PrLZ regulates EMT and invasion in prostate cancer via the TGF-ß1/p-smad2/miR-200 family/ZEB1 axis.

BACKGROUND: Prostate leucine zipper (PrLZ) is a prostate-specific protein, and our previous study demonstrated that PrLZ enhances the malignant progression of prostate cancer (Pca). However, the roles of PrLZ in epithelial to mesenchymal transition (EMT) remain unknown. METHODS: Quantitative real-time PCR (qRT-PCR), immunohistochemical (IHC) staining, hematoxylin-eosin (HE) staining, and western blotting were used to analyze the expression of protein and genes level in human PCa cell lines. Invasion assay was used to examine the effect of PrLZ, miR-200a, miR-200b, miR-200c, miR-141, miR-429, miR-205, and ZEB1 on PCa cell line invasion in vitro. Prostate cancer metastasis animal model was designed to assess the effect of PrLZ on PCa cell line invasion in vivo. RESULTS: We proved that high PrLZ expression initiates EMT, which was shown by the downregulation of E-cadherin and upregulation of vimentin in PC-3/PrLZ and ARCaP-E/PrLZ cells. Mechanistic analysis revealed that PrLZ regulates EMT by activating TGF-ß1/p-smad2 signaling and further inhibiting the expression of miR-200 family members, which negatively regulates ZEB1 expression and causes EMT in Pca. Moreover, using two of orthotopic mouse model and tail vein injection of human prostate cancer cells mouse model, we observed that PC-3/PrLZ cells led to the development of distant organ metastases in vivo. CONCLUSIONS: Our results show the mechanism by which PrLZ regulates EMT and metastasis and suggest that PrLZ may be a potential therapeutic target for Pca metastasis.

Bisphenol F induced hyperglycemia via activation of oxidative stress-responsive miR-200 family in the pancreas.

Bisphenol F (BPF), BPS and BPAF are gaining popularity as main substitutes to BPA, but there is no clear evidence that these compounds disrupt glycemic homeostasis in the same way. In this study, four bisphenols were administered to C57BL/6 J mice, and showed that the serum insulin was elevated in the BPA and BPS exposed mice, whereas BPF exposed mice exhibited lower serum insulin and higher blood glucose. BPF decreased oxidized glutathione/reduced glutathione ratio (GSSG/GSH) and N6-methyladenosine (m6A) levels, which was responsible for pancreatic apoptosis in mice. Additionally, the downregulation of Nrf2 and the aberrant regulation of the p53-lncRNA H19 signaling pathway further increased miR-200 family in the BPF-exposed pancreas. The miR-200 family directly suppressed Mettl14 and Xiap by targeting their 3' UTR, leading to islet apoptosis. Antioxidant treatment not only elevated m6A levels and insulin contents but also suppressed the miR-200 family in the pancreas, ultimately improving BPF-induced hyperglycemia. Taken together, miR-200 family could serve as a potential oxidative stress-responsive regulator in the pancreas. And moreover, we demonstrated a novel toxicological mechanism in that BPF disrupted the Keap1-Nrf2 redox system to upregulate miR-141/200b/c which controlled pancreatic insulin production and apoptosis via Mettl14 and Xiap, respectively. As the major surrogates of BPA in various applications, BPF was also diabetogenic, which warrants attention in future research.

Diagnostic potential of miR-200 family members in gingival crevicular fluid for chronic periodontitis: correlation with clinical parameters and therapeutic implications.

