MiR-34a modulates ErbB2 in breast cancer.

Breast cancer is the second highest cause of carcinoma-related death caused by distant metastasis in women. Estrogen receptor (ER), human epidermal growth factor receptor 2, (HER2) and progesterone receptor (PR) are three classified makers of breast cancer, which are defined as ER+, HER2+, and the most serious ER-PR-HER2- (triple-negative). It is well known that ErbB2 (V-Erb-B2 avian erythroblastic leukemia viral oncogene homolog 2) plays an important part in breast cancer. However, the molecular mechanisms underlying ErbB2 action needs to be well studied. In this report, we discovered that the decreased expression levels of miR-34a were inversely correlated with the increased ErbB2 levels in breast cancer. A luciferase reporter assay was done to understand the potential correlation between ErbB2 and miR-34a. Over-expression of miR-34a reduces ErbB2 expression and suppresses breast cancer cell invasion and growth in vitro. What's more, reduced expression of ErbB2 inhibits breast Cancer cell proliferation and re-expression of ErbB2 reversed miR-34a-dependent tumor suppression. Meanwhile, miR-34a levels were correlated inversely with breast cancer malignancy. Our study demonstrates that miR-34a, like ErbB2, might be a diagnostic target in breast cancer.

MiR-145 directly targets p70S6K1 in cancer cells to inhibit tumor growth and angiogenesis.

MiR-145 can regulate cell apoptosis, proliferation, neural development and stem cell differentiation. Previous studies indicate that miR-145 is downregulated in human colon cancer cells. However, the molecular mechanisms of miR-145 used to regulate colon carcinogenesis and angiogenesis remain to be clarified. Here, we show that the expression of miR-145 is downregulated in colon and ovarian cancer tissues and cell lines. MiR-145 inhibits p70S6K1 post-transcriptional expression by binding to its 3'-UTR. The angiogenic factors hypoxia-inducible factor 1 (HIF-1) and vascular endothelial growth factor (VEGF), which are downstream molecules of p70S6K1, are decreased by miR-145 overexpression. P70S6K1 rescues miR-145-suppressed HIF-1 and VEGF levels, tumorigenesis and tumor angiogenesis. Furthermore, the miR-145 level is inversely correlated with the amount of p70S6K1 protein in colon cancer tissues. Taken together, these studies suggest that miR-145 serves as a tumor suppressor which downregulates HIF-1 and VEGF expression by targeting p70S6K1, leading to the inhibition of tumor growth and angiogenesis. The miR-145 rescue could be a rationale for therapeutic applications in colon cancer in the future.

The Effectiveness of the Moving to Emptiness Technique on Clients Who Need Help During the COVID-19 Pandemic: A Real-World Study.

With Western therapeutic techniques prevailing in Chinese therapies, some techniques that include Chinese traditional cultural features are required since some cultural factors are not considered in the Western method. Our study introduced a new technique, the moving to emptiness technique (MET), which combines Western structural progress and core factors of Chinese culture. Seventeen therapists treated 107 clients with the MET. Clients reported their target symptoms initially, and therapists helped them transfer invisible symptoms to perceivable stuff and remove their jarring stuff using the psychological emptiness area. At the end of the consultations, we found that MET could eliminate symptoms immediately. By grouping target symptoms according to their frequency, the results showed that clients in the high-frequency symptom group had higher rehabilitation rates than those in the low-frequency symptom group. Additionally, the results of the bereavement group were better than those of the non-bereavement group, indicating that the MET can significantly alleviate clients' target symptoms. In future studies, the replication and stability of the MET can be assessed by integrating questionnaires, experimental designs, and neurological equipment.

Predicting miRNA's target from primary structure by the nearest neighbor algorithm.

