Down-regulation of ATF1 leads to early neuroectoderm differentiation of human embryonic stem cells by increasing the expression level of SOX2.

We reveal by high-throughput screening that activating transcription factor 1 (ATF1) is a novel pluripotent regulator in human embryonic stem cells (hESCs). The knockdown of ATF1 expression significantly up-regulated neuroectoderm (NE) genes but not mesoderm, endoderm, and trophectoderm genes. Of note, down-regulation or knockout of ATF1 with short hairpin RNA (shRNA), small interfering RNA (siRNA), or clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) was sufficient to up-regulate sex-determining region Y-box (SOX)2 and paired box 6 (PAX6) expression under the undifferentiated or differentiated conditions, whereas overexpression of ATF1 suppressed NE differentiation. Endogenous ATF1 was spontaneously down-regulated after d 1-3 of neural induction. By double-knockdown experiments, up-regulation of SOX2 was critical for the increase of PAX6 and SOX1 expression in shRNA targeting Atf1 hESCs. Using the luciferase reporter assay, we identified ATF1 as a negative transcriptional regulator of Sox2 gene expression. A novel function of ATF1 was discovered, and these findings contribute to a broader understanding of the very first steps in regulating NE differentiation in hESCs.-Yang, S.-C., Liu, J.-J., Wang, C.-K., Lin, Y.-T., Tsai, S.-Y., Chen, W.-J., Huang, W.-K., Tu, P.-W. A., Lin, Y.-C., Chang, C.-F., Cheng, C.-L., Lin, H., Lai, C.-Y., Lin, C.-Y., Lee, Y.-H., Chiu, Y.-C., Hsu, C.-C., Hsu, S.-C., Hsiao, M., Schuyler, S. C., Lu, F. L., Lu, J. Down-regulation of ATF1 leads to early neuroectoderm differentiation of human embryonic stem cells by increasing the expression level of SOX2.

Determinants of satisfaction following total hip arthroplasty in patients with ankylosing spondylitis.

BACKGROUND: The aim was to explore the factors contributing to a high degree of satisfaction following total hip arthroplasty in patients with ankylosing spondylitis and hip joint fusion. METHODS: From January 2001 to June 2015, 43 patients with ankylosing spondylitis were treated with total hip arthroplasty. The patients were divided into two groups, including 20 patients in non-fusion groups, with 25 cases of hip non-fusion, and 23 patients in fusion groups, with 40 cases of hip fusion. Patients were followed for an average of 27 months. Harris hip scores (HHSs) and Barthel scores were recorded pre-operatively and post-operatively. The improvement rates of both groups were calculated, and the data were compared and analyzed. The Self-Administered Patient Satisfaction Scale (SAPS) was used to assess the degree of satisfaction in each group. RESULTS: The post-operative HHS and Barthel scores of two groups both significantly improved. In non-fusion groups compared with fusion groups, there were significant differences in the pre-operative HHSs, the improvement of the HHSs, the pre-operative Barthel score, the improvement of the Barthel score, and the satisfaction score. However, there were no significant differences in the post-operative HHSs and Barthel scores. CONCLUSION: Patients in the hip fusion group had higher satisfaction scores. The improvements in joint function and self-care ability after total hip arthroplasty are the decisive factors in determining patient satisfaction for patients with ankylosing spondylitis combined with hip fusion.

Spatial sensitivity distribution assessment and Monte Carlo simulations for needle-based bioimpedance imaging during venipuncture using the finite element method.

Despite being among the most common medical procedures, needle insertions suffer from a high error rate. Impedance measurements using electrode-equipped needles offer promise for improved tissue targeting and reduced errors. Impedance visualization usually requires an extensive pre-measured impedance dataset for tissue differentiation and knowledge of the electric fields contributing to the resulting impedances. This work presents two finite element simulation approaches for both problems. The first approach describes the generation of a multitude of impedances with Monte Carlo simulations for both, homogeneous and inhomogeneous tissue to circumvent the need to rely on previously measured data. These datasets could be used for tissue discrimination. The second method describes the simulation of the spatial sensitivity distribution of an electrode layout. Two singularity analysis methods were employed to determine the bulk of the sensitivity within a finite volume, which in turn enables consistent 3D visualization. The modeled electrode layout consists of 12 electrodes radially placed around a hypodermic needle. Electrical excitation was simulated using two neighboring electrodes for current carriage and voltage pickup, which resulted in 12 distinct bipolar excitation states. Both, the impedance simulations and the respective singularity analysis methods were compared with each other. The results show that the statistical spread of impedances is highly dependent on the tissue type and its inhomogeneities. The bounded bulk of sensitivities of both methods are of similar extent and symmetry. Future models should incorporate more detailed tissue properties such as anisotropy or changing material properties due to tissue deformation to gain more accurate predictions.