OBJECTIVE: The purpose of this study was to evaluate the potential of miR-200 family members in gingival crevicular fluid (GCF) as diagnostic biomarkers for chronic periodontitis (CP), aiming to provide valuable insights for the early detection and management of the disease. METHODS: GSE89081 dataset profiled miRNAs in GCF derived from 5 healthy and 5 periodontitis was analyzed by GEO2R. Quantitative real-time PCR was used to quantify the expression levels of miR-200 family members (miR-200a-3p, miR-200a-5p, miR-200b-3p, miR-200b-5p, miR-200c-3p, miR-200c-5p, miR-141-3p, miR-141-5p, and miR-429) in the GCF samples from 103 CP patients and 113 healthy controls. Receiver operating characteristic (ROC) curve analysis was used to evaluate the diagnostic potential of miR-200 family members in differentiating CP patients from healthy controls. RESULTS: By analyzing the GSE89081 dataset, miR-200a-5p, miR-200b-5p and miR-200c-5p were significantly upregulated in GCF of the CP patients compared to the healthy control. In this study, miR-200a-3p, miR-200a-5p, miR-200b-3p, miR-200b-5p, miR-200c-3p, miR-200c-5p were significantly increased in GCF of CP patients compared to the healthy control, while miR-141 and miR-429 did not show significant differences. MiR-200a, -200b and 200c had good diagnostic value, and when these miRNAs were combined, they demonstrated excellent diagnostic value for CP with an AUC of 0.997, sensitivity of 99.03%, and specificity of 98.23%. MiR-200a, -200b and 200c in GCF showed significant and positive correlation with plaque index (PI), gingival index (GI), bleeding on probing (BOP), clinical attachment level (CAL), and probing pocket depth (PPD). CONCLUSION: MiR-200a, -200b and 200c in GCF may serve as potential biomarkers for the early diagnosis of CP, which was correlated with clinical parameters, being therapeutic targets for CP.

Circulating miR-200 family as predictive markers during systemic therapy of metastatic breast cancer.

PURPOSE: Circulating miRNAs can provide valid prognostic and predictive information for breast cancer diagnosis and subsequent management. They may comprise quintessential biomarkers that can be obtained minimally invasively from liquid biopsy in metastatic breast cancer patients. Therefore, they would be clinically crucial for monitoring therapy response, with the goal of detecting early relapse. This study investigated miRNA expression in patients with early and/or late relapse, and the predictive value for assessing overall (OS) and progression-free survival (PFS). METHODS: Forty-seven patients with metastatic breast cancer from the University Women's Hospital Heidelberg were enrolled in this study. Expression of miR-200a, miR-200b, miR-200c, miR-141, and miR-429 was analyzed by RT-qPCR before a new line of systemic therapy and after the first cycle of a respective therapy. Tumor response was assessed every 3 months using the RECIST criteria. Statistical analysis focused on the relation of miR-200s expression and early vs. late cancer relapse in relation to systemic treatment. The association of miRNAs with PFS and OS was investigated. RESULTS: Before starting a new line of systemic therapy, miR-429 (p = 0.024) expression was significantly higher in patients with early relapse (PFS ≤ 4 months) than in patients with late relapse (PFS > 4 months). After one cycle of systemic therapy, miR-200a (p = 0.039), miR-200b (p = 0.003), miR-141 (p = 0.017), and miR-429 (p = 0.010) expression was higher in early than in late progressive cancer. In addition, 4 out of 5 miR-200 family members (miR-200a, miR-200b, miR-141, and miR-429) predicted PFS (p = 0.048, p = 0.008, p = 0.026, and p = 0.016, respectively). Patients with heightened miRNA levels showed a significant reduction in OS and PFS. CONCLUSION: Circulating miR-200s were differentially expressed among patients with late and/or early relapse. 4 of 5 members of the miR-200 family predicted significantly early relapse after systemic treatment. Our results encourage the use of circulating miR-200s as valuable prognostic biomarkers during metastatic breast cancer therapy.

Circulating miR-200 Family and CTCs in Metastatic Breast Cancer before, during, and after a New Line of Systemic Treatment.