We used a machine learning method, the nearest neighbor algorithm (NNA), to learn the relationship between miRNAs and their target proteins, generating a predictor which can then judge whether a new miRNA-target pair is true or not. We acquired 198 positive (true) miRNA-target pairs from Tarbase and the literature, and generated 4,888 negative (false) pairs through random combination. A 0/1 system and the frequencies of single nucleotides and di-nucleotides were used to encode miRNAs into vectors while various physicochemical parameters were used to encode the targets. The NNA was then applied, learning from these data to produce a predictor. We implemented minimum redundancy maximum relevance (mRMR) and properties forward selection (PFS) to reduce the redundancy of our encoding system, obtaining 91 most efficient properties. Finally, via the Jackknife cross-validation test, we got a positive accuracy of 69.2% and an overall accuracy of 96.0% with all the 253 properties. Besides, we got a positive accuracy of 83.8% and an overall accuracy of 97.2% with the 91 most efficient properties. A web-server for predictions is also made available at http://app3.biosino.org:8080/miRTP/index.jsp.

MicroRNA-15b regulates cell cycle progression by targeting cyclins in glioma cells.

MicroRNAs (miRNAs) are non-protein-coding RNAs that function as post-transcriptional gene regulators. Recent evidence has shown that miRNA plays a pivotal role in the development of many cancers including glioma, a lethal brain cancer. We have recently compared the miRNA expression profiles between normal brain and glioma tissues from Chinese patients by miRNA microarray and identified a panel of differentially expressed miRNAs. Here, we studied the function of one miRNA, miR-15b, in glioma carcinogenesis and elucidated its downstream targets. Over-expression of miR-15b resulted in cell cycle arrest at G0/G1 phase while suppression of miR-15b expression resulted in a decrease of cell populations in G0/G1 and a corresponding increase of cell populations in S phase. We further showed that CCNE1 (encoding cyclin E1) is one of the downstream targets of miR-15b. Taken together, our findings indicate that miR-15b regulates cell cycle progression in glioma cells by targeting cell cycle-related molecules.

Two-dimensional differential gel electrophoresis/analysis of diethylnitrosamine induced rat hepatocellular carcinoma.

Diethylnitrosamine (DEN) is a known carcinogen that can alkylate DNA molecules. In rats, DEN-induced hepatocellular carcinoma (HCC) model is well established. In this study, we used a two-dimensional differential gel electrophoresis (2D-DIGE) system and liquid chromatography/mass spectrometry/mass spectrometry to identify the differential expression protein profiles between the DEN-induced HCC and healthy liver cells. Western blotting and semiquantitative RT-PCR were used to further confirm the results. Seventeen differentially expressed spots were identified in DEN-induced HCC cells. Among all, the most prominent upregulated proteins include the members of the glutathione S-transferase super family, aldo-keto reductase superfamily and proteins involved in the response to oxidative stress. Downregulation was observed in 2 proteins that were known to contribute to hepatic dysfunction. This study provides the first comprehensive protein profiling of the DEN-induced HCC in rats. This model simulates the differential protein expression of human HCC and may be useful for further understanding the mechanism of HCC tumorigenesis.

EFA6A enhances glioma cell invasion through ADP ribosylation factor 6/extracellular signal-regulated kinase signaling.

EFA6A, or Pleckstrin and Sec7 domain protein, is a member of guanine nucleotide exchange factors for ADP ribosylation factor 6 (ARF6). Whereas EFA6A is specifically expressed in the brain, little is known about its function in glial cells or glioma. Here we show that elevated EFA6A expression is detectable in both low-grade and high-grade human glioma tissues samples. To investigate the role of EFA6A in glioma carcinogenesis, we generated a human glioblastoma cell line which conditionally overexpresses EFA6A (U373-EFA6A). We showed that overexpression of EFA6A had no effect on cell proliferation, apoptosis, or cell cycle control. However, as shown by wound healing and in vitro cell invasion assays, it significantly enhanced the cell motility and invasiveness whereas silencing EFA6A by its dominant negative mutant EFA6A(E242K) produced opposite effects. We further showed that ARF6/extracellular signal-regulated kinase (ERK) signaling is required for the EFA6A-mediated cell invasion because both EFA6A(E242K) and ARF6 dominant negative mutant ARF6(T27N) markedly reduced the phosphorylated ERK level and EFA6A-mediated invasive capacity. Consistently, mitogen-activated protein kinase/ERK kinase inhibitor U0126 could abolish the EFA6A-induced cell invasion. These results suggest for the first time a potential role of EFA6A/ARF6/ERK signal cascade in glioma cell migration and invasion.

miR-195 inhibits tumor growth and angiogenesis through modulating IRS1 in breast cancer.