Paternally inherited gsα mutation impairs adipogenesis and potentiates a lean phenotype in vivo.

Paternally inherited inactivating mutations of the GNAS gene have been associated with a rare and disabling genetic disorder, progressive osseous heteroplasia, in which heterotopic ossification occurs within extraskeletal soft tissues, such as skin, subcutaneous fat, and skeletal muscle. This ectopic bone formation is hypothesized to be caused by dysregulated mesenchymal progenitor cell differentiation that affects a bipotential osteogenic-adipogenic lineage cell fate switch. Interestingly, patients with paternally inherited inactivating mutations of GNAS are uniformly lean. Using a mouse model of Gsα-specific exon 1 disruption, we examined whether heterozygous inactivation of Gnas affects adipogenic differentiation of mesenchymal precursor cells from subcutaneous adipose tissues (fat pad). We found that paternally inherited Gsα inactivation (Gsα(+/p-) ) impairs adipogenic differentiation of adipose-derived stromal cells (ASCs). The Gsα(+/p-) mutation in ASCs also decreased expression of the adipogenic factors CCAAT-enhancer-binding protein (C/EBP)ß, C/EBPα, peroxisome proliferator-activated receptor gamma, and adipocyte protein 2. Impaired adipocyte differentiation was rescued by an adenylyl cyclase activator, forskolin, and provided evidence that Gsα-cAMP signals are necessary in early stages of this process. Supporting a role for Gnas in adipogenesis in vivo, fat tissue weight and expression of adipogenic genes from multiple types of adipose tissues from Gsα(+/p-) mice were significantly decreased. Interestingly, the inhibition of adipogenesis by paternally inherited Gsα mutation also enhances expression of the osteogenic factors, msh homeobox 2, runt-related transcription factor 2, and osteocalcin. These data support the hypothesis that Gsα plays a critical role in regulating the balance between fat and bone determination in soft tissues, a finding that has important implications for a wide variety of disorders of osteogenesis and adipogenesis.

Needle-Based Electrical Impedance Imaging Technology for Needle Navigation.

Needle insertion is a common procedure in modern healthcare practices, such as blood sampling, tissue biopsy, and cancer treatment. Various guidance systems have been developed to reduce the risk of incorrect needle positioning. While ultrasound imaging is considered the gold standard, it has limitations such as a lack of spatial resolution and subjective interpretation of 2D images. As an alternative to conventional imaging techniques, we have developed a needle-based electrical impedance imaging system. The system involves the classification of different tissue types using impedance measurements taken with a modified needle and the visualization in a MATLAB Graphical User Interface (GUI) based on the spatial sensitivity distribution of the needle. The needle was equipped with 12 stainless steel wire electrodes, and the sensitive volumes were determined using Finite Element Method (FEM) simulation. A k-Nearest Neighbors (k-NN) algorithm was used to classify different types of tissue phantoms with an average success rate of 70.56% for individual tissue phantoms. The results showed that the classification of the fat tissue phantom was the most successful (60 out of 60 attempts correct), while the success rate decreased for layered tissue structures. The measurement can be controlled in the GUI, and the identified tissues around the needle are displayed in 3D. The average latency between measurement and visualization was 112.1 ms. This work demonstrates the feasibility of using needle-based electrical impedance imaging as an alternative to conventional imaging techniques. Further improvements to the hardware and the algorithm as well as usability testing are required to evaluate the effectiveness of the needle navigation system.

