LIN28A inhibits lysosome­associated membrane glycoprotein 1 protein expression in embryonic stem and bladder cancer cells.

Tumor cells and embryonic stem cells (ESCs) have similar transcription mechanisms. LIN28A is an important factor in tumor cells and ESCs, it is an inhibitor of intracellular endoplasmic reticulum (ER)­related protein translation in ESCs. The present study aimed to examine the effects of LIN28A on an ER­related protein, lysosome­associated membrane glycoprotein 1 (LAMP1), in human bladder cancer cells and mouse (m)ESCs, using reverse transcription­quantitative polymerase chain reaction and western blotting to detect the expression of LAMP1 mRNA and protein, respectively, following LIN28A knockdown. LIN28A was revealed to promote the proliferation, migration and invasion in human bladder cancer cells. These data suggested similarities between ESC cells and cancer cells and may provide novel ideas for the use of induced embryonic stem cell differentiation to treat tumors.

miR­30a­5p in the tumorigenesis of renal cell carcinoma: A tumor suppressive microRNA.

Renal cell carcinoma (RCC) is the most common type of malignant tumor of the adult kidney and has a poor prognosis. MicroRNAs (miRs) are important in a wide range of biological and pathological processes, including cell differentiation, migration, growth, proliferation, apoptosis and metabolism. The present study aimed to determine the role exerted by miR­30a­5p in the tumorigenesis of RCC. The expression levels of miR­30a­5p in RCC tissues and RCC­derived cells were demonstrated to be significantly downregulated by real­time quantitative polymerase chain reaction (RT­qPCR). Wound scratch assay, cell proliferation assay and flow cytometric analysis revealed that the abilities of migration and proliferation of the RCC­derived cells were suppressed, whereas cell apoptosis was promoted, when miR­30a­5p was overexpressed in these cells. N­acetylgalactosaminyltransferase 7 (GALNT7) was predicted to be one target gene of miR­30a­5p by bioinformatics analysis. Luciferase reporter assay, RT­qPCR and western blotting were performed to confirm that GALNT7 is the direct conserved target of miR­30a­5p. These results suggested that miR­30a­5p has a tumor­suppressive role in the tumorigenesis of RCC.

Oncogenic microRNA-142-3p is associated with cellular migration, proliferation and apoptosis in renal cell carcinoma.

MicroRNAs (miRNAs/miRs) serve an important role in the regulation of carcinogenic pathways. RCC is the most prevalent kidney cancer that occurs in adults. miRNAs have gained increasing attention due to their association with RCC tumorigenesis, serving as biomarkers for early detection and progression monitoring, and as potential targets for molecular therapy. Upregulation of miRNA-142-3p has been previously identified in RCC tissues by microarray profile, however, its expression and function in RCC have not yet been validated. In the present study, quantitative polymerase chain reaction was performed to quantify the relative expression of miR-142-3p in 53 paired RCC and adjacent normal tissues. Furthermore, wound healing, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and flow cytometry assays were performed to analyze the impacts of miR-142-3p on cellular migration, proliferation and apoptosis. The results demonstrated that miR-142-3p was significantly upregulated in RCC tissues compared with adjacent normal tissues. Downregulation of miR-142-3p, induced by chemically synthesized miR-142-3p inhibitor, significantly suppressed cell migration and proliferation, and promoted cell apoptosis in 786-O and ACHN cells, supporting the theory that miR-142-3p may function as an oncogene in RCC. The potential clinical significance of miR-142-3p, as a biomarker and therapeutic target, provides rationale for further investigation into the miR-142-3p-mediated molecular pathway and how it is associated with RCC development.

Identification of miR­130b as an oncogene in renal cell carcinoma.