The extracellular circulating microRNA (miR)-200 regulates epithelial-mesenchymal transition and, thus, plays an essential role in the metastatic cascade and has shown itself to be a promising prognostic and predictive biomarker in metastatic breast cancer (MBC). Expression levels of the plasma miR-200 family were analyzed in relationship to systemic treatment, circulating tumor cells (CTC) count, progression-free survival (PFS), and overall survival (OS). Expression of miR-200a, miR-200b, miR-200c, miR-141, and miR-429, and CTC status (CTC-positive ≥ 5 CTC/7.5 mL) was assessed in 47 patients at baseline (BL), after the first completed cycle of a new line of systemic therapy (1C), and upon the progression of disease (PD). MiR-200a, miR-200b, and miR-141 expression was reduced at 1C compared to BL. Upon PD, all miR-200s were upregulated compared to 1C. At all timepoints, the levels of miR-200s were elevated in CTC-positive versus CTC-negative patients. Further, heightened miR-200s expression and positive CTC status were associated with poorer OS at BL and 1C. In MBC patients, circulating miR-200 family members decreased after one cycle of a new line of systemic therapy, were elevated during PD, and were indicative of CTC status. Notably, increased levels of miR-200s and elevated CTC count correlated with poorer OS and PFS. As such, both are promising biomarkers for optimizing the clinical management of MBC.

Crosstalk between NRF2 and Dicer through metastasis regulating MicroRNAs; mir-34a, mir-200 family and mir-103/107 family.

Oxidative stress due to generation of reactive oxygen species (ROS) can cause damage to cellular proteins, lipids and DNA, which is one of crucial causes responsible for cancer. Nuclear factor erythroid 2 [NF-E2]-related factor 2 (NRF2) is a transcription factor of a variety of antioxidant and cytoprotective enzymes, so that it reduces the levels of damaging ROS in the cell. Over expression of NRF2 in cancer cells can enhance cancer progression, confer resistance to chemo and radiotherapy, and metastasis through the process of epithelial-to mesenchymal transition (EMT); which is a hallmark of cancer-related death. Dicer, a key component of the microRNAs biogenesis, is a ribonuclease enzyme which involves in maturation of microRNAs that have a role in distinct steps of metastasis cascade. Moreover, Dicer was found to be regulated by ROS/NRF2 interaction to contribute to activation of DNA damage repair mechanism. In addition, Dicer is directly reduced by mir-103/107 family that confers migratory capacity through down-regulation of mir-200 family (mir-200b/mir-200c/mir-429). Mir-200c and mir-34a were predicted to target the repressor of NRF2; Sirt1. On the other hand, mir-200a and mir-141 (mir-200 family) were detected to regulate NRF2 expression. This review highlights the regulation of redox homeostasis that is mediated by NRF2 could be modulated by metastasis regulating microRNAs under the control of Dicer. In addition, NRF2 may indirectly control DNA damage repair and microRNAs processing machinery through the crosstalk between NRF2 and Dicer. Understanding such interrelations could provide and shed light on the significance of microRNA-based therapies that will improve the action of clinically used cancer treatments.

DNA methylation of miR-200 clusters promotes epithelial to mesenchymal transition in human conjunctival epithelial cells.