Catechol-O-methyltransferase-mediated metabolism of 4-hydroxyestradiol inhibits the growth of human renal cancer cells through the apoptotic pathway.

Long-term exposure to estrogen and its metabolites may play an important role in renal cell carcinogenesis. Catechol-O-methyltransferase (COMT) participates in the estrogen metabolism pathway by neutralizing toxic substances. Although reduced COMT activity has been suggested to be a risk factor for estrogen-associated cancers, no studies have investigated the biological significance of COMT in the pathogenesis of human renal cell cancers (RCCs). We initially found that COMT levels are significantly decreased in human RCC tissues and cells suggesting it plays a suppressive role in tumor development. However, transient overexpression of COMT has no functional effect on RCC cell lines. In contrast, when cells overexpressing COMT are treated with its substrate 4-hydroxyestradiol (4-OHE(2)), growth is inhibited by apoptotic cell death. We also found that COMT overexpression combined with 4-OHE(2) induces upregulation of growth arrest- and DNA damage-inducible protein α (GADD45α). We further show that downregulation of GADD45α by a small interfering RNA-mediated approach inhibits cell death, indicating the essential role of GADD45α in the underlying mechanism of COMT action in response to 4-OHE(2). Finally, 4-methoxyestradiol fully reproduces the antiproliferative function of COMT with 4-OHE(2) by promoting GADD45α induction. Together, these findings show that COMT in the presence of 4-OHE(2) prevents RCC cell proliferation by enhancing apoptosis and that GADD45α plays a critical role in the COMT-mediated inhibition of RCC.

Semi-supervised learning with natural language processing for right ventricle classification in echocardiography-a scalable approach.

We created a deep learning model, trained on text classified by natural language processing (NLP), to assess right ventricular (RV) size and function from echocardiographic images. We included 12,684 examinations with corresponding written reports for text classification. After manual annotation of 1489 reports, we trained an NLP model to classify the remaining 10,651 reports. A view classifier was developed to select the 4-chamber or RV-focused view from an echocardiographic examination (n = 539). The final models were two image classification models trained on the predicted labels from the combined manual annotation and NLP models and the corresponding echocardiographic view to assess RV function (training set n = 11,008) and size (training set n = 9951. The text classifier identified impaired RV function with 99% sensitivity and 98% specificity and RV enlargement with 98% sensitivity and 98% specificity. The view classification model identified the 4-chamber view with 92% accuracy and the RV-focused view with 73% accuracy. The image classification models identified impaired RV function with 93% sensitivity and 72% specificity and an enlarged RV with 80% sensitivity and 85% specificity; agreement with the written reports was substantial (both κ = 0.65). Our findings show that models for automatic image assessment can be trained to classify RV size and function by using model-annotated data from written echocardiography reports. This pipeline for auto-annotation of the echocardiographic images, using a NLP model with medical reports as input, can be used to train an image-assessment model without manual annotation of images and enables fast and inexpensive expansion of the training dataset when needed.

MicroRNA-145 is regulated by DNA methylation and p53 gene mutation in prostate cancer.

MiR-145 is downregulated in various cancers including prostate cancer. However, the underlying mechanisms of miR-145 downregulation are not fully understood. Here, we reported that miR-145 was silenced through DNA hypermethylation and p53 mutation status in laser capture microdissected (LCM) prostate cancer and matched adjacent normal tissues. In 22 of 27 (81%) prostate tissues, miR-145 was significantly downregulated in the cancer compared with the normal tissues. Further studies on miR-145 downregulation mechanism showed that miR-145 is methylated at the promoter region in both prostate cancer tissues and 50 different types of cancer cell lines. In seven cancer cell lines with miR-145 hypermethylation, 5-aza-2'-deoxycytidine treatment dramatically induced miR-145 expression. Interestingly, we also found a significant correlation between miR-145 expression and the status of p53 gene in both LCM prostate tissues and 47 cancer cell lines. In 29 cell lines with mutant p53, miR-145 levels were downregulated in 28 lines (97%), whereas in 18 cell lines with wild-type p53 (WT p53), miR-145 levels were downregulated in only 6 lines (33%, P < 0.001). Electrophoretic mobility shift assay showed that p53 binds to the p53 response element upstream of miR-145, but the binding was inhibited by hypermethylation. To further confirm that p53 binding to miR-145 could regulate miR-145 expression, we transfected WT p53 and MUT p53 into PC-3 cells and found that miR-145 is upregulated by WT p53 but not with MUTp53. The apoptotic cells are increased after WT p53 transfection. In summary, this is the first report documenting that downregulation of miR-145 is through DNA methylation and p53 mutation pathways in prostate cancer.