Angiogenesis has been found as an attractive target for drug therapy as it is necessary for tumor growth. Accumulating evidences show that microRNAs (miRNAs), which are a group of highly conserved, single-stranded, short non-coding RNAs, play important roles through directly targeting angiogenic factors and protein kinases. The purpose of this study is to investigate the role of miR-195 in breast cancer development and angiogenesis through targeting IRS1. We show that miR-195 is inversely related with Insulin receptor substrate 1 (IRS1) in both breast cancer cells and breast cancer tissues. Induction of miR-195 could suppress IRS1 protein expression through binding to its 3'UTR regions either by transfection with miR-195 oligo or by infection with lentivirus encoding miR-195 gene. Moreover, re-expression of IRS1 reverses miR-195-mediated repression of tumor cell growth and miR-195 inhibits tumor angiogenesis through suppressing IRS1-VEGF axis. These data suggest that miR-195 mimics are potential therapeutic agents for breast cancer diagnose.

A preliminary evaluation of recombinant adeno-associated virus biodistribution in rhesus monkeys after intrahepatic inoculation in utero.

The ability to deliver genes to fetuses in utero may prove crucial for those genetic diseases that are associated with severe fetal morbidity and for which there is no effective postnatal therapy. In utero therapy may be especially useful in diseases that affect the central nervous system because the immature blood-brain barrier may facilitate gene delivery to neural target cells. We investigated whether in utero inoculation of recombinant adeno-associated virus (rAAV) into rhesus monkey fetuses would be a useful method of gene delivery, especially to the central nervous system. When the monkeys were sacrificed after birth, we found vector genomes distributed in many tissues, including the brain and peripheral blood. Pericapillary astrocytes expressing transgene products were detected by immunohistochemistry. In addition, we occasionally found vector genomes in the maternal blood. No adverse clinical or pathologic effects were observed in the inoculated monkeys. We concluded that (1) in utero intrahepatic inoculation of rAAV is a potentially safe and useful method of delivering genes to many fetal tissues; (2) astrocytes may be the cell type most easily targeted in the central nervous system (CNS) after systemic administration; and (3) the potential of inadvertent gene transfer to the mother must be considered.

MicroRNA-143 inhibits tumor growth and angiogenesis and sensitizes chemosensitivity to oxaliplatin in colorectal cancers.

Colorectal cancer (CRC) is one of the leading cancer-related causes of death in the world. Recently, downregulation of microRNA-143 (miR-143) has been observed in CRC tissues. Here in this study, we found that miR-143 expression was downregulated both in CRC patients' blood samples and tumor specimens. MiR-143 expression levels were strongly correlated with clinical stages and lymph node metastasis. Furthermore, insulin-like growth factor-I receptor (IGF-IR), a known oncogene, was a novel direct target of miR-143, whose expression levels were inversely correlated with miR-143 expression in human CRC specimens. Overexpression of miR-143 inhibited cell proliferation, migration, tumor growth and angiogenesis and increased chemosensitivity to oxaliplatin treatment in an IGF-IR-dependent manner. Taken together, these results revealed that miR-143 levels in human blood and tumor tissues are associated with CRC cancer occurrence, metastasis and drug resistance, and miR-143 levels may be used as a new diagnostic marker and therapeutic target for CRC in the future.

MiR-145 inhibits tumor angiogenesis and growth by N-RAS and VEGF.

MiR-145 is known as a tumor suppressor in numerous human cancers. However, its role in tumor angiogenesis remains poorly defined. In this study, we found that miR-145 was significantly downregulated in breast cancer tissues by using 106 cases of normal and cancer tissues as well as in breast cancer cells. MiR-145 exhibited inhibitory role in tumor angiogenesis, cell growth and invasion and tumor growth through the post-transcriptional regulation of the novel targets N-RAS and VEGF-A. In addition, we provide evidence that the expression levels of miR-145 correlate inversely with malignancy stages of breast tumors, although there is no association between miR-145 levels and hormone receptor levels in breast cancer. Taken together, these results demonstrate that miR-145 plays important inhibitory role in breast cancer malignancy by targeting N-RAS and VEGF-A, which may be potential therapeutic and diagnostic targets.