Epithelial to mesenchymal transition (EMT) contributes to fibrosis associated pathologies including scarring of different ocular tissues. Recently targeting EMT is seen as an appropriate therapeutic approach for different fibrosis related eye diseases such as macular degeneration or glaucoma surgery related fibrosis. Nevertheless, for ocular surface diseases, target genes specific for particular cell type or condition are still undefined. This study aimed to expose the complex regulatory mechanisms that trigger EMT in human conjunctival epithelial (HCjE) cells. EMT was induced by prolonged treatment with two TGF-ß isoforms, TGF-ß1 and TGF-ß2, and their combination. TGF-ß1 showed the strongest potential for initiating EMT in HCjE cells, reflected on morphological changes, cell migration and the levels of mRNA expression of different epithelial (CDH1, OCLN, DSP) and mesenchymal (CDH2, FN1, VIM, SNAI1, ZEB2, TWIST1) marker genes. Co-treatment with the DNA demethylating agent 5-Azacytidine (5-AzaC) was capable of stopping the transition of HCjE cells towards a mesenchymal phenotype, based on morphological features, reduced cell mobility and mRNA and protein expression levels of epithelial and mesenchymal marker genes. An EMT qRT-PCR-based array revealed that EMT induced considerable alterations in gene expression, with downregulation of the majority of epithelial marker genes and upregulation of genes specific for the mesenchymal state. The major effect of 5-AzaC treatment was observed as a suppression of mesenchymal marker genes, suggesting the involvement of upstream negative regulator(s) whose promoter demethylation and subsequent expression will in turn promote EMT switch off. The expression level of miRNAs potentially important for EMT induction was determined using qRT-PCR-based array which pointed at members of miR-200 family as main regulators of EMT process in HCjE cells. 5-AzaC treatment induced increased expression of miR-200a, -200b, -200c and miR-141 towards the control level, indicating important role of DNA methylation in their regulation. The DNA methylation status of both miR-200 family clusters, analyzed with high-resolution melting (HRM) and bisulfite sequencing (Bis-Seq), revealed that TGF-ß1-induced EMT was accompanied by increase in promoter CpG methylation of both miR-200 loci, which was reverted after 5-AzaC treatment. In conclusion, our results indicate that DNA demethylation of promoters of miR-200 loci is critically important for stopping and reverting the EMT in human conjunctival epithelial cells, suggesting the potential for the development of novel epigenetic-based therapeutic strategies for treating conjunctival conditions associated with EMT.

The regulatory network of miR-141 in the inhibition of angiogenesis.

The miR-200 family, consisting of miR-200a/b/c, miR-141, and miR-429, is well known to inhibit epithelial-to-mesenchymal transition (EMT) in cancer invasion and metastasis. Among the miR-200 family members, miR-200a/b/c and miR-429 have been reported to inhibit angiogenesis. However, the role of miR-141 in angiogenesis remains elusive, as contradicting results have been found in different cancer types and tumor models. Particularly, the effect of miR-141 in vascular endothelial cells has not been defined. In this study, we used several in vitro and in vivo models to demonstrate that miR-141 in endothelial cells inhibits angiogenesis. Additional mechanistic studies showed that miR-141 suppresses angiogenesis through multiple targets, including NRP1, GAB1, CXCL12ß, TGFß2, and GATA6, and bioinformatics analysis indicated that miR-141 and its targets comprise a powerful and precise regulatory network to modulate angiogenesis. Taken together, these data not only demonstrate an anti-angiogenic effect of miR-141, further strengthening the critical role of miR-200 family in the process of angiogenesis, but also provides a valuable cancer therapeutic target to control both angiogenesis and EMT, two essential steps in tumor growth and metastasis.

The miR-200b/200a/429 cluster prevents metastasis and induces dormancy in a murine claudin-low mammary tumor cell line.

The miR-200 family of microRNAs consisting of miR-141, miR-200a, miR-200b, miR-200c and miR-429 are emerging as important regulators of breast cancer progression. This family of microRNAs maintain mammary epithelial identity and downregulation of miR-200 expression has been associated with epithelial-to-mesenchymal transition in mammary tumors. Therefore, re-expression of one or more miR-200 family members in mammary tumor cells with mesenchymal characteristics may restore an epithelial phenotype including growth and metastasis suppression. To test this hypothesis, the miR-200b/200a/429 cluster was re-expressed in a murine claudin-low cell line, RJ423. Re-expression of the miR-200b/200a/429 cluster in RJ423 cells significantly suppressed the expression of Vim, Snai1, Twist1, Twist2 and Zeb1, reverted RJ423 cells to a more epithelial morphology and significantly inhibited proliferation in vitro. Moreover, the miR-200b/200a/429 cluster prevented lung metastasis in an experimental metastasis model and although tumor initiation was not prevented, re-expression of the miR-200b/200a/429 cluster induced a dormancy-like state where mammary tumors failed to grow beyond ~150 mm3 or grew extremely slowly following intra-mammary injection. These dormant tumors contained elevated levels of collagen and were highly vascularized. Therefore, re-expression of the miR-200b/200a/429 cluster in the claudin-low mammary tumor cell line, RJ423, is sufficient to alter cell morphology, impair metastasis and induce tumor dormancy.

