p mimic. Further analyses demonstrated that miR-338-3p inhibitor-induced EMT in HCC cells was blocked by treatment with a small interfering RNA (siRNA) targeting Snail1, that the SHH signaling pathway was required for both miR-338-3p inhibitor-induced EMT and up-regulation of Snail1, and that miR-338-3p targeted a sequence within the 3´-untranslated region of N-cadherin mRNA. Notably, miR-338-3p expression was significantly down-regulated in HCC samples from patients with metastases and was associated with poor metastasis-free survival rates. Lastly, correlations between the expression levels of miR-338-3p and E-cadherin, Smoothened (SMO), Gli1, Snail1, N-cadherin, and vimentin were confirmed in HCC xenograft tumors and HCC patient specimens. Our findings suggest that miR-338-3p suppresses EMT and metastasis via both inhibition of the SHH/Gli1 pathway and direct binding of N-cadherin. miR-338-3p is a potential therapeutic target for metastatic HCC.

As important disease diagnostic markers, circulating miRNAs have been put on an urgent agenda in recent years with the focus on their highly sensitive and specific multiplex detection in one reaction. In this article, we proposed a unique miRNAs detection method based on tailored-designed stem-loop structure ligation strategy to realize ideal detection performance. The stem-loop ligation probe had a target miRNA mediated quick ligation, which the relative ligation efficiency of stem-loop structure was superior evidently to straight chain structure about 76% at 10 min on account of the preferentially formed partial dimer. Moreover, the streptavidin-coated magnetic microspheres combined with optimized ligation probes concentration at 10 nM were utilized to purify ligation products and completely eliminated nonspecific ligation substantially. Due to the stem-loop structure ligation products contained universal primer regions, let-7 family as model was used to evaluate the detection performance, demonstrating high sensitivity as the minimum detection limit of 2.5 fM with a five-orders of magnitude dynamic detection range. To some degree, the stem-loop structure-based ligation may inspire the flexible design strategy in the future application and provide a significant quick and efficient platform to miRNAs detection.

Purpose : To investigate the anti-tumor effect of microRNA-194 (miR-194) and possible relationship with profilin 2. Methods : The expressions of miR-194 and profilin 2 were investigated via bioinformatic analysis and were further verified using quantitative real-time polymerase chain reaction (qRT-PCR) in both normal and non-small cell lung cancer (NSCLC) cells. Dual luciferase reporter assay, together with transfection, was applied to determine the effect of miR-194 and profilin 2 on proliferation, migration and invasion, and its underlying mechanisms. Results : Relatively low expression of miR-194 and high expression of profilin 2 in cancerous lung tissue were found through bioinformatic analysis and further confirmed by qRT-PCR on normal and NSCLC cell lines. Profilin 2 was the direct target of miR-194. Also, miR-194 overexpression inhibited the proliferation, migration and invasion of NSCLC cells, and this suppressive effect was partially reversed by transfection of pcDNA3.1-PFN2 plasmid. Conclusion: The results show that miR-194 plays a tumor suppressor role in NSCLC development. It inhibits the proliferation, migration and invasion of NSCLC cells via profilin 2 suppression. Interventions targeting the miR-194/profilin 2 axis may therefore serve as a clinical strategy for the prevention and treatment of NSCLC metastases. Keywords: Non-small cell lung cancer, MicroRNA-194, Profilin 2, Proliferation, Migration, Invasion

Ovarian cancer has gradually become one of the commonest gynecological tumor in the world. Although various therapies have been developed by researchers, the chemoresistance of ovarian cancer is still a huge challenge. MircroRNAs (miRNAs) have been widely studied due to their anti-oncogenic functions. MiR-378a-3p has been reported to sensitize breast cancer cells to chemotherapy. Here, we hypothesized that miR-378a-3p is a potential chemosensitizer in ovarian cancer. Firstly, miR-378a-3p was uncovered to down-regulated in ovarian cancer tissues and cell lines through using qRT-PCR analysis and northern blot analysis. According to the result of Kaplan Meier analysis, low expression of miR-378a-3p is closely associated with unfavorable prognosis of ovarian cancer patients. Subsequently, gain-of function assays indicated that miR-378a-3p suppressed cell proliferation and promoted cell apoptosis. Moreover, miR-378a-3p was found to enhance cisplatin sensitivity of ovarian cancer cells. Mechanism investigations suggested that MAPK1 and GRB2 are two targets of miR-378a-3p. Finally, rescue assays revealed that MAPK1 and GRB2 can reverse the effects of miR-378a-3p on chemosensitivity of ovarian cancer cells. In conclusion, miR-378a-3p enhanced the sensitivity of ovarian cancer cells to cisplatin through targeting MAPK1 and GRB2.

