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The revelation that microRNAs (miRNAs) exist within the human genome uncovered an underappreciated mechanism of gene expression. For cells to regulate expression of their genes, miRNA molecules and argonaute proteins bind to mRNAs and interfere with efficient translation of the RNA transcript. Although miRNAs have important roles in normal tissues, miRNAs may adopt aberrant functions in malignant cells depending on their classification as either a tumor suppressor or oncogenic miRNA. Within this review, the current status of miRNA regulation is described in the context of signaling through the lysophosphatidic acid receptors, including the lysophosphatidic acid-producing enzyme, autotaxin. Thus far, research has revealed miRNAs that increase in response to lysophosphatidic acid stimulation, such as miR-21, miR-30c-2-3p, and miR-122. Other miRNAs inhibit the translation of lysophosphatidic acid receptors, such as miR-15b, miR-23a, and miR200c, or proteins that are downstream of lysophosphatidic acid signaling, such as miR-146 and miR-21. With thousands of miRNAs still uncharacterized, it is anticipated that the complex regulation of lysophosphatidic acid signaling by miRNAs will continue to be elucidated. RNA-based therapeutics have entered the clinic with enormous potential in precision medicine. This exciting field is rapidly emerging and it will be fascinating to witness its expansion in scope.

Background Hypoxia is a key feature of breast cancer, which affects cancer development, metastasis and metabolism. Previous studies suggested that circular RNAs (circRNAs) could participate in cancer progression and hypoxia regulation. This study aimed to investigate the role of circRNA differentially expressed in normal cells and neoplasia domain containing 4C (circDENND4C) in breast cancer progression under hypoxia. Methods Forty-three patients with breast cancer were involved in this study. Breast cancer cell lines MDA-MB-453 and SK-BR-3 were cultured under hypoxia (1% O2) for experiments in vitro. The expression levels of circDENND4C, microRNA-200b (miR-200b) and miR-200c were measured by quantitative real-time polymerase chain reaction. Glycolysis was investigated by glucose consumption, lactate production and hexokinase II (HK2) protein level. Migration and invasion were evaluated via trans-well assay and protein levels of matrix metallopeptidase 9 (MMP9) and MMP2. The interaction between circDENND4C and miR-200b or miR-200c was explored by bioinformatics analysis, luciferase assay and RNA immunoprecipitation. Murine xenograft model was established to investigate the anti-cancer role of circDENND4C in vivo. Results circDENND4C highly expressed in breast cancer was up-regulated in response to hypoxia. Knockdown of circDENND4C decreased glycolysis, migration and invasion in breast cancer cells under hypoxia. circDENND4C was validated as a sponge of miR-200b and miR-200c. Deficiency of miR-200b or miR-200c reversed the suppressive effect of circDENND4C knockdown on breast cancer progression. Moreover, silence of circDENND4C reduced xenograft tumor growth by increasing miR-200b and miR-200c. Conclusion circDENND4C silence suppresses glycolysis, migration and invasion in breast cancer cells under hypoxia by increasing miR-200b and miR-200c. Electronic supplementary material The online version of this article (10.1186/s13046-019-1398-2) contains supplementary material, which is available to authorized users.

Background Accumulating evidence indicates that long non-coding RNAs (lncRNAs) are potential biomarkers and key regulators of tumour development and progression. SOX2 overlapping transcript (SOX2OT) is a novel lncRNA that acts as a potential biomarker and is involved in the development of cancer and cancer stem cells. However, the clinical significance and molecular mechanism of SOX2OT in bladder cancer are still unknown. Methods The expression level of SOX2OT was determined by RT-qPCR in a total of 106 patients with urothelial bladder cancer and in different bladder cancer cell (BCC) lines. Bladder cancer stem cells (BCSCs) were isolated from BCCs using flow cytometry based on the stem cell markers CD44 and ALDH1. Loss-of-function experiments were performed to investigate the biological roles of SOX2OT in the stemness phenotype of BCSCs. Comprehensive transcriptional analysis, RNA FISH, dual-luciferase reporter assays and western blots were performed to explore the molecular mechanisms underlying the functions of SOX2OT. Results SOX2OT was highly expressed in bladder cancer, and increased SOX2OT expression was positively correlated with a high histological grade, advanced TNM stage and poor prognosis. Further experiments demonstrated that knockdown of SOX2OT inhibited the stemness phenotype of BCSCs. Moreover, inhibition of SOX2OT delayed xenograft tumour growth and decreased metastases in vivo. Mechanistically, we found that SOX2OT was mainly distributed in the cytoplasm and positively regulated SOX2 expression by sponging miR-200c. Furthermore, SOX2 overexpression reversed the SOX2OT silencing-induced inhibition of the BCSC stemness phenotype. Conclusion This study is the first to demonstrate that SOX2OT plays an important regulatory role in BCSCs and that SOX2OT may serve as a potential diagnostic biomarker and therapeutic target in bladder cancer.