Epithelial down-regulation of the miR-200 family in fibrostenosing Crohn's disease is associated with features of epithelial to mesenchymal transition.

Intestinal mesenchymal cells deposit extracellular matrix in fibrotic Crohn's disease (CD). The contribution of epithelial to mesenchymal transition (EMT) to the mesenchymal cell pool in CD fibrosis remains obscure. The miR-200 family regulates fibrosis-related EMT in organs other than the gut. E-cadherin, cytokeratin-18 and vimentin expression was assessed using immunohistochemistry on paired strictured (SCD) and non-strictured (NSCD) ileal CD resections and correlated with fibrosis grade. MiR-200 expression was measured in paired SCD and NSCD tissue compartments using laser capture microdissection and RT-qPCR. Serum miR-200 expression was also measured in healthy controls and CD patients with stricturing and non-stricturing phenotypes. Extra-epithelial cytokeratin-18 staining and vimentin-positive epithelial staining were significantly greater in SCD samples (P = 0.04 and P = 0.03, respectively). Cytokeratin-18 staining correlated positively with subserosal fibrosis (P < 0.001). Four miR-200 family members were down-regulated in fresh SCD samples (miR-141, P = 0.002; miR-200a, P = 0.002; miR-200c, P = 0.001; miR-429; P = 0.004); miR-200 down-regulation in SCD tissue was localised to the epithelium (P = 0.001-0.015). The miR-200 target ZEB1 was up-regulated in SCD samples (P = 0.035). No difference in serum expression between patient groups was observed. Together, these observations suggest the presence of EMT in CD strictures and implicate the miR-200 family as regulators. Functional studies to prove this relationship are now warranted.

Interplay Between Long Noncoding RNA ZEB1-AS1 and miR-200s Regulates Osteosarcoma Cell Proliferation and Migration.

In our previous study, we found long noncoding RNA ZEB1-AS1 is upregulated and functions as an oncogene in osteosarcoma. MiR-200 family (miR-200s) functions as tumor suppressor via directly targeting ZEB1 in various cancers. In this study, we further investigate the potential interplay between ZEB1-AS1, miR-200s, and ZEB1 in osteosarcoma. Our results showed that ZEB1-AS1 functions as a molecular sponge for miR-200s and relieves the inhibition of ZEB1 caused by miR-200s. ZEB1-AS1 and miR-200s reciprocally negatively regulate each other. MiR-200s are downregulated in osteosarcoma tissues, and negatively correlated with ZEB1-AS1 and ZEB1 expression levels in osteosarcoma. Functional experiments showed that consistent with ZEB1-AS1 depletion, miR-200s overexpression and ZEB1 depletion both inhibit osteosarcoma cell proliferation and migration. Overexpression of miR-200s partially abolished the effects of ZEB1-AS1 on osteosarcoma cell proliferation and migration. Moreover, the combination of ZEB1-AS1 depletion and miR-200s overexpression significantly inhibits osteosarcoma cell proliferation and migration. In conclusion, this study revealed a novel regulatory mechanism between ZEB1-AS1, miR-200s, and ZEB1. The interplay between ZEB1-AS1 and miR-200s contributes to osteosarcoma cell proliferation and migration, and targeting this interplay could be a promising strategy for osteosarcoma treatment. J. Cell. Biochem. 118: 2250-2260, 2017. © 2017 Wiley Periodicals, Inc.

Plasma miR-122 and miR-200 family are prognostic markers in colorectal cancer.