Intention Long noncoding RNAs, transcribed from a recently discovered class of noncoding genes, may play a critical role in regulating cellular processes, such as cell proliferation and apoptosis, as well as in cancer progression and metastasis. We previously detected the induction of growth arrest-specific 5 (GAS5) during glioma cell death. However, the function and underlying mechanism of GAS5 in human gliomas remain to be elucidated. Methods Cell proliferation was detected using CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay (MTS) and tumorigenicity assay in nude mice. Wound-healing assay and transwell assay were utilized to examine the effects of GAS5 expression on glioma cells migration and invasion. In situ hybridization (ISH) was performed to evaluate GAS5 and microRNA (miR)-18a-5p levels in tissue microarrays. The relationship between GAS5 and miR-18a-5p was evaluated by quantitative reverse-transcription polymerase chain reaction and RNA precipitation. Results In this study, we demonstrated that overexpression of GAS5 inhibits malignant phenotypes in glioma cells, including proliferation, migration, and invasion, whereas GAS5 knockdown enhances these phenotypes. We further observed Argonaute 2-dependent reciprocal repression between GAS5 and miR-18a-5p in glioma cells. Downregulation of GAS5 and upregulation of miR-18a-5p were observed in glioma tissue microarrays relative to normal brain tissue by ISH. By deletion analysis, we identified one miR-18a-5p-binding site within exon 2 of GAS5 that is partially responsible for the tumor-suppressor functions of GAS5. Conclusion Taken together, our findings suggest that GAS5 is a tumor suppressor in human gliomas that acts in part by repressing miR-18a-5p.

The aim of the present study was to screen out the biomarkers associated with chemoresistance in ovarian carcinomas and to investigate the molecular mechanisms. microRNA (miRNA) expression data was obtained from published microarray data of the GSE43867 dataset from Gene Expression Omnibus (GEO), including the data of 86 chemotherapy-treated patients with serous epithelial ovarian carcinomas (response group, 36 complete response cases and 12 partial response cases; non-response group, 10 stable cases and 28 progressive disease cases), and identification of differentially-expressed miRNAs were conducted with a GEO2R online tool based on R language. TargetScan 6.2 was used to predict the targets of differentially-expressed miRNAs. Protein-protein interaction network analysis was conducted by STRING 9.1, while functional enrichment [Gene Ontology (GO) biological process terms] and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were conducted by GeneCodis3 for the target genes. A total of 6 differentially-expressed miRNAs were screened out, with 317 target genes obtained. It was found that 67 interactions existed among 76 genes/proteins through the PPI network analysis, and that 6 of these were potential key genes (PIK3R5, MAPK3, PTEN, S1PR3, BDKRB2 and NCBP2). The main biological processes involved in chemoresistant ovarian carcinoma were apoptosis, programmed cell death, cell migration, cell death and cell motility. The miRNA target genes were found to be associated with the ErbB signaling pathway, the gonadotropin-releasing hormone signaling pathway and other pathways in cancer. IK3R5, MAPK3 and PIK3R5 are involved in the majority of GO terms and KEGG pathways associated with chemoresistance in ovarian carcinoma.