Effect of miRNA-200c (miR-200c) on the proliferation, invasion and apoptosis of prostate cancer cell line LNCaP was investigated. The difference in miR-200c expression was observed using RT-qPCR in the NC group (transfected empty plasmid), simulation group (simulation sequence) and inhibition group (transferred inhibition sequence), which were established by transfecting LNCaP cells with a kit. The proliferation, invasion and apoptosis of cells after transfection were detected using the cell counting kit-8 (CCK-8) method, Transwell chamber and flow cytometry. RT-qPCR detection showed that the relative expression of miR-200c in LNCaP cells significantly increased compared with RWPE-1 cells (P0.05). Flow cytometry detection of the apoptosis ability of each group of cells showed that there was a difference in the apoptotic rate in the NC, simulation and inhibition groups (P

Background: Metformin has been reported to inhibit the growth of various types of cancers, including breast cancer. Yet the mechanisms underlying the anticancer effects of metformin are not fully understood. Growing evidence suggests that metformin's anticancer effects are mediated at least in part by modulating microRNAs, including miR-200c, which has a tumor suppressive role in breast cancer. We hypothesized that miR-200c has a role in the antitumorigenic effects of metformin on breast cancer cells. Methods: To delineate the role of miR-200c in the effects of metformin on breast cancer, plasmids containing pre-miR-200c or miR-200c inhibitor were transfected into breast cancer cell lines. The MDA-MB-231, BT549, MCF-7, and T-47-D cells' proliferation, apoptosis, migration, and invasion were assessed. The antitumor role of metformin in vivo was investigated in a MDA-MB-231 xenograft tumor model in SCID mice. Results: Metformin significantly inhibited the growth, migration, and invasion of breast cancer cells, and induced their apoptosis; these effects were dependent on both dose and time. Metformin also suppressed MDA-MB-231 tumor growth in SCID mice in vivo. Metformin treatment was associated with increased miR-200c expression and decreased c-Myc and AKT2 protein expression in both breast cancer cells and tumor tissues. Overexpression of miR-200c exhibited effects on breast cancer cells similar to those of metformin treatment. In contrast, inhibiting the expression of miR-200c increased the growth, migration, and invasion of MCF-7 and MDA-MB-231 cells. Conclusion: Metformin inhibits the growth and invasiveness of breast cancer cells by upregulation of miR-200c expression by targeting AKT2. These findings provide novel insight into the molecular functions of metformin that suggest its potential as an anticancer agent.

P27Kip1, also known as Cyclin-dependent kinase inhibitor 1B, is an important check-point protein in the cell cycle. It has been identified that although as a tumor suppressor, P27Kip1 is expressed in different cancer cell types, which shows the therapeutic potential in tumor genesis. In this study, we examined the upstream regulatory mechanism of P27Kip1 at the microRNA (miRNA) level in gastric carcinogenesis. We used bioinformatics to predict that microRNA-200c (miR-200c) might be a direct upstream regulator of P27Kip1. It was also verified in gastric epithelial-derived cell lines that overexpression of miR-200c significantly inhibited the expression levels of P27Kip1, whereas knockdown of miR-200c promoted P27Kip1 expression in AGS and BGC-823 cells. Furthermore, we identified the direct binding of miR-200c on the P27Kip1 3′ -UTR sequence by luciferase assay. MiR-200c could enhance the colony formation of cells by repressing P27Kip1 expression. In addition, the negative correlation between P27Kip1 and miR-200c in human gastric cancer tissues and matched normal tissues further supported the tumor-promoting action of miR-200c in vivo. Our finding suggested that miR-200c directly regulates the expression of P27Kip1 and promotes cell growth in gastric cancer as an oncogene, which may provide new clues to treatment.

Yidong Cheng,* Xiaolei Zhang,* Peng Li,* Chengdi Yang, Jinyuan Tang, Xiaheng Deng, Xiao Yang, Jun Tao, Qiang Lu, Pengchao Li Department of Urology, The First Affiliated Hospital of Nanjing Medical University, Nanjing, People’s Republic of China *These authors contributed equally tothis work Background: Increasing evidence suggests that the dysregulation of certain microRNAs plays an important role in tumorigenesis and metastasis. MiR-200c exhibits a disordered expression in many tumors and presents dual roles in bladder cancer (BC). Therefore, the definite role of miR-200c in BC needs to be investigated further.Materials and methods: Quantitative reverse transcription polymerase chain reaction was used to assess miR-200c expression. Cell invasion and migration were evaluated using wound healing and transwell assays. The luciferase reporter assay was used to identify the direct target of miR-200c. The expression of reversion-inducing cysteine-rich protein with kazal motifs (RECK) in BC tissues and adjacent nontumor tissues, as well as in BC cell lines, was detected through quantitative reverse transcription polymerase chain reaction, Western blot assay, and immunohistochemistry.Results: The miR-200c expression was significantly upregulated in the BC tissues compared with the adjacent nontumor tissues. The downregulation of miR-200c significantly inhibited cell migration and invasion in the BC cell lines. The luciferase reporter assay showed that RECK was a direct target of miR-200c. The knockdown of RECK in the BC cell lines treated with anti-miR-200c elevated the previously attenuated cell migration and invasion.Conclusion: Our findings indicated that miR-200c functions as oncogenes in BC and may provide a novel therapeutic strategy for the treatment of BC. Keywords: miR-200c, bladder cancer, migration, invasion, RECK

Yidong Cheng,* Xiaolei Zhang,* Peng Li,* Chengdi Yang, Jinyuan Tang, Xiaheng Deng, Xiao Yang, Jun Tao, Qiang Lu, Pengchao Li Department of Urology, The First Affiliated Hospital of Nanjing Medical University, Nanjing, People’s Republic of China *These authors contributed equally tothis work Background: Increasing evidence suggests that the dysregulation of certain microRNAs plays an important role in tumorigenesis and metastasis. MiR-200c exhibits a disordered expression in many tumors and presents dual roles in bladder cancer (BC). Therefore, the definite role of miR-200c in BC needs to be investigated further.Materials and methods: Quantitative reverse transcription polymerase chain reaction was used to assess miR-200c expression. Cell invasion and migration were evaluated using wound healing and transwell assays. The luciferase reporter assay was used to identify the direct target of miR-200c. The expression of reversion-inducing cysteine-rich protein with kazal motifs (RECK) in BC tissues and adjacent nontumor tissues, as well as in BC cell lines, was detected through quantitative reverse transcription polymerase chain reaction, Western blot assay, and immunohistochemistry.Results: The miR-200c expression was significantly upregulated in the BC tissues compared with the adjacent nontumor tissues. The downregulation of miR-200c significantly inhibited cell migration and invasion in the BC cell lines. The luciferase reporter assay showed that RECK was a direct target of miR-200c. The knockdown of RECK in the BC cell lines treated with anti-miR-200c elevated the previously attenuated cell migration and invasion.Conclusion: Our findings indicated that miR-200c functions as oncogenes in BC and may provide a novel therapeutic strategy for the treatment of BC. Keywords: miR-200c, bladder cancer, migration, invasion, RECK