MiR-21 induced angiogenesis through AKT and ERK activation and HIF-1α expression.

MicroRNAs (miRNAs) are endogenous, small noncoding RNAs that play important roles in various cellular functions and tumor development. Recent studies have indicated that miR-21 is one of the important miRNAs associated with tumor growth and metastasis, but the role and molecular mechanism of miR-21 in regulating tumor angiogenesis remain to be elucidated. In this study, miR-21 was overexpressed by transfecting pre-miR-21 into human prostate cancer cells and tumor angiogenesis was assayed using chicken chorioallantoic membrane (CAM). We found that overexpression of miR-21 in DU145 cells increased the expression of HIF-1α and VEGF, and induced tumor angiogenesis. AKT and extracellular regulated kinases (ERK) 1/2 are activated by miR-21. Inhibition of miR-21 by the antigomir blocked this process. Overexpression of the miR-21 target, PTEN, also inhibited tumor angiogenesis by partially inactivating AKT and ERK and decreasing the expression of HIF-1 and VEGF. The AKT and ERK inhibitors, LY294002 and U0126, suppressed HIF-1α and VEGF expression and angiogenesis. Moreover, inhibition of HIF-1α expression alone abolished miR-21-inducing tumor angiogenesis, indicating that HIF-1α is required for miR-21-upregulated angiogenesis. Therefore, we demonstrate that miR-21 induces tumor angiogenesis through targeting PTEN, leading to activate AKT and ERK1/2 signaling pathways, and thereby enhancing HIF-1α and VEGF expression; HIF-1α is a key downstream target of miR-21 in regulating tumor angiogenesis.

Surface-modified gold nanoshells for enhanced cellular uptake.

Gold nanoshells have shown a great potential for use as agents in a wide variety of biomedical applications, and some of which require the delivery of large numbers of gold nanoshells onto or into the cells. Here, we develop a ready method to enhance the cellular uptake of gold nanoshells by modifying with meso-2,3-dimercaptosuccinic acid (DMSA). The quantifiable technique of inductively coupled plasma atomic emissions spectroscopy (ICP-AES) and transmission electron microscopy (TEM) were used to investigate the cellular uptake of unmodified and DMSA-modified gold nanoshells. Three cell lines (RAW 264.7, A549, and BEL-7402) were involved and the results indicated that the cellular uptake of the DMSA-modified gold nanoshells was obviously enhanced versus the unmodified gold nanoshells. The reason possibly lies in the nonspecific adsorption of serum protein on the DMSA-modified gold nanoshells (DMSA-GNs), which consequently enhanced the cellular uptake. As a continued effort, in vitro experiments with endocytic inhibitors suggested the DMSA-GNs internalized into cells via receptor-mediated endocytosis (RME) pathway. This study has provided a valuable insight into the effects of surface modification on cellular uptake of nanoparticles.

Analysis of MiR-195 and MiR-497 expression, regulation and role in breast cancer.

PURPOSE: To investigate expression, regulation, potential role and targets of miR-195 and miR-497 in breast cancer. EXPERIMENTAL DESIGN: The expression patterns of miR-195 and miR-497 were initially examined in breast cancer tissues and cell lines by Northern blotting and quantitative real-time PCR. Combined bisulfite restriction analysis and bisulfite sequencing were carried out to study the DNA methylation status of miR-195 and miR-497 genes. Breast cancer cells stably expressing miR-195 and miR-497 were established to study their role and targets. Finally, normal, fibroadenoma and breast cancer tissues were employed to analyze the correlation between miR-195/497 levels and malignant stages of breast tumor tissues. RESULTS: MiR-195 and miR-497 were significantly downregulated in breast cancer. The methylation state of CpG islands upstream of the miR-195/497 gene was found to be responsible for the downregulation of both miRNAs. Forced expression of miR-195 or miR-497 suppressed breast cancer cell proliferation and invasion. Raf-1 and Ccnd1 were identified as novel direct targets of miR-195 and miR-497. miR-195/497 expression levels in clinical specimens were found to be correlated inversely with malignancy of breast cancer. CONCLUSIONS: Our data imply that both miR-195 and miR-497 play important inhibitory roles in breast cancer malignancy and may be the potential therapeutic and diagnostic targets.