Yanmei Qi,1 Fengqiang Zhou,2 Lu Zhang,2 Lei Liu,2 Hong Xu,2 Huiguang Guo2 1Department of Gastroenterology, 2Department of General Surgery, Binzhou People’s Hospital, Binzhou, Shandong, People’s Republic of China Background: Prior study indicates that abnormal protein expression and functional changes in the development and progression of colorectal cancer is related to gene expression. The aim of this study was to construct an interference plasmid targeting the Ep-CAM gene and to investigate its effects on the proliferation of colorectal cancer cells. Methods: In this study, HT-29 and HCT-116 colorectal cancer cell lines were selected as cell models. The double-stranded micro (mi)RNA oligo was inserted into the pcDNATM6.2-GW/EmGFPmiR vector, which is an expression of miRNA. Lipofectamine™ 2000 was used to transfer plasmid into the empty plasmid group (transfected pcDNATM6.2-GW/EmGFPmiR-neg) and the interference group (transfected pcDNATM6.2-GW/EmGFPmiR-Ep-CAM-1), respectively. Meanwhile, the nontransferred HT-29 and HCT-116 acts as the blank control group. Reverse transcription polymerase chain reaction (RT-PCR) was used to detect the transfection efficiency. Western blot was used to detect Ep-CAM protein expression. The cell proliferation in each group was detected by using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Results:The results indicated that the Ep-CAM messenger (m)RNA expression in the interference group was lower significantly compared with that of the empty plasmid group and control group (P

Although microRNAs (miRs) have been implicated in the pathogenesis of various human malignancies, limited information is available regarding mechanisms by which these noncoding RNAs contribute to initiation and progression of tobacco-induced esophageal cancers. In this study, array and quantitative reverse transcriptase-PCR techniques were used to examine miR expression in immortalized esophageal epithelia (IEE) and esophageal adenocarcinoma (EAC) cells cultured in normal media with or without cigarette smoke condensate (CSC). Under relevant exposure conditions, CSC significantly decreased miR-217 expression in these cells. Endogenous levels of miR-217 expression in cultured EAC cells (EACC)/primary EACs were significantly lower than those observed in IEE/ paired normal esophageal tissues. RNA crosslink immunoprecipitation, quantitative reverse transcriptase-PCR (qRT-PCR) and immunoblot experiments demonstrated direct interaction of miR-217 with kallikrein 7 (KLK7), encoding a putative oncogene not previously implicated in EAC. Repression of miR-217 correlated with increased levels of KLK7 in primary EACs, particularly those from smokers. Chromatin and methylated DNA immunoprecipitation experiments demonstrated that CSC-mediated repression of miR-217 coincided with DNMT3b-dependent hypermethylation and decreased occupancy of nuclear factor 1 within the miR-217 genomic locus. Deoxyazacytidine induced miR-217 expression and downregulated KLK7 in EACC; deoxyazacytidine also attenuated CSC-mediated miR-217 repression and upregulation of KLK7 in IEE and EACC. Overexpression of miR-217 significantly decreased, whereas overexpression of KLK7 increased proliferation, invasion and tumorigenicity of EACC. Collectively, these data demonstrate that epigenetic repression of miR-217 contributes to the pathogenesis of EAC via upregulation of KLK7 and suggest that restoration of miR-217 expression may be a novel treatment strategy for these malignancies.

Background Hypoxia plays an important role in the development of various cancers. MicroRNAs (miRNAs) act as post-transcriptional regulators of gene expression and modulate the tumorigenesis, including gastric cancer. However, the roles and molecular mechanism of miR-224 in gastric cancer under hypoxia remain poorly understood. Method Real-time PCR and Northern blot assay were used to examine the effects of hypoxia and HIF-1α on miR-224 expression. Luciferase and ChIP assays were performed to determine whether miR-224 was a transcriptional target of HIF-1α. Then MTT, colony formation, in vitro scratch and invasion assays were used to detect the effects of miR-224 on cell growth, migration and invasion under hypoxia, as well as the in vivo animal study. Luciferase assay and Western blot were performed to validate the targets of miR-224. Functional studies were performed to determine the roles of RASSF8 as that of miR-224 under hypoxia. The effects of RASSF8 knockdown on the transcriptional activity and translocation of NF-κB were investigated using Luciferase assay and Western blot, respectively. Finally, the expression levels of miR-224 and RASSF8 were detected using real-time PCR in gastric cancer tissues as well as lymph node metastasis tissues. Results We demonstrated that miR-224 was upregulated by hypoxia and HIF-1α. HIF-1α affected miR-224 expression at the transcriptional level. MiR-224 inhibition suppressed cell growth, migration and invasion induced by hypoxia, while miR-224 overexpression resulted in opposite effects. MiR-224 inhibition also suppressed tumor growth in vivo. We then validated that RASSF8 was a direct target of miR-224. RASSF8 overexpression inhibited cell growth and invasion, while RASSF8 knockdown ameliorated the inhibitory effects of miR-224 inhibition on cell growth and invasion. Furthermore, we found that RASSF8 knockdown enhanced the transcriptional activity of NF-κB and p65 translocation, while RASSF8 overexpression resulted in opposite effects. Inhibition of NF-κB activity by PDTC attenuated the effects of RASSF8 knockdown on cell proliferation and invasion. Finally, miR-224 was upregulated in both gastric cancer tissues and lymph node metastasis positive tissues, while RASSF8 expression was opposite to that of miR-224. Conclusion These results indicate that hypoxia-inducible miR-224 promotes gastric cancer cell growth, migration and invasion by downregulating RASSF8 and acts as an oncogene, implying that inhibition of miR-224 may have potential as a therapeutic target for patients with hypoxic gastric tumors.