// Jun Guo 1, 3, * , Weiwei Fang 1, 2, * , Libo Sun 4 , Yonggang Lu 1 , Lin Dou 1 , Xiuqing Huang 1 , Mingxiao Sun 1 , Cheng Pang 1, 2 , Jing Qu 5, 6 , Guanghui Liu 3, 6 , Jian Li 1, 2 1 The Key Laboratory of Geriatrics, Beijing Hospital and Beijing Institute of Geriatrics, Ministry of Health, Beijing 100730, China 2 Graduate School of Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing 100730, China 3 National Laboratory of Biomacromolecules, Institute of Biophysics, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Beijing 100101, China 4 Department of Hepatobiliay Surgery and You-An Liver Transplantation Center, Beijing You-An Hospital, Capital Medical University, Beijing 100069, China 5 State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China 6 University of Chinese Academy of Sciences, Beijing 100049, China * These authors contributed equally to this work Correspondence to: Jian Li, email: lijian@bjhmoh.cn Guanghui Liu, email: ghliu@ibp.ac.cn Jing Qu, email: qujing@ioz.ac.cn Keywords: miR-200b, miR-200c, lipogenesis, JUN, SREBP1 Received: September 22, 2015 Accepted: April 22, 2016 Published: May 05, 2016 ABSTRACT Previous studies indicated that miR-200s participated in IL-6-induced hepatic insulin resistance. However, the role of miR-200s in hepatic lipid accumulation has not been elucidated. Here we found that miR-200b and miR-200c were reduced in the steatotic livers of mice fed a high-fat diet (HFD) and patients with nonalcoholic fatty liver disease. This down-regulation was accompanied by an increase in the expression of lipogenic proteins such as sterol regulatory element-binding protein 1 (SREBP1) and fatty acid synthase (FAS). The suppression of miR-200b and miR-200c in Hep1-6 and NCTC1469 hepatocytes enhanced intracellular triglyceride levels, which were associated with increased SREBP-1 and FAS protein levels. In contrast, the over-expression of miR-200b and miR-200c suppressed lipid accumulation and reduced the expression of SREBP1 and FAS in Hep1-6 and NCTC1469 cells transfected with miR-200b or miR-200c mimics. Importantly, the up-regulation of miR-200b and miR-200c could reverse oleic acid/palmitic acid-induced lipid accumulation in hepatocytes. A luciferase reporter assay identified that miR-200b and miR-200c could directly bind the 3’UTR of jun . JUN activated the transcription of srebp1 to increase lipid accumulation. The data also demonstrated that increased miR-200b and miR-200c expression might be associated with sitagliptin-reduced hepatic lipid accumulation in mice fed a HFD. These findings suggest, for the first time, that reduced miR-200b and miR-200c expression contributes to abnormal hepatic lipid accumulation by stimulating JUN expression and activating the transcription of srebp1 .

BACKGROUND In prostate cancer (PCa), abnormal expression of several microRNAs (miRNAs) has been previously reported. Increasing evidence shows that aberrant epigenetic regulation of miRNAs is a contributing factor to their altered expression in cancer. In this study, we investigate whether expression of miR‐200c and miR‐141 in PCa is related to the DNA methylation status of their promoter. METHODS PCR analysis of miR‐200c and miR‐141, and CpG methylation analysis of their common promoter, was performed in PCa cell‐lines and in archived prostate biopsy specimens. The biological significance of miR‐200c and miR‐141 expression in prostate cancer cells was assessed by a series of in vitro bioassays and the effect on proposed targets DNMT3A and TET1/TET3 was investigated. The effect on promoter methylation status in cells treated with demethylating agents was also examined. RESULTS miR‐200c and miR‐141 are both highly elevated in LNCaP, 22RV1, and DU145 cells, but significantly reduced in PC3 cells. This correlates inversely with the methylation status of the miR‐200c/miR‐141 promoter, which is unmethylated in LNCaP, 22RV1, and DU145 cells, but hypermethylated in PC3. In PC3 cells, miR‐200c and miR‐141 expression is subsequently elevated by treatment with the demethylating drug decitabine (5‐aza‐2′deoxycytidine) and by knockdown of DNA methyltransferase 1 (DNMT1), suggesting their expression is regulated by methylation. Expression of miR‐200c and miR‐141 in prostate biopsy tissue was inversely correlated with methylation in promoter CpG sites closest to the miR‐200c/miR‐141 loci. In vitro, over‐expression of miR‐200c in PC3 cells inhibited growth and clonogenic potential, as well as inducing apoptosis. Expression of the genes DNMT3A and TET1/TET3 were down‐regulated by miR‐200c and miR‐141 respectively. Finally, treatment with the soy isoflavone genistein caused demethylation of the promoter CpG sites closest to the miR‐200c/miR‐141 loci resulting in increased miR‐200c expression. CONCLUSIONS Our findings provide evidence that miR‐200c and miR‐141 are under epigenetic regulation in PCa cells. We propose that profiling their expression and methylation status may have potential as a novel biomarker or focus of therapeutic intervention in the diagnosis and prognosis of PCa. Prostate 76:1146–1159, 2016. © 2016 The Authors. The Prostate published by Wiley Periodicals, Inc.