Circulating microRNAs (miRNAs) have been proposed as minimally invasive prognostic markers for various types of cancers, including colorectal cancer (CRC), the third most diagnosed cancer worldwide. We aimed to evaluate the levels of circulating miRNAs that might serve as markers for CRC prognosis and survival. We included plasma samples of 543 CRC patients with stage I-IV disease from a population-based study carried out in Germany. After comprehensive evaluation of current literature, 95 miRNAs were selected and measured with Custom TaqMan® Array MicroRNA Cards. Plasma samples of non-metastatic and metastatic colon cancer patients, each group consisting of ten patients with 'good' and ten patients with 'bad' prognosis were screened. Identified candidate miRNAs were further validated by RT-qPCR in the whole study cohort. The association of the miRNA levels with patients' survival and the prognostic subtypes was analyzed with uni- and multivariate logistic regression and Cox proportional hazards regression models. Increased miR-122 levels were associated with a 'bad' prognostic subtype in metastatic CRC (Odds ratio: 1.563, 95% confidence interval (CI): 1.038-2.347) and a shorter relapse-free survival and overall survival for non-metastatic (Hazard ratio (HR): 1.370, 95% CI: 1.028-1.825; HR: 1.353, 95% CI: 1.002-1.828) and metastatic (HR: 1.264, 95% CI: 1.050-1.520; HR: 1.292, 95% CI: 1.078-1.548) CRC patients. Additionally, several members of the miR-200 family showed associations with patients' prognosis and correlations to clinicopathological characteristics. The here identified miRNA markers, miR-122 and the miR-200 family members, could be of use in the development of a multi-marker blood test for CRC prognosis.

The Pitx2:miR-200c/141:noggin pathway regulates Bmp signaling and ameloblast differentiation.

The mouse incisor is a remarkable tooth that grows throughout the animal's lifetime. This continuous renewal is fueled by adult epithelial stem cells that give rise to ameloblasts, which generate enamel, and little is known about the function of microRNAs in this process. Here, we describe the role of a novel Pitx2:miR-200c/141:noggin regulatory pathway in dental epithelial cell differentiation. miR-200c repressed noggin, an antagonist of Bmp signaling. Pitx2 expression caused an upregulation of miR-200c and chromatin immunoprecipitation assays revealed endogenous Pitx2 binding to the miR-200c/141 promoter. A positive-feedback loop was discovered between miR-200c and Bmp signaling. miR-200c/141 induced expression of E-cadherin and the dental epithelial cell differentiation marker amelogenin. In addition, miR-203 expression was activated by endogenous Pitx2 and targeted the Bmp antagonist Bmper to further regulate Bmp signaling. miR-200c/141 knockout mice showed defects in enamel formation, with decreased E-cadherin and amelogenin expression and increased noggin expression. Our in vivo and in vitro studies reveal a multistep transcriptional program involving the Pitx2:miR-200c/141:noggin regulatory pathway that is important in epithelial cell differentiation and tooth development.

CRKL oncogene is downregulated by p53 through miR-200s.

Tumor suppressive miRNAs that target oncogenes are frequently downregulated in cancers, and this downregulation leads to oncogene pathway activation. Thus, tumor suppressive miRNAs and their target oncogenes have been proposed as useful targets in cancer treatment. miR-200 family downregulation has been reported in cancer progression and metastasis. The miR-200 family consists of two gene clusters, miR-200b/200a/429 and miR-200c/141, which are located on human chromosomes 1 and 12, respectively. Here, we identified that p53 response elements are located around both clusters of the miR-200 family and confirmed that miR-200s are transcriptional targets of the p53 family. In silico analyses of miRNA targets established the CRKL oncogene as a potential target for miR-200b/200c/429. Moreover, miR-200b/200c/429 inhibited CRKL mRNA and protein expression by directly targeting its 3'-UTR region. Importantly, endogenous CRKL expression was decreased in cancer cells through the introduction of p53 family and endogenous p53 activation. Moreover, the downregulation of CRKL by siRNA inhibited cancer cell growth. The Oncomine database demonstrates that CRKL is overexpressed in a subset of cancer types. Furthermore, CRKL is significantly overexpressed in primary breast cancer tissues harboring mutant TP53. Our results demonstrate that the p53 target miR-200b/200c/429 miRNAs are negative regulators of the CRKL oncogene.