Central nervous system (CNS) is an increasingly common site of isolated metastasis for patients with Stage 4 neuroblastoma. To explore the microRNA (miRNA) profile of this metastatic process, miRNA sequencing was performed to identify miRNA sequence families with differential expression between tumor pairs (pre-CNS primary and CNS metastasis) from 13 patients with Stage 4 neuroblastoma. Seven miRNA sequence families had distinct expression in CNS metastases when compared with their corresponding pre-CNS primaries. MiR-7 was upregulated (3.75-fold), and miR-21, miR-22, miR-29a, miR-143, miR-199a-1-3p, and miR-199a-1-5p were downregulated (3.5-6.1-fold), all confirmed by quantitative reverse transcription-PCR. MiR-29a, previously shown to be downregulated in a broad spectrum of solid tumors including neuroblastoma, had the most significant decrease in all 13 CNS metastases (P = 0.001). Its known onco-targets CDC6, CDK6, and DNMT3A, as well as B7-H3, an inhibitory ligand for T cells, and natural killer cells, were found to have higher differential expression in these 13 CNS metastases when compared with their paired primaries. Additionally, miR-29a expression in primary tumors was significantly lower among patients who eventually relapsed in the CNS. Irrespective of the amplification status of MYCN, which is known to be associated with metastasis, pre-CNS primaries, and CNS metastases had significantly lower miR-29a expression than non-CNS primary tumors. Among MYCN amplified cell lines, those from CNS relapse also had lower miR-29a expression than non-CNS relapse. These findings raised the hypothesis that miR-29a could be a biomarker for neuroblastoma CNS metastasis, and its downregulation may play a pivotal role in CNS progression.

microRNA-944 (miR-944) has been reported to be deregulation and play either tumor suppressive or oncogenic function in human malignancies. Previous studies have reported that miR-944 is overexpressed in gynecological malignancies including cervical cancer and breast cancer. While, the clinical significance of miR-944 and its function in human endometrial carcinoma (EC) keep unknown. The levels of miR-944 were analyzed in 68 EC tissues and 20 normal endometrial tissues. Results showed that miR-944 was significantly overexpressed in EC tissues compared to normal endometrial tissues. In addition, increased levels of miR-944 also observed in EC cell lines. Clinicopathological analysis verified that miR-944 expression was associated with international federation of gynecology and obstetrics (FIGO) stages and pathology classification of EC. Moreover, Kaplan-Meier analysis found that miR-944 highly expressing EC patients showed a notable shorter survival. Further experiments revealed that miR-944 knockdown significantly inhibited growth and cell cycle progression while facilitated apoptosis in Ishikawa cells. In turn, miR-944 overexpression prominently promoted proliferation and cell cycle progression, and suppressed apoptosis in KLE cells. In vivo experiments further confirmed that miR-944 silencing notably restrained the tumor growth of EC in nude mice. Mechanically, cell adhesion molecule 2 (CADM2) was recognized as a direct downstream target of miR-944 in EC cells. An inverse correlation between miR-944 and CADM2 expression was observed in EC tissues. Interestingly, CADM2 overexpression showed similar effects to miR-944 knockdown in Ishikawa cells with decreased growth, cell cycle arrest at G1 phase and increased apoptosis. Taken together, this work support the first evidence that miR-944 can be potentially used as a promising biomarker and novel therapeutic target for human EC.