Lung metastasis is the major cause of death in osteosarcoma patients. However, molecular mechanisms underlying this metastasis remain poorly understood. To identify key molecules related with pulmonary metastasis of pediatric osteosarcomas, we analyzed high-throughput miRNA expression in a cohort of 11 primary tumors and 15 lung metastases. Results were further validated with an independent cohort of 10 primary tumors and 6 metastases. In parallel, we performed immunohistochemical analysis of activated signaling pathways in 36 primary osteosarcomas. Only phospho-AKT associated with lower overall survival in primary tumors, supporting its role in osteosarcoma progression. CTNNB1 expression also associated with lower overall survival but was not strong enough to be considered an independent variable. Interestingly, miR-200c was overexpressed in lung metastases, implicating an inhibitory feed-back loop to PI3K-AKT. Moreover, transfection of miR200c-mimic in U2-OS cells reduced phospho-AKT levels but increased cellular migration and proliferation. Notably, miR-200c expression strongly correlated with miR-141 and with the osteogenic inhibitor miR-375, all implicated in epithelial to mesenchymal transition. These findings contrast epithelial tumors where reduced miR-200c expression promotes metastasis. Indeed, we noted that osteosarcoma cells in the lung also expressed the epithelial marker CDH1, revealing a change in their mesenchymal phenotype. We propose that miR-200c upregulation occurs late in osteosarcoma progression to provide cells with an epithelial phenotype that facilitates their integration in the metastatic lung niche. Thus, our findings identify phospho-AKT in the primary tumor and miR-200c later during tumor progression as prognostic molecules and potential therapeutic targets to prevent progression and metastasis of pediatric osteosarcomas.

Migration of glioblastoma cells into surrounding tissue is one of the main features that makes this tumor incurable. We evaluated whole-genome miRNA expression profiling associated with different EGFR amplification patterns in 30 cases of primary glioblastoma. From the 64 miRNAs that showed differential expression between tumors with a high level of EGFR amplification and tumors without EGFR amplification, 40% were related with cell migration, being miR-200c the most differentially expressed between these two groups. We investigated the effect of miR-200c on ZEB1 expression and cell migration in an in vitro transfection model with a miR-200c mimic, a miR-200c inhibitor and siRNA targeting EGFR in three short-term cultures with different levels of EGFR amplification obtained from resected glioblastomas. The cell culture with the highest EGFR amplification level presented the lowest miR-200c expression and the status of EGFR modulated the effect of miR-200c on ZEB1 expression. Silencing EGFR led to miR-200c upregulation and ZEB1 downregulation in transfected cultures, except in the presence of high levels of EGFR. Likewise, miR-200c upregulation decreased ZEB1 expression and inhibited cell migration, especially when EGFR was not amplified. Our results suggest that modulating miR-200c may serve as a novel therapeutic approach for glioblastoma depending on EGFR status.

Background The management of pancreatic cancer (PanCa) is exceptionally difficult due to poor response to available therapeutic modalities. Tubulins play a major role in cell dynamics, thus are important molecular targets for cancer therapy. Among various tubulins, βIII and βIV-tubulin isoforms have been primarily implicated in PanCa progression, metastasis and chemo-resistance. However, specific inhibitors of these isoforms that have potent anti-cancer activity with low toxicity are not readily available. Methods We determined anti-cancer molecular mechanisms and therapeutic efficacy of a novel small molecule inhibitor (VERU-111) using in vitro (MTS, wound healing, Boyden chamber and real-time xCELLigence assays) and in vivo (xenograft studies) models of PanCa. The effects of VERU-111 treatment on the expression of β-tubulin isoforms, apoptosis, cancer markers and microRNAs were determined by Western blot, immunohistochemistry (IHC), confocal microscopy, qRT-PCR and in situ hybridization (ISH) analyses. Results We have identified a novel small molecule inhibitor (VERU-111), which preferentially represses clinically important, βIII and βIV tubulin isoforms via restoring the expression of miR-200c. As a result, VERU-111 efficiently inhibited tumorigenic and metastatic characteristics of PanCa cells. VERU-111 arrested the cell cycle in the G2/M phase and induced apoptosis in PanCa cell lines via modulation of cell cycle regulatory (Cdc2, Cdc25c, and Cyclin B1) and apoptosis - associated (Bax, Bad, Bcl-2, and Bcl-xl) proteins. VERU-111 treatment also inhibited tumor growth (P

The bidirectional regulation of epithelial–mesenchymal transitions (EMT) is key in tumorigenesis. Rho GTPases regulate this process via canonical pathways that impinge on the stability of cell-to-cell contacts, cytoskeletal dynamics, and cell invasiveness. Here, we report that the Rho GTPase activators Vav2 and Vav3 utilize a new Rac1-dependent and miR-200c-dependent mechanism that maintains the epithelial state by limiting the abundance of the Zeb2 transcriptional repressor in breast cancer cells. In parallel, Vav proteins engage a mir-200c-independent expression prometastatic program that maintains epithelial cell traits only under 3D culture conditions. Consistent with this, the depletion of endogenous Vav proteins triggers mesenchymal features in epithelioid breast cancer cells. Conversely, the ectopic expression of an active version of Vav2 promotes mesenchymal-epithelial transitions using E-cadherin-dependent and independent mechanisms depending on the mesenchymal breast cancer cell line used. In silico analyses suggest that the negative Vav anti-EMT pathway is operative in luminal breast tumors. Gene signatures from the Vav-associated proepithelial and prometastatic programs have prognostic value in breast cancer patients., XRB is supported by grants from the Castilla-León Government (CSI049U16), the Spanish Ministry of Economy and Competitiveness (MINECO) (SAF2015-64556-R), Worldwide Cancer Research (14-1248), the Ramón Areces Foundation, and the Spanish Association against Cancer (GC16173472GARC). MD (BFU2014-52729-P), JMP (SAF2015-66015-R, PIE15/00076), and PMF-S (SAF2014-51813-R) are supported by MINECO grants. Spanish funding is partially supported by the European Regional Development Fund. LFL-M, JC, and SR-F were supported by funding from the Spanish Ministry of Education, Culture and Sports (LFL-M, FPU13/02923) and the MINECO (JC, CD15/00113; SR-F, BES-2013-063573).