Shp2 acts downstream of SDF-1alpha/CXCR4 in guiding granule cell migration during cerebellar development.

Shp2 is a non-receptor protein tyrosine phosphatase containing two Src homology 2 (SH2) domains that is implicated in intracellular signaling events controlling cell proliferation, differentiation and migration. To examine the role of Shp2 in brain development, we created mice with Shp2 selectively deleted in neural stem/progenitor cells. Homozygous mutant mice exhibited early postnatal lethality with defects in neural stem cell self-renewal and neuronal/glial cell fate specification. Here we report a critical role of Shp2 in guiding neuronal cell migration in the cerebellum. In homozygous mutants, we observed reduced and less foliated cerebellum, ectopic presence of external granule cells and mispositioned Purkinje cells, a phenotype very similar to that of mutant mice lacking either SDF-1alpha or CXCR4. Consistently, Shp2-deficient granule cells failed to migrate toward SDF-1alpha in an in vitro cell migration assay, and SDF-1alpha treatment triggered a robust induction of tyrosyl phosphorylation on Shp2. Together, these results suggest that although Shp2 is involved in multiple signaling events during brain development, a prominent role of the phosphatase is to mediate SDF-1alpha/CXCR4 signal in guiding cerebellar granule cell migration.

Polymorphisms of MLH1 in benign prostatic hyperplasia and sporadic prostate cancer.

Mismatch repair is one of several DNA repair pathways of which defects may lead to cancer. We hypothesize that polymorphisms of the MLH1 gene can be a risk factor for benign prostatic hyperplasia (BPH) and prostate cancer. The genetic distribution of MLH1 polymorphisms that lead to amino acid changes at codons 132, 219, 384, and 723 were analyzed in BPH and sporadic prostate cancer patients, and compared to healthy controls from an Asian population. These experiments demonstrate a protective role for the codon 384 variant allele against prostate cancer (P=0.031) but not BPH when compared to normal controls and furthermore, an inverse association was observed with stage (P=0.074) and grade (P=0.056) of cancer. This is the first report that demonstrates a protective effect for the race-related MLH1 polymorphism at codon 384 against prostate cancer and these results are important in understanding their role in this disease.

The Role of Primary Care Physicians in Providing End-of-Life Care.

BACKGROUND: Primary care physicians (PCPs) frequently have long-term relationships with patients as well as their families. As such they are well positioned to care for their patients at the end of their lives. As the number of patients in need of end-of-life care continues to grow, it is critical to understand how PCPs can fulfill that need. The purpose of our study is to perform a narrative review of the literature and develop a theoretical model delineating the overarching roles played by PCPs in caring for patients at the end of life. METHODS: For this narrative review, the authors searched Medline (PubMed), Embase, Cochrane Library, and Scopus up to March 22, 2017. Articles were not limited by geography. RESULTS: Review of existing literature generally supports 4 broad categories as the primary roles for PCP involvement in end of life: pain and symptom management; information management, including transmitting and clarifying information, setting care priorities, and assisting patients with treatment decisions; coordinating care and collaborating with other providers; and addressing patients' social, emotional, and spiritual needs. CONCLUSIONS: Based on the results of this review, PCPs provide a wide range of services to patients at the end of life. Promoting the provision of the full scope of services by PCPs will help ensure improved continuity of care while providing the highest quality of care for patients, both in the United States and around the world.

Diagnostic Performance Using Automated Breast Ultrasound System for Breast Cancer in Chinese Women Aged 40 Years or Older: A Comparative Study.