Non Coding RNA Molecules as Potential Biomarkers in Breast Cancer.

The pursuit of minimally invasive biomarkers is a challenging but exciting area of research. Clearly, such markers would need to be sensitive and specific enough to aid in the detection of breast cancer at an early stage, would monitor progression of the disease, and could predict the individual patient's response to treatment. Unfortunately, to date, markers with such characteristics have not made it to the clinic for breast cancer. Past years, many studies indicated that the non-coding part of our genome (the so called 'junk' DNA), may be an ideal source for these biomarkers. In this chapter, the potential use of microRNAs and long non-coding RNAs as biomarkers will be discussed.

The miR-200 Family: Versatile Players in Epithelial Ovarian Cancer.

The role of microRNAs (miRNAs or miRs) in the pathology of epithelial ovarian cancer (EOC) has been extensively studied. Many miRNAs differentially expressed in EOC as compared to normal controls have been identified, prompting further inquiry into their role in the disease. miRNAs belonging to the miR-200 family have repeatedly surfaced over multiple profiling studies. In this review, we attempt to consolidate the data from different studies and highlight mechanisms by which these miRNAs influence progression of metastasis and chemo-resistance in EOC.

Generation of Artificial Blastoids Combining miR-200-Mediated Reprogramming and Mechanical Cues.

In vitro-generated blastocyst-like structures are of great importance since they recapitulate specific features or processes of early embryogenesis, thus avoiding ethical concerns as well as increasing scalability and accessibility compared to the use of natural embryos. Here, we combine cell reprogramming and mechanical stimuli to create 3D spherical aggregates that are phenotypically similar to those of natural embryos. Specifically, dermal fibroblasts are reprogrammed, exploiting the miR-200 family property to induce a high plasticity state in somatic cells. Subsequently, miR-200-reprogrammed cells are either driven towards the trophectoderm (TR) lineage using an ad hoc induction protocol or encapsulated into polytetrafluoroethylene micro-bioreactors to maintain and promote pluripotency, generating inner cell mass (ICM)-like spheroids. The obtained TR-like cells and ICM-like spheroids are then co-cultured in the same micro-bioreactor and, subsequently, transferred to microwells to encourage blastoid formation. Notably, the above protocol was applied to fibroblasts obtained from young as well as aged donors, with results that highlighted miR-200's ability to successfully reprogram young and aged cells with comparable blastoid rates, regardless of the donor's cell age. Overall, the approach here described represents a novel strategy for the creation of artificial blastoids to be used in the field of assisted reproduction technologies for the study of peri- and early post-implantation mechanisms.

GLI family zinc finger protein 2 promotes skin fibroblast proliferation and DNA damage repair by targeting the miR-200/ataxia telangiectasia mutated axis in diabetic wound healing.

Diabetic foot ulcer (DFU) is a serious complication of diabetic patients which negatively affects their foot health. This study aimed to estimate the role and mechanism of the miR-200 family in DNA damage of diabetic wound healing. Human foreskin fibroblasts (HFF-1 cells) were stimulated with high glucose (HG). Db/db mice were utilized to conduct the DFU in vivo model. Cell viability was evaluated using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assays. Superoxide dismutase activity was determined using detection kits. Reactive oxygen species determination was conducted via dichlorodihydrofluorescein-diacetate assays. Enzyme-linked immunosorbent assay was used to evaluate 8-oxo-7,8-dihydro-2'deoxyguanosine levels. Genes and protein expression were analyzed by quantitative real-time polymerase chain reaction, western blotting, or immunohistochemical analyses. Luciferase reporter gene and RNA immunoprecipitation assays determined the interaction with miR-200a/b/c-3p and GLI family zinc finger protein 2 (GLI2) or ataxia telangiectasia mutated (ATM) kinase. HG repressed cell proliferation and DNA damage repair, promoted miR-200a/b/c-3p expression, and suppressed ATM and GLI2. MiR-200a/b/c-3p inhibition ameliorated HG-induced cell proliferation and DNA damage repair repression. MiR-200a/b/c-3p targeted ATM. Then, the silenced ATM reversed the miR-200a/b/c-3p inhibition-mediated alleviative effects under HG. Next, GLI2 overexpression alleviated the HG-induced cell proliferation and DNA damage repair inhibition via miR-200a/b/c-3p. MiR-200a/b/c-3p inhibition significantly promoted DNA damage repair and wound healing in DFU mice. GLI2 promoted cell proliferation and DNA damage repair by regulating the miR-200/ATM axis to enhance diabetic wound healing in DFU.