Objective: microRNAs (miRNAs) play important role in progression of tumorigenesis. They can target self-renewal and epithelial-mesenchymal transition (EMT) abilities in tumor cells, especially in cancer stem cells (CSCs). The objective of this study was to implement data mining to identify important miRNAs for targeting both self-renewal and EMT. We also aimed to evaluate these factors in mammospheres as model of breast cancer stem cells (BCSCs) and metastatic tumor tissues. Materials and Methods: In this experimental study, mammospheres were derived from MCF-7 cells and characterized for the CSCs properties. Then expression pattern of the selected miRNAs in spheroids were evaluated, using the breast tumor cells obtained from seven patients. Correlation of miRNAs with self-renewal and EMT candidate genes were assessed in mammospheres and metastatic tumors. Results: The results showed that mammospheres represented more colonogenic and spheroid formation potential than MCF-7 cells (P

Polycystic ovary syndrome (PCOS) is one of the most common endocrine disorders in reproductive-aged women, affecting 6–8% of women and characterized by hyperandrogenism, ovulatory dysfunction, and polycystic ovarian morphology. Accumulating evidence demonstrates that different microRNAs (miRNAs) expressions may contribute to the pathogenesis of PCOS. Therefore, the goal of this study is to compare the expression levels of miR-141 and miR-200c in granulosa cells isolated from PCOS patients and also evaluate their predictive values for pregnancy complications. First, RNA extraction, reverse transcription, and reverse transcription-polymerase chain reaction (RT-PCR) were performed to assess the expression levels of miR-141 and miR-200c in granulosa cells isolated from 62 PCOS patients and 61 controls. Second, according to each mean of miR-141 and miR-200c measured values in all patients, PCOS, and controls were divided into low-expression group and high-expression group to better evaluate their predictive values for pregnancy complications. Significantly elevated expressions of miR-141 and miR-200c were observed in PCOS patients compared with the controls (p < 0.001 and p = 0.002, respectively). Furthermore, PCOS patients had a significantly increased incidence of pregnancy complications in low-expression groups of miR-141 and miR-200c (p = 0.007 and p = 0.002, respectively). Our findings demonstrated that the expressions of both miR-141 and miR-200c were significantly increased in PCOS patients, which might contribute to the pathogenesis of PCOS. PCOS patients had an increased risk of pregnancy complications in low-expression groups of both miR-141 and miR-200c.

Background Cancer treatment is based on tumor staging. Curative intent is only applied to localized tumors. Recent studies show that oligometastatic patients who have limited number of metastases may benefit from metastasis-directed local treatments to achieve long-term survival. However, mechanisms underlying oligometastatic to polymetastatic progression remains elusive. Methods The effects of miR-200c and Sec23a on tumor metastasis were verified both in vitro and in vivo. The secretome changes were detected by mass spectrometry. Findings We established a pair of homologous lung-metastasis derived oligometastatic and polymetastatic cell lines from human melanoma cancer cell line M14. Using the two cell lines, we have identified Sec23a, a gene target of miR-200c, suppresses miR-200c augmented oligometastatic to polymetastatic progression via its secretome. Firstly, miR-200c over-expression and Sec23a interference accelerated oligometastatic to polymetatic progression. Secondly, Sec23a functions downstream of miR-200c. Thirdly, mass spectrometric analysis of the secretory protein profile suggests that Sec23a-dependent secretome may impact metastatic colonization by modifying tumor microenvironment. Fourthly, the survival analysis using The Cancer Genome Atlas database shows Sec23a as a favorable prognostic marker for skin cutaneous melanoma, supporting the clinical relevance of our findings. Interpretation The finding that Sec23a is a suppressor of oligometastatic to polymetastatic progression has clinical implications. First, it provides a new theoretical framework for the development of treatments that prevent oligometastasis to polymetastasis. Second, Sec23a may be used as a favorable prognostic marker for the selection of patients with stable oligometastatic disease for oligometastasis-based local therapies of curative intent. Fund National Natural Science Foundations of China.

Yunxia Zhang,1,2 Jing Wang,2 Di Wu,1 Miao Li,1 Fenshu Zhao,1 Mulan Ren,2 Yunlong Cai,2 Jun Dou1 1Department of Pathogenic Biology and Immunology, School of Medicine, Southeast University, Nanjing, People’s Republic of China; 2Department of Gynecology & Obstetrics, Zhongda Hospital, School of Medicine, Southeast University, Nanjing, People’s Republic of China Background: Epithelial ovarian cancer (EOC) with insidious characteristic manifests no symptoms in its early onset but most patients have advanced and distant cancer metastasis at diagnosis. Innovative early diagnosis and effective treatment of EOC are urgently needed. Methods: In the study, we developed a novel agent of IL-21-secreting human umbilical cord mesenchymal stem cells (hUCMSCs) combined with miR-200c to evaluate its effects on SKOV3 EOC in vitro and in vivo.Results: hUCMSCs-LV-IL-21 combined with miR-200c significantly inhibited the SKOV3 cell mobility and tumorigenesis compared with hUCMSCs-LV-IL-21, hUCMSCs- LV-vector, and hUCMSCs, respectively. These were reflected in decreasing the tumor sizes and elongating the tumor bearing nude mouse survival, accompanied with increasing the serum cytokine levels of IFN-γ, IL-21 and TNF-α as well as the splenocyte cytotoxicity. In addition, the expression of β-catenin, cyclin-D1, Gli1, Gli2, and ZEB1 was decreased but the E-cadherin expression was increased in tumor tissues of mice treated with hUCMSCs-LV-IL-21 plus miR-200c.Conclusion: We demonstrated that the synergistic effect of fighting SKOV3 EOC is attributable to repression of Wnt/β-catenin signaling and epithelial-mesenchymal transition in SKOV3 EOC. The findings may provide a new strategy for therapy of EOC. Keywords: epithelial ovarian cancer, umbilical cord mesenchymal stem cells, IL-21, miR-200c, Wnt/β-catenin signaling, epithelial–mesenchymal transition