The purpose of this study was to investigate the diagnostic performance of the automated breast ultrasound system (ABUS) compared with hand-held ultrasonography (HHUS) and mammography (MG) for breast cancer in women aged 40 y or older. A total of 594 breasts in 385 patients were enrolled in the study. HHUS, ABUS and MG exams were performed for these patients. Follow-up and pathologic findings were used as the reference standard. Based on the reference standard, 519 units were benign or normal and 75 were malignant. The sensitivity, specificity, accuracy and Youden index were 97.33%, 89.79%, 90.74% and 0.87 for HHUS; 90.67%, 92.49%, 92.26% and 0.83 for ABUS; 84.00%, 92.87%, 91.75% and 0.77 for MG, respectively. The specificity of ABUS was significantly superior to that of HHUS (p = 0.024). The area under the receiver operating characteristic curve was 0.936 for HHUS, which was the highest, followed by 0.916 for ABUS and 0.884 for MG. However, the difference was not statistically significant (p > 0.05). In conclusion, the diagnostic performance of ABUS for breast cancer was equivalent to HHUS and MG and potentially can be used as an alternative method for breast cancer diagnosis.

Dual roles of Cripto as a ligand and coreceptor in the nodal signaling pathway.

The EGF-CFC gene Cripto encodes an extracellular protein that has been implicated in the signaling pathway for the transforming growth factor beta (TGF beta) ligand Nodal. Although recent findings in frog and fish embryos have suggested that EGF-CFC proteins function as coreceptors for Nodal, studies in cell culture have implicated Cripto as a growth factor-like signaling molecule. Here we reconcile these apparently disparate models of Cripto function by using a mammalian cell culture assay to investigate the signaling activities of Nodal and EGF-CFC proteins. Using a luciferase reporter assay, we found that Cripto has activities consistent with its being a coreceptor for Nodal. However, Cripto can also function as a secreted signaling factor in cell coculture assays, suggesting that it may also act as a coligand for Nodal. Furthermore, we found that the ability of Cripto to bind to Nodal and mediate Nodal signaling requires the addition of an O-linked fucose monosaccharide to a conserved site within EGF-CFC proteins. We propose a model in which Cripto has dual roles as a coreceptor as well as a coligand for Nodal and that this signaling interaction with Nodal is regulated by an unusual form of glycosylation. Our findings highlight the significance of extracellular modulation of ligand activity as an important means of regulating TGF beta signaling pathways during vertebrate development.

Stem cell therapy for hearing loss: Math1 overexpression in VOT-E36 cells.

HYPOTHESIS: VOT-E36 cells acquire mechanosensitivity after mammalian atonal homolog 1 (Math1) overexpression. BACKGROUND: VOT-E36 cells are derived from a population of epithelial cells in the ventral region of the otocyst at embryonic Day 10.5, before hair cell differentiation. These cells express a number of specific molecular markers for hair cells under both proliferation and differentiation states. Overexpression of Math1 can convert nonsensory epithelial cells into hair cells in the cochlea. Based on this information, we tested whether VOT-E36 cells can be converted into hair cells by Math1 overexpression. METHODS: Using reverse transcriptase-polymerase chain reaction-based analysis, we first compared the expression patterns of various molecular markers for hair cell development in VOT-E36 cells between proliferation and differentiation states, and also before and after overexpression of Math1. Subsequently, with a standard calcium imaging method, we examined whether VOT-E36 cells overexpressing Math1 could detect mechanical vibrations and activate spiral ganglion neurons in a coculture model. In addition, using confocal and scanning electron microscopes, we examined morphologic changes of VOT-E36 cells after Math1 overexpression. RESULTS: Consistent with previous reports, this study has shown that VOT-E36 cells express a number of specific molecular markers for hair cells in both proliferation and differentiation states. Under appropriate culture conditions, Math1 is transiently expressed in this cell line during conditional differentiation. In VOT-E36 cells overexpressing Math1, the normal expression pattern of certain molecular markers for mature hair cells is partially restored. Interestingly, after coculture with spiral ganglion neurons, VOT-E36 cells overexpressing Math1 are able to respond to mechanical vibrations and activate spiral ganglion neurons. Possible molecular mechanisms underlying this novel finding have been explored. CONCLUSION: Math1 overexpression can partially restore presumably downstream signaling cascades for normal hair cell differentiation in VOT-E36 cells, which are able to detect mechanical vibrations after being cocultured with spiral ganglion neurons.