Adenosine diphosphate-ribosylation factor 6 is required for epidermal growth factor-induced glioblastoma cell proliferation.

BACKGROUND: : Epidermal growth factor (EGF) signaling plays a pivotal role in gliomagenesis. The authors previously demonstrated that adenosine diphospate-ribosylation factor 6 (ARF6), a member of the Ras-related small guanosine-5'-triphospate-binding protein family, is required for EFA6A-induced glioma cell migration and invasion. However, the role of ARF6 in EGF signaling is unknown. METHODS: : The authors analyzed messenger RNA (mRNA) levels of ARF6 and EGF receptor (EGFR) in 16 high-grade glioma samples and in 6 low-grade glioma samples by reverse transcriptase-polymerase chain reaction analysis. To determine whether EGF induces ARF6 expression in human glioblastoma U87 cells through transcriptional regulation and EGFR activation, the levels of ARF6 were assayed in EGF-treated U87 cells that were preincubated with a transcriptional inhibitor (actinomycin D) and an EGFR tyrosine kinase inhibitor (PD153035), respectively. The downstream signaling of EGFR-mediated ARF6 up-regulation also was investigated using specific inhibitors of mitogen-activated protein kinase (MEK), phosphatidylinositol 3' kinase (PI3K), and Janus kinase 2. The involvement of SP1 in the downstream signaling was studied by using an SP1 inhibitor (mithramycin A). Small-interfering RNAs (siRNAs) targeting ARF6 were used to investigate the effects of ARF6 on EGF-mediated glioma cell proliferation. RESULTS: : The results demonstrated that ARF6 and EGFR mRNA levels were elevated in glioma tissues. Furthermore, EGF stimulated ARF6 expression in U87 cells in a dose-dependent and time-dependant manner. This stimulation was caused by increased transcription of ARF6 and by activation of the MEK/extracellular signal-regulated kinase 1 and 2 (ERK1/2) and PI3K signaling pathways. It is noteworthy that SP1 was essential for EGF-induced ARF6 up-regulation. Finally, EGF-induced glioblastoma cell proliferation depended on ARF6, because the suppression of ARF6 by siRNA or by a dominant-negative mutant significantly inhibited EGF-induced cell proliferation. CONCLUSIONS: : The results of the current study suggested that EGF-induced ARF6 expression plays a significant role in glioma cell proliferation. Cancer 2009. (c) 2009 American Cancer Society.

microRNA-146b inhibits glioma cell migration and invasion by targeting MMPs.

MicroRNAs (miRNAs) are a class of endogenous, small non-protein coding single-stranded RNA molecules, which are crucial post-transcriptional regulators of gene expression. Previous studies have shown that miRNAs participate in a wide range of biological functions and play important roles in various human diseases including glioma. However, the role of miRNAs in mediating glioblastoma cell migration and invasion has not been elucidated. Using miRNA microarray, we identified miR-146b as one of the miRNAs that is significantly dysregulated in human glioblastoma tissue. We showed that miR-146b overexpression by transfection with the precursor miR-146b, or knock-down by Locked Nucleic Acid (LNA)-modified anti-miR-146b, has no effect on the growth of human glioblastoma U373 cells. However, precursor miR-146b transfection significantly reduced the migration and invasion of U373 cells, while LNA-anti-miR-146b transfection generated the opposite result. Furthermore, we discovered that a matrix metalloproteinase gene, MMP16, is one of the downstream targets of miR-146b. Taken together, our findings suggest that miR-146b is involved in glioma cell migration and invasion by targeting MMPs, and implicate miR-146b as a metastasis-inhibiting miRNA in glioma.

Hepatocyte nuclear factor 1 binding element within the promoter of microsomal triglyceride transfer protein (MTTP) gene is crucial for MTTP basal expression and insulin responsiveness.