5-FU is a chemotherapeutic agent that has been used in colorectal cancer (CRC) treatment. However, it is usually associated with the acquired resistance, which decreases the therapeutic effects of 5-FU. miR-200c is involved in chemotherapeutic drug resistance, but its mechanism is not fully understood. In this study, the effect of inhibition of miR-200c in sensitivity of HCT-116 CRC cells to 5-FU was evaluated. HCT-116 cells were transfected with LNA-anti- miR-200c for 48 h. mRNA expression of miR-200c was evaluated using quantitative real- time PCR. The protein expression of phosphatase and tensin homolog (PTEN) and E-cadherin were analyzed by western blotting. Annexin V and propidium iodide staining assay were applied for apoptosis detection. The caspase-3 activation was evaluated by an enzymatic assay. The results showed LNA-anti-miR-200c inhibited the expression of PTEN and E-cadherin protein, apoptosis and activation of caspase 3 compared with control cells. 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The microRNA, miR-200c, is involved in the tumorigenesis and progression of a variety of cancers. The purpose of this study was to investigate the expression, mechanism and prognostic roles of miR-200c in breast cancer. We found that miR-200c was downregulated in both breast cancer tissue and cell lines using quantitative real-time PCR (qRT-PCR). In situ hybridization (ISH) and microarrays showed that low miR-200c expression was associated with poor patient overall survival (OS) and disease free survival (DFS). We used luciferase reporter plasmids to find that miR-200c inhibited the AKT and ERK pathways by directly targeting KRAS. Repression of KRAS by miR-200c suppressed the proliferation and survival of breast cancer cells in vitro and in vivo. miR-200c also had an anti-tumor effect by negatively regulating KRAS in a xenograft mouse model. Our findings provide clues regarding the role of miR-200c as a tumor suppressor in breast cancer through the inhibition of KRAS translation both in vitro and in vivo. miR-200c could be a potential therapeutic target in breast cancer.

// Florian Christoph Sigloch 1, 2, * , Ulrike Christina Burk 1, 3, * , Martin Lothar Biniossek 1 , Thomas Brabletz 4 , Oliver Schilling 1, 3, 5 1 Institute of Molecular Medicine and Cell Research, Albert-Ludwigs-University Freiburg, Freiburg, Germany 2 Faculty of Biology, Albert-Ludwigs-University Freiburg, Freiburg, Germany 3 BIOSS Centre for Biological Signaling Studies, University of Freiburg, Freiburg, Germany 4 Experimental Medicine I, Nikolaus-Fiebiger-Center for Molecular Medicine, University Erlangen-Nurnberg, Erlangen, Germany 5 German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Heidelberg, Germany * These authors have contributed equally to this work Correspondence to: Oliver Schilling, e-mail: oliver.schilling@mol-med.uni-freiburg.de Keywords: miR-200c, miRNA target cluster, PKA, cofilin, proteomics Received: February 09, 2015 Accepted: June 19, 2015 Published: June 29, 2015 ABSTRACT Expression of miR-200c is a molecular switch to determine cellular fate towards a mesenchymal or epithelial phenotype. miR-200c suppresses the early steps of tumor progression by preventing epithelial-mesenchymal transition (EMT) and intravasation of tumor cells. Unraveling the underlying molecular mechanisms might pinpoint to novel therapeutic options. To better understand these mechanisms it is crucial to identify targets of miR-200c. Here, we employ a combination of quantitative proteomic and bioinformatic strategies to identify novel miR-200c targets. We identify and confirm two subunits of the central cellular kinase protein kinase A (PKA), namely PRKAR1A and PRKACB, to be directly regulated by miR-200c. Notably, siRNA-mediated downregulation of both proteins phenocopies the migratory behavior of breast cancer cells after miR-200c overexpression. Patient data from publicly accessible databases supports a miR-200c-PKA axis. Thus, our study identifies the PKA heteroprotein as an important mediator of miR-200c induced repression of migration in breast cancer cells. By bioinformatics, we define a miRNA target cluster consisting of PRKAR1A, PRKAR2B, PRKACB, and COF2, which is targeted by a group of 14 miRNAs.

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

Claudin-low tumors are a highly aggressive breast cancer subtype with no targeted treatments and a clinically documented resistance to chemotherapy. They are significantly enriched in cancer stem cells (CSCs), which makes claudin-low tumor models particularly attractive for studying CSC behavior and developing novel approaches to minimize CSC therapy resistance. One proposed mechanism by which CSCs arise is via an epithelial-mesenchymal transition (EMT), and reversal of this process may provide a potential therapeutic approach for increasing tumor chemosensitivity. Therefore, we investigated the role of known EMT regulators, miR-200 family of microRNAs in controlling the epithelial state, stem-like properties and therapeutic response in an in vivo primary, syngeneic p53(null) claudin-low tumor model that is normally deficient in miR-200 expression. Using an inducible lentiviral approach, we expressed the miR-200c cluster in this model and found that it changed the epithelial state, and consequently, impeded CSC behavior in these mesenchymal tumors. Moreover, these state changes were accompanied by a decrease in proliferation and an increase in the differentiation status. miR-200c expression also forced a significant reorganization of tumor architecture, affecting important cellular processes involved in cell-cell contact, cell adhesion and motility. Accordingly, induced miR200c expression significantly enhanced the chemosensitivity and decreased the metastatic potential of this p53(null) claudin-low tumor model. Collectively, our data suggest that miR-200c expression in claudin-low tumors offers a potential therapeutic application to disrupt the EMT program on multiple fronts in this mesenchymal tumor subtype, by altering tumor growth, chemosensitivity and metastatic potential in vivo.