Reprogramming of single-cell-derived mesenchymal stem cells into hair cell-like cells.

HYPOTHESIS: Adult mesenchymal stem cells (MSCs) can be converted into hair cell-like cells by transdetermination. BACKGROUND: Given the fundamental role sensory hair cells play in sound detection and the irreversibility of their loss in mammals, much research has focused on developing methods to generate new hair cells as a means of treating permanent hearing loss. Although MSCs can differentiate into multiple cell lineages, no efficient means of reprogramming them into sensory hair cells exists. Earlier work has shown that the transcription factor Atoh1 is necessary for early development of hair cells, but it is not clear whether Atoh1 can be used to convert MSCs into hair cells. METHODS: Clonal MSC cell lines were established and reprogrammed into hair cell-like cells by a combination of protein transfer, adenoviral based gene transfer, and co-culture with neurons. During transdetermination, inner ear molecular markers were analyzed using reverse transcriptase-polymerase chain reaction, and cell structures were examined using immunocytochemistry. RESULTS: Atoh1 overexpression in MSCs failed to convert MSCs into hair cell-like cells, suggesting that the ability of Atoh1 to induce hair cell differentiation is context dependent. Because Atoh1 overexpression successfully transforms VOT-E36 cells into hair cell-like cells, we modified the cell context of MSCs by performing a total protein transfer from VOT-E36 cells before overexpressing Atoh1. The modified MSCs were transformed into hair cell-like cells and attracted contacts from spiral ganglion neurons in a co-culture model. CONCLUSION: We established a new procedure, consisting of VOT-E36 protein transfer, Atoh1 overexpression, and co-culture with spiral ganglion neurons, which can transform MSCs into hair cell-like cells.

Effects of Salivae Miltiorrhizae Liguspyragine Hydrochloride and Glucose Injection on the levels of main platelet thrombin receptors in chronic haemodialysis patients.

OBJECTIVE: To investigate the effects of Salvia Miltiorrhiza Liguspyragine Hydrochloride and Glucose Injection (, SLGI) on the expression of platelet membrane receptors proteinase-activated receptor-1 (PAR1) and proteinase-activated receptor-4 (PAR4) in end-stage renal disease (ESRD) patients on chronic haemodialysis (HD). METHODS: Eighty-six ESRD patients on HD (treated group) were treated with SLGI, 7 days as one therapeutic course, for two successive courses. The previous therapies were unchanged. Flow cytometry was used to assess the expression of platelet PAR1 and PAR4 in the patients, and turbidity method was used to determine the platelet maximum aggregation rate (MAR). Meanwhile, renal function was measured. The final data were compared with those before treatment and with those in the normal control group (54 healthy subjects). RESULTS: Compared with the normal control group, the expressions of PAR1 and PAR4 and platelet MAR in ESRD patients on HD was significantly higher before treatment (P=0.001, P=0.006, and P=0.008); after treatment with SLGI, the above indices in patients were remarkably decreased (P=0.036 and P=0.046), except PAR4 (P=0.067), but still higher than those in the normal control group, however, it was not statistically significant. CONCLUSIONS: (1) The overexpression of PAR1 and PAR4 might lead to increased platelet aggregation and this could be one of the reasons for the thrombotic events in ESRD patients on HD. (2) SLGI was able to down-regulate the expression of PAR1 in ESRD patients on HD, improve platelet function, and regulate platelet activation.

MicroRNAs 221/222 and genistein-mediated regulation of ARHI tumor suppressor gene in prostate cancer.