MicroRNAs are critical mediators of stem cell pluripotency, differentiation, and malignancy. Limited information exists regarding microRNA alterations that facilitate initiation and progression of human lung cancers. In this study, array techniques were used to evaluate microRNA expression in normal human respiratory epithelia and lung cancer cells cultured in the presence or absence of cigarette smoke condensate (CSC). Under relevant exposure conditions, CSC significantly repressed miR-487b. Subsequent experiments demonstrated that miR-487b directly targeted SUZ12, BMI1, WNT5A, MYC, and KRAS. Repression of miR-487b correlated with overexpression of these targets in primary lung cancers and coincided with DNA methylation, de novo nucleosome occupancy, and decreased H2AZ and TCF1 levels within the miR-487b genomic locus. Deoxy-azacytidine derepressed miR-487b and attenuated CSC-mediated silencing of miR-487b. Constitutive expression of miR-487b abrogated Wnt signaling, inhibited in vitro proliferation and invasion of lung cancer cells mediated by CSC or overexpression of miR-487b targets, and decreased growth and metastatic potential of lung cancer cells in vivo. Collectively, these findings indicate that miR-487b is a tumor suppressor microRNA silenced by epigenetic mechanisms during tobacco-induced pulmonary carcinogenesis and suggest that DNA demethylating agents may be useful for activating miR-487b for lung cancer therapy.

MicroRNAs act at the post-transcriptional level and guide Argonaute proteins to cleave their corresponding target transcripts. However, little attention has been paid to arm selection in miRNA precursors. In this study, small RNA high-throughput sequencing data from 29 different rice libraries were pooled to investigate tissue- and abiotic stress-specific dynamic expression of miRNAs. We found that more than half of pre-miRNAs showed changes in arm selection in different tissues and/or under different abiotic stresses. Our findings suggest that miRNA selection is remarkably prevalent in plants, providing new insights into the role of miRNAs in plant growth and development.

Small RNAs represented by microRNA (miRNA) plays important roles in plant development and responds to biotic and abiotic stresses. Previous studies have placed special emphasis on gene-repression mediated by miRNA. In this work, the DNA methylation pattern of microRNA genes (MIRs) was interrogated. Full-length cDNA and EST were used to confirm the entity of pri-miRNA. In parallel, miRNA in 24 nucleotides (nt) was pooled to detect chromatin modification effect by using bisulfite sequencing data. 97 MIRs were supported by full-length cDNA and 30 more were hit by EST. Notably, methylation levels of conserved MIRs were significantly lower than the non-conserved at all contexts (CG, CHG, and CHH). Additionally, a substantial part of 24-nt miRNA was able to induce target site methylation, providing a broader perspective for researchers.

miR393, which is encoded by MIR393a and MIR393b in Arabidopsis, post-transcriptionally regulates mRNAs for the F-box auxin receptors TIR1 (Transport Inhibitor Response Protein 1), AFB1 (Auxin Signaling F-box Protein 1), AFB2 and AFB3. However, biological functions of the miR393-TIR1/AFBs module in auxin response and plant development is not fully understood. In the study herein, we demonstrate that miR393 accumulated in response to exogenous IAA treatment, and its induction was due to enhanced MIR393b transcription but not MIR393a. Overexpression of a miR393-resistant form of TIR1 (mTIR1) enhanced auxin sensitivity and led to pleiotropic effects on plant development including inhibition of primary root growth, overproduction of lateral roots, altered leave phenotype and delayed flowering. Furthermore, miR393 level was increased in 35S:mTIR1 plant, suggesting that TIR1 promoted the expression of miR393 by a feedback loop. The interaction between miR393 and its target indicates a fine adjustment to the roles of the miR393-TIR1 module, which is required for auxin responses in plant development.