This study was aimed to investigate the function and mechanism of microRNA-200c (miR-200c) in the progression of non-small cell lung cancer (NSCLC). A total of 76 patients diagnosed as having NSCLC were enrolled in this study. The expression level of miR-200c in NSCLC tissues and cell lines was investigated using the quantitative real-time polymerase chain reaction (RT-qPCR) method. We found that the expression of miR-200c was significantly reduced in NSCLC tissues and cell lines compared with normal lung tissues and the human bronchial epithelial cell line. Overexpression of miR-200c using the miR-200c mimic significantly suppressed cell proliferation and migration of NSCLC cell lines. The results of the luciferase reporter assay identified lactate dehydrogenase A (LDHA) as a direct target of miR-200c. The expression of LDHA was shown to be suppressed in NSCLC cell lines with miR-200c mimic transfection. Furthermore, the transfection of small interfering RNA (siRNA) targeting LDHA suppressed the proliferation and migration of NSCLC cell lines. In summary, our results presented in this study suggested that miR-200c was able to inhibit the proliferation and migration of NSCLC cells by downregulating LDHA. Therefore, miR-200c may be considered as a potential candidate for the treatment of NSCLC.

Scientists are increasingly focusing attention on natural products of plant origin for use as agents in cancer protection and treatment. Cyclamen L. tuber extracts contain saponin glycosides that have been shown to have anti-cancer and other biological activities. The epithelial-to-mesenchymal transition (EMT) is thought to enhance malignant tumour progress. The transcriptional repressor zinc-finger E-box binding homeobox 1 (ZEB1) is an important inducer of EMT in different human tumours and has recently been shown to boost invasion by tumour cells. In this study, we investigated the effects of endemic Cyclamen pseudibericum (CP) saponin-rich tuber extract on the capacity of non-small cell lung cancer line A549 cells to proliferate, invade and migrate and also examined the expression levels of several invasion–migration-related microRNAs (miRNAs) to identify those which directly targeted ZEB1. The cytotoxicity effect of the CP extract on the A549 cancer cells was determined by the luminometric method. The half-minimal (50%) inhibitory concentration dose in the A549 cells was determined to be 41.64 ± 2.35 µg/mL. Using the Matrigel invasion chamber system and the wound healing assay we observed that the CP extract suppressed the invasion and migration capacity of A549 cells, respectively. The expression of miRNAs in A549 cells was evaluated by real-time PCR. Our data showed that overexpression of miRNA miR-200c hindered the EMT by increasing the expression of E-cadherin and decreasing the expression of both N-cadherin and vimentin through the direct targeting of ZEB1. These findings suggest that the saponin-rich tuber extract of CP may have considerable anti-cancer properties in lung cancer. Further studies are required to examine in detail the molecular-based mechanism involved in the EMT process of the extract along with isolation and identification of active saponin components. © 2018, The Japanese Society of Pharmacognosy and Springer Japan KK, part of Springer Nature.

Scientists are increasingly focusing attention on natural products of plant origin for use as agents in cancer protection and treatment. Cyclamen L. tuber extracts contain saponin glycosides that have been shown to have anti-cancer and other biological activities. The epithelial-to-mesenchymal transition (EMT) is thought to enhance malignant tumour progress. The transcriptional repressor zinc-finger E-box binding homeobox 1 (ZEB1) is an important inducer of EMT in different human tumours and has recently been shown to boost invasion by tumour cells. In this study, we investigated the effects of endemic Cyclamen pseudibericum (CP) saponin-rich tuber extract on the capacity of non-small cell lung cancer line A549 cells to proliferate, invade and migrate and also examined the expression levels of several invasion-migration-related microRNAs (miRNAs) to identify those which directly targeted ZEB1. The cytotoxicity effect of the CP extract on the A549 cancer cells was determined by the luminometric method. The half-minimal (50%) inhibitory concentration dose in the A549 cells was determined to be 41.64 +/- 2.35 A mu g/mL. Using the Matrigel invasion chamber system and the wound healing assay we observed that the CP extract suppressed the invasion and migration capacity of A549 cells, respectively. The expression of miRNAs in A549 cells was evaluated by real-time PCR. Our data showed that overexpression of miRNA miR-200c hindered the EMT by increasing the expression of E-cadherin and decreasing the expression of both N-cadherin and vimentin through the direct targeting of ZEB1. These findings suggest that the saponin-rich tuber extract of CP may have considerable anti-cancer properties in lung cancer. Further studies are required to examine in detail the molecular-based mechanism involved in the EMT process of the extract along with isolation and identification of active saponin components.