ARHI is an imprinted tumor suppressor gene and is downregulated in various malignancies. However, ARHI expression, function, and mechanisms of action in prostate cancer have not been reported. Here, we report that ARHI mRNA and protein levels were downregulated in prostate cancer tissues compared with adjacent normal tissues. Overexpression of ARHI inhibited cell proliferation, colony formation, invasion, and induced apoptosis. Further studies on a new mechanism of ARHI downregulation showed a significant inverse relationship between ARHI and miR-221 and 222, which were upregulated in prostate cancer cell lines. Transfection of miR-221 and 222 inhibitors into PC-3 cells caused a significant induction of ARHI expression. A direct interaction of miR-221 or 222 with a target site on the 3'UTR of ARHI was confirmed by a dual luciferase pMIR-REPORT assay. Finally, we also found that genistein upregulates ARHI by downregulating miR-221 and 222 in PC-3 cells. In conclusion, ARHI is a tumor suppressor gene downregulated in prostate cancer, and overexpression of ARHI can inhibit cell proliferation, colony formation, and invasion. This study demonstrates for the first time that prostate cancer cells have decreased level of ARHI which could be caused by direct targeting of 3'UTR of ARHI by miR221/222. Genistein, a potential nontoxic chemopreventive agent, restores expression of ARHI and may be an important dietary therapeutic agent for treating prostate cancer.

Regulation of minichromosome maintenance gene family by microRNA-1296 and genistein in prostate cancer.

The minichromosome maintenance (MCM) gene family is essential for DNA replication and is frequently upregulated in various cancers. Here, we examined the role of MCM2 in prostate cancer and the effect of microRNA-1296 (miR-1296), genistein, and trichostatin A (TSA) on the MCM complex. Profiling results showed that expression of MCM genes was higher in tumor samples. Genistein and TSA significantly downregulated the expression of all MCM genes. Genistein, TSA, and small interfering RNA duplexes caused a significant decrease in the S phase of the cell cycle. There was also downregulation of CDT1, CDC7, and CDK2 genes, which govern loading of the MCM complex on chromatin. We also found that miR-1296 was significantly downregulated in prostate cancer samples. In PC3 cells, inhibition of miR-1296 upregulated both MCM2 mRNA and protein, whereas overexpression caused a significant decrease in MCM2 mRNA, protein, and the S phase of the cell cycle. MCM genes are excellent anticancer drug targets because they are essential DNA replication factors that are highly expressed in cancer cells. This is the first report showing anti-MCM effect by miR-1296, genistein, and TSA. TSA is undergoing clinical trials as a prostate cancer treatment but has high toxicity. Genistein, a natural, nontoxic dietary isoflavone, may be an advantageous therapeutic agent for treating prostate cancer. The use of RNA interference is currently being implemented as a gene-specific approach for molecular medicine. The specific downregulation of oncogenes by miR may contribute to novel therapeutic approaches in the treatment of prostate cancer.

Functional significance of secreted Frizzled-related protein 1 in metastatic renal cell carcinomas.

The secreted Frizzled-related protein 1 (SFRP1) is a Wingless-type (Wnt) antagonist that has been associated with various malignancies, including renal cell carcinomas (RCC). However, the functional significance of SFRP1 has never been investigated in metastatic RCC. Here, we investigated the role of this molecule in kidney cancer progression and metastasis. Using Wnt pathway-focused cDNA expression profiling in normal renal, primary RCC, and metastatic RCC cell lines, we identified that SFRP1 is up-regulated in metastatic RCC. SFRP1 overexpression in metastatic RCC was confirmed by immunostaining in renal tissues. We explored the molecular mechanisms underlying SFRP1 up-regulation by analyzing DNA methylation and histone modification patterns on SFRP1 promoter. We found that this gene is unmethylated/hypomethylated and enriched in activating histone modifications in metastatic RCC. To understand the functional significance of SFRP1 overexpression in metastatic RCC with regard to tumorigenesis, we used a small interfering RNA-mediated approach to knockdown the gene and monitored cellular proliferation, apoptosis, and metastatic behavior. Proliferation was unaltered and apoptosis increased on attenuation of SFRP1 expression. Also, SFRP1 depletion decreased the invasive potential of the metastatic RCC cell line, suggesting that the overexpression of this Wnt antagonist may be related to invasiveness and metastatic behavior in RCC. We investigated the molecular basis of the role of SFRP1 in invasion and metastasis and found that matrix metalloproteinase MMP10 is regulated by SFRP1. In conclusion, our data suggest that SFRP1 plays a role in the metastatic potential of RCC. The present findings may be important in the design of treatment modalities for metastatic RCC.