Insulin inhibits the transcription of the microsomal triglyceride transfer protein (MTTP), which plays a pivotal role in lipoprotein assembly and secretion. Here, we provide evidence that a hepatocyte nuclear factor 1 binding element (HNF1A element) within the MTTP promoter serves as a novel negative insulin-responsive element. Deletion/mutation mapping of the MTTP gene promoter identified a modified HNF1A element that is crucial to the negative insulin effect. Chimeric promoter containing this HNF1A element and minimal TEAD1 promoter also responded negatively toward insulin treatment. Gel shift assay demonstrated that HNF1A but not HNF1B binds to this element. Enforced expression of HNF1A was sufficient to reconstitute the negative insulin responsiveness of MTTP promoter in TM4SF1 myocytes that are HNF1A negative. Furthermore, replacing this element with consensus HNF1A element preserved the negative insulin response, suggesting that negative insulin responsiveness is a generic characteristic of HNF1A element. Given that many genes implicated in diabetes contain HNF1A element, the potential regulation of these genes by insulin via HNF1A element may provide important clues for the manifestation and treatment of diabetic metabolic syndromes.

Preparation and biomedical application of a non-polymer coated superparamagnetic nanoparticle.

We report the preparation of a non-polymer coated superparamagnetic nanoparticle that is stable and biocompatible both in vitro and in vivo. The non-polymer, betaine, is a natural methylating agent in mammalian liver with active surface property. Upon systemic administration, the nanoparticle has preferential biodistribution in mammalian liver and exhibits good reduction of relaxivity time and negative enhancement for the detection of hepatoma nodules in rats using MRI. Our data demonstrate that the non-polymer coated superparamagnetic nanoparticle should have potential applications in biomedicine.

Cell cycle-related kinase: a novel candidate oncogene in human glioblastoma.

BACKGROUND: Median survival for patients with glioblastoma multiforme, the most aggressive glioma, is only 12-15 months, despite multimodal treatment that includes surgery, chemotherapy, and radiotherapy. Thus, identification of genes that control the progression of glioblastoma multiforme is crucial for devising new therapies. We investigated the involvement of cell cycle-related kinase (CCRK), a novel protein kinase that is homologous to cyclin-dependent kinase 7, in glioblastoma multiforme carcinogenesis. METHODS: We analyzed the expression levels of CCRK in 26 glioma patient samples (19 high-grade and seven low-grade) and normal brain by semiquantitative reverse transcription-polymerase chain reaction assays. CCRK expression was knocked down in human glioma U-373 MG and U-87 MG cells with small-interfering RNAs and short hairpin RNAs (siCCRK and shCCRK, respectively), and cell proliferation, cell cycle distribution, and cyclin-dependent kinase 2 (CDK2) phosphorylation were examined. A subcutaneous nude mouse xenograft model (n = 4 mice per group) was used to study the effect of CCRK knockdown and overexpression on tumorigenicity and growth of glioblastoma multiforme cells in vivo. All statistical tests were two-sided. RESULTS: CCRK mRNA was elevated at least 1.5-fold and as much as 3.7-fold in 14 (74%) of 19 high-grade glioblastoma multiforme patient samples and in four (80%) of five glioma cell lines examined compared with normal brain tissue. Suppression of CCRK by siCCRK inhibited the proliferation of U-373 MG and U-87 MG glioblastoma cells in a time- and dose-dependent manner. The growth-inhibiting effect of siCCRK was mediated via G1- to S-phase cell cycle arrest and reduced CDK2 phosphorylation. CCRK knockdown statistically significantly suppressed glioma cell growth in vivo as indicated by the mean tumor volumes at week 6 after tumor cell injection (U-373-control = 1352 mm3, U-373-shCCRK = 294 mm3, difference = 1058 mm3, 95% confidence interval [CI] = 677 to 1439 mm3, P<.001; U-87-control = 1910 mm3, U-87-shCCRK = 552 mm3, difference = 1358 mm3, 95% CI = 977 to 1739 mm3, P<.001). CONCLUSIONS: CCRK is a candidate oncogene in glioblastoma multiforme tumorigenesis.