Background Limited information is available regarding mechanisms by which miRNAs contribute to pulmonary carcinogenesis. The present study was undertaken to examine expression and function of miRNAs induced by cigarette smoke condensate (CSC) in normal human respiratory epithelia and lung cancer cells. Methodology Micro-array and quantitative RT-PCR (qRT-PCR) techniques were used to assess miRNA and host gene expression in cultured cells, and surgical specimens. Software-guided analysis, RNA cross-link immunoprecipitation (CLIP), 3′ UTR luciferase reporter assays, qRT-PCR, focused super-arrays and western blot techniques were used to identify and confirm targets of miR-31. Chromatin immunoprecipitation (ChIP) techniques were used to evaluate histone marks and transcription factors within the LOC554202 promoter. Cell count and xenograft experiments were used to assess effects of miR-31 on proliferation and tumorigenicity of lung cancer cells. Results CSC significantly increased miR-31 expression and activated LOC554202 in normal respiratory epithelia and lung cancer cells; miR-31 and LOC554202 expression persisted following discontinuation of CSC exposure. miR-31 and LOC554202 expression levels were significantly elevated in lung cancer specimens relative to adjacent normal lung tissues. CLIP and reporter assays demonstrated direct interaction of miR-31 with Dickkopf-1 (Dkk-1) and DACT-3. Over-expression of miR-31 markedly diminished Dkk-1 and DACT3 expression levels in normal respiratory epithelia and lung cancer cells. Knock-down of miR-31 increased Dkk-1 and DACT3 levels, and abrogated CSC-mediated decreases in Dkk-1 and DACT-3 expression. Furthermore, over-expression of miR-31 diminished SFRP1, SFRP4, and WIF-1, and increased Wnt-5a expression. CSC increased H3K4Me3, H3K9/14Ac and C/EBP-β levels within the LOC554202 promoter. Knock-down of C/EBP-β abrogated CSC-mediated activation of LOC554202. Over-expression of miR-31 significantly enhanced proliferation and tumorigenicity of lung cancer cells; knock-down of miR-31 inhibited growth of these cells. Conclusions Cigarette smoke induces expression of miR-31 targeting several antagonists of cancer stem cell signaling in normal respiratory epithelia and lung cancer cells. miR-31 functions as an oncomir during human pulmonary carcinogenesis.

Limited information is available regarding the mechanisms by which miRNA alternations contribute to initiation and progression of lung cancer. In order to examine this issue, array techniques were used to assess miRNA expression profiles in short term normal human bronchial epithelia (NHBE) and small airway epithelial cells (SAEC), as well as A549 and Calu-6 lung cancer cells cultured in normal media (NM) with or without cigarette smoke condensate (CSC). Under relevant exposure conditions, CSC consistently mediated a 3.4 − 5.5 fold decrease in has-miR-487b expression in cultured normal as well as lung cancer cells. Quantitative RT-PCR experiments demonstrated that expression levels of has-miR-487b were significantly downregulated (2.9 − 21 fold) in a panel of resected lung cancers relative to adjacent normal tissues. Software-guided analysis revealed numerous potential targets for has-miR-487b including H2AZ, encoding a histone variant modulating expression of developmentally regulated genes as well as SUZ12, which encodes a component of polycomb repressor complexes implicated in aberrant silencing of tumor suppressor genes such as Dickkopf-1 (Dkk-1) in lung cancer cells. Quantitative RT-PCR experiments revealed that constitutive over-expression of has-miR-487b mediated 3-35 fold reductions in H2AZ and SUZ12 expression in SAEC, A549, and Calu-6 cells. RNA cross-link immunoprecipitation (CLIP) experiments confirmed direct interference of has-miR-487b with H2AZ and SUZ12 transcripts. Overexpression of has-miR-487b enhanced sensitivity to cisplatin in lung cancer cells. Collectively, these data suggest that CSC-mediated downregulation of has-miR-487b promotes pulmonary carcinogenesis, in part, via activation of genes encoding proteins implicated in epigenetic regulation of stem cell gene expression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2057.

Background The purpose of the present study was to examine the expression levels of microRNA-9 (miR-9) in osteosarcoma tissues and normal bone tissues, and investigate the relationships between miR-9 expression, clinicopathological features and the prognosis of patients with osteosarcoma. Methods The expression levels of miR-9 in osteosarcoma tissues and corresponding non-cancerous tissues were detected using a real-time quantitative assay. Differences in patient survival were determined using the Kaplan–Meier method and a log-rank test. A Cox proportional hazards regression analysis was used for univariate and multivariate analyses of prognostic values. Results Compared to non-cancerous bone tissues, the expression levels of miR-9 in osteosarcoma tissues were significantly elevated (P