Duchenne muscular dystrophy (DMD) is a genetic disease associated with mutations of Dystrophin gene that regulate myofiber integrity and muscle degeneration, characterized by oxidative stress increase. We previously published that reactive oxygen species (ROS) induce miR-200c that is responsible for apoptosis and senescence. Moreover, we demonstrated that miR-200c increases ROS production and phosphorylates p66Shc in Ser-36. p66Shc plays an important role in muscle differentiation; we previously showed that p66Shc−/− muscle satellite cells display lower oxidative stress levels and higher proliferation rate and differentiated faster than wild-type (wt) cells. Moreover, myogenic conversion, induced by MyoD overexpression, is more efficient in p66Shc−/− fibroblasts compared to wt cells. Herein, we report that miR-200c overexpression in cultured myoblasts impairs skeletal muscle differentiation. Further, its overexpression in differentiated myotubes decreases differentiation indexes. Moreover, anti-miR-200c treatment ameliorates myogenic differentiation. In keeping, we found that miR-200c and p66Shc Ser-36 phosphorylation increase in mdx muscles. In conclusion, miR-200c inhibits muscle differentiation, whereas its inhibition ameliorates differentiation and its expression levels are increased in mdx mice and in differentiated human myoblasts of DMD. Therefore, miR-200c might be responsible for muscle wasting and myotube loss, most probably via a p66Shc-dependent mechanism in a pathological disease such as DMD.

Background Endometriosis is a common, benign, and estrogen-dependent disease characterized by pelvic pain and infertility. To date, the pathogenesis of endometriosis remains unclear. Recent studies have demonstrated that noncoding RNAs, including microRNAs and long noncoding RNAs, play important roles in the development of endometriosis. Methods Expression profiling of miRNAs in endometrial tissue was characterized using microarrays. The most differentially expressed miRNAs were confirmed using quantitative reverse transcriptase-polymerase chain reaction analysis in additional ectopic endometrial (n = 27) and normal endometrial (n = 12) tissues. For in-vitro functional studies, 5-ethynyl-2′-deoxyuridine incorporation assay, Transwell assay, and dual-luciferase reporter assay were used to measure the proliferation, migration, and luciferase activity of miR-200c and the predicted targets of miR-200c in primary endometrial stromal cells (HESCs) derived from human endometrial biopsies, respectively. For in-vivo therapeutic interventions, polymeric nanoparticles of polyethylenimine–polyethylene glycol–arginine–glycine–aspartic acid were used for delivery of miR-200c mimic and inhibitor to determine the therapeutic effect of miR-200c in a rat model of endometriosis. Results Exogenous overexpression of miR-200c inhibited the proliferation and migration of HESCs, which were mainly regulated by metastasis-associated lung adenocarcinoma transcript 1 (MALAT1). In contrast, inhibition of miR-200c promoted the proliferation and migration of HESCs, while the simultaneous silencing of MALAT1 expression exerted the opposite effects. We demonstrated that expression of MALAT1 in ectopic endometrial specimens was negatively correlated with that of miR-200c and that MALAT1 knockdown increased the level of miR-200c in HESCs. Moreover, the transfection of endometrial stromal cells with the miR-200c mimic or MALAT1 siRNAs decreased the protein levels of mesenchymal markers ZEB1, ZEB2, and N-cadherin and increased the protein levels of the epithelial marker E-cadherin. Furthermore, using a rat endometriosis model, we showed that local delivery of the miR-200c mimic significantly inhibited the growth of ectopic endometriotic lesions. Conclusions The MALAT1/miR-200c sponge may be a potential therapeutic target for endometriosis. Electronic supplementary material The online version of this article (doi:10.1186/s13287-017-0706-z) contains supplementary material, which is available to authorized users.

// Jiayu Li 1 , Xuefei Li 2 , Shengxiang Ren 1 , Xiaoxia Chen 1 , Yishi Zhang 1 , Fei Zhou 1 , Mingchuan Zhao 1 , Chao Zhao 2 , Xiu Chen 3 , Ningning Cheng 1 , Yinmin Zhao 4 , Caicun Zhou 1 , Fred R.Hirsch 5 1 Department of Medical Oncology, Shanghai Pulmonary Hospital, Tongji University School of Medicine, Tongji University Medical School Cancer Institute, Shanghai, China 2 Department of Lung Cancer and Immunology, Shanghai Pulmonary Hospital, Tongji University, Tongji University Medical School Cancer Institute, Shanghai, China 3 Department of Respiration, Zaozhuang Municipal Hospital, Zaozhuang, Shandong, China 4 Department of Central Laboratory, Shanghai Pulmonary Hospital, Tongji University, Shanghai, China 5 Departments of Medicine and Pathology, University of Colorado Cancer Center, Aurora, Colorado, USA. Correspondence to: Dr. Caicun Zhou, e-mail: caicunzhoudr@163.com Key Words: Non-small cell lung cancer, MiR-200c, Epidermal growth factor receptor, Wild type, Tyrosine-kinase inhibitor Received: July 05, 2014 Accepted: July 29, 2014 Published: August 08, 2014 ABSTRACT Several randomized trials have demonstrated non-small cell lung cancer (NSCLC) patients with activating epidermal growth factor receptor (EGFR) mutations can achieve favorable clinical outcomes on treatment with EGFR tyrosine kinase inhibitors (TKIs). EGFR mutation is considered as a predictive marker for efficacy of EGFR-TKIs in NSCLC. Here we show miR-200c overexpression was correlated with the epithelial phenotype and sensitivity to gefitinib in EGFR wild-type NSCLC cell lines. Up-regulated miR-200c could regain the sensitivity to gefitinib in the EGFR wild-type cell lines and miR-200c could regulate epithelial to mesenchymal transition through PI3K/AKT and MEK/ERK pathways. NSCLC patients at advanced stage (N=150) who received EGFR-TKIs (gefitinib or erlotinib) as second- or third-line therapy from September 2008 to December 2012 were included in the study. In 66 NSCLC patients with wild-type EGFR , high levels of miR-200c expression was associated with higher disease control rate (DCR), longer progression-free survival (PFS) and longer overall survival (OS) compared with low miR-200c expression subgroup. In the subgroup with EGFR mutation, the trend remained the same but not statistically significant. Overall, these findings indicated that miR-200c might be a predictive biomarker for sensitivity to EGFR-TKIs in advanced NSCLC patients with wild-type EGFR .