miRNA and mRNA expression profiling identifies members of the miR-200 family as potential regulators of epithelial-mesenchymal transition in pterygium.

The current study investigates whether microRNA (miRNA) regulators of epithelial-mesenchymal transition (EMT), tissue fibrosis, and angiogenesis are differentially expressed in human primary pterygium. Genome-wide miRNA and mRNA expression profiling of paired pterygium and normal conjunctiva was performed in the context of conventional excision of pterygium with autotransplantation of conjunctiva (n = 8). Quantitative real time polymerase chain reaction (qRT-PCR) was used to validate the expression of key molecules previously detected by microarray. In pterygium, 25 miRNAs and 31 mRNAs were significantly differentially expressed by more than two-fold compared to normal conjunctiva. 14 miRNAs were up-regulated (miR-1246, -486, -451, -3172, -3175, -1308, -1972, -143, -211, -665, -1973, -18a, 143, and -663b), whereas 11 were down-regulated (miR-675, -200b-star, -200a-star, -29b, -200b, -210, -141, -31, -200a, -934, and -375). Unsupervised hierarchical cluster analysis demonstrated that members of the miR-200 family were coexpressed and down-regulated in pterygium. The molecular and cellular functions that were most significant to the miRNA data sets were cellular development, cellular growth and proliferation, and cellular movement. qRT-PCR confirmed the expression of 15 of the 16 genes tested and revealed that miR-429 was down-regulated by more than two-fold in pterygium. The concerted down-regulation of four members from both clusters of the miR-200 family (miR-200a/-200b/-429 and miR-200c/-141), which are known to regulate EMT, and up-regulation of the predicted target and mesenchymal marker fibronectin (FN1), suggest that EMT could potentially play a role in the pathogenesis of pterygium and might constitute promising new targets for therapeutic intervention in pterygium.

Synergistic Effect of miR-200 and Young Extracellular Matrix-based Bio-scaffolds to Reduce Signs of Aging in Senescent Fibroblasts.

Aging is defined as a complex, multifaceted degenerative process that causes a gradual decline of physiological functions and a rising mortality risk with time. Stopping senescence or even rejuvenating the body represent one of the long-standing human dreams. Somatic cell nuclear transfer as well as cell reprogramming have suggested the possibility to slow or even reverse signs of aging. We exploited miR-200 family ability to induce a transient high plasticity state in human skin fibroblasts isolated from old individuals and we investigated whether this ameliorates cellular and physiological hallmarks of senescence. In addition, based on the assumption that extracellular matrix (ECM) provides biomechanical stimuli directly influencing cell behavior, we examine whether ECM-based bio-scaffolds, obtained from decellularized ovaries of young swine, stably maintain the rejuvenated phenotype acquired by cells after miR-200 exposure. The results show the existence of multiple factors that cooperate to control a unique program, driving the cell clock. In particular, miR-200 family directly regulates the molecular mechanisms erasing cell senescence. However, this effect is transient, reversible, and quickly lost. On the other hand, the use of an adequate young microenvironment stabilizes the miR-200-mediated rejuvenating effects, suggesting that synergistic interactions occur among molecular effectors and ECM-derived biomechanical stimuli. The model here described is a useful tool to better characterize these complex regulations and to finely dissect the multiple and concurring biochemical and biomechanical cues driving the cell biological clock.