Renal cell carcinoma (RCC) is the most common type of renal tumor, which has a poor prognosis. Improvements in understanding the underlying molecular biology of RCC has led to systemic treatments, which have markedly improved patient outcomes. Therefore, it is necessary and worthwhile to identify novel biomarkers for RCC. MicroRNAs (miRNAs) have been found to be important in a wide range of biological and pathological processes, including cell differentiation, migration, growth, proliferation, apoptosis and metabolism. Aberrant expression of miRNA­130b has previously been reported in tumors, however, its role in RCC remains to be elucidated. In the present study, the upregulation of miR­130b was observed in RCC tissues and cell lines using reverse transcription­quantitative polymerase chain reaction analysis, which was consistent with previous microRNA profiling in RCC. Furthermore, the effects of miR­130b on cell migration, proliferation and apoptosis were examined using a wound scratch assay, an MTT assay and flow cytometric analysis, respectively. The results demonstrated that the downregulation of miR­130b by a synthesized inhibitor inhibited cell migration, suppressed cell proliferation and induced RCC cell apoptosis. The present study was the first, to the best of our knowledge, to suggest that miR­130b may be a promising biomarker for diagnosis and a therapeutic target for the treatment of RCC. Further investigations are required to examine the roles and target genes of miR­130b in RCC.

Oncogenic cAMP responsive element binding protein 1 is overexpressed upon loss of tumor suppressive miR-10b-5p and miR-363-3p in renal cancer.

Renal cell carcinoma (RCC) is the most common kidney cancer in adults and has a poor prognosis. cAMP responsive element binding protein 1 (CREB1) is a proto­oncogenic transcription factor involved in malignancies of various organs. However, its functional role(s) have not yet been elucidated in RCC. We investigated the expression pattern, function and regulation of CREB1 in RCC. CREB1 was overexpressed in the RCC tissues and cell lines. Downregulation of CREB1 inhibited RCC tumorigenesis by affecting cell proliferation, migration and apoptosis. Multiple computational algorithms predicted that the 3'­untranslated region (3'­UTR) of human CREB1 mRNA is a target for miR­10b­5p and miR­363­3p. Luciferase reporter assay, qPCR and western blot analysis confirmed that miR­10b­5p and miR­363­3p bind directly to the 3'­UTR of CREB1 mRNA and inhibit mRNA and protein expression of CREB1. qPCR data also revealed a significantly lower expression of miR­10b­5p and miR­363­3p in RCC tissues. Introduction of miR­10b­5p and miR­363­3p mimics led to suppressed expression of CREB1 and inhibited cell proliferation, migration and apoptosis reduction. Taken together, we propose that CREB1 is an oncogene in RCC and that upregulation of CREB1 by loss of tumor suppressive miR­10b­5p and miR­363­3p plays an important role in the tumorigenesis of RCC.

MicroRNA-106b functions as an oncogene in renal cell carcinoma by affecting cell proliferation, migration and apoptosis.

Kidney cancer is the 14th most common cancer in the world and its prognosis remains poor due to difficult early detection and treatment. Therefore, the identification of biomarkers for early-stage renal cell carcinoma (RCC) is important. MicroRNA-106b (miR-106b) has been described as an oncogene in several types of human cancer. Previous microarray studies have suggested that miR-106b was significantly upregulated in RCC tissues compared with paired normal kidney tissues and may be a promising biomarker for the prediction of early metastasis following nephrectomy. The present study aimed to determine the expression and function of miR-106b in RCC. The expression of miR-106b in RCC tissues and cells, and in paired normal tissues and cells was determined by reverse transcription quantitative polymerase chain reaction, based on the previous sequencing results of miRNAs. Furthermore, a wound scratch assay, MTT assay and flow cytometry were performed to examine the functions of miR-106b on cell migration, proliferation and apoptosis. The results demonstrated that miR-106b was upregulated in RCC tissues and cell lines compared with control normal tissues and cell lines. Downregulation of miR-106b with a synthesized inhibitor suppressed cell migration and proliferation and induced renal cancer cell apoptosis, suggesting that miR-106b can be characterized as an oncogene in RCC. To the best of our knowledge, the present study was the first to reveal that miR-106b is upregulated and affects cellular migration, proliferation and apoptosis in RCC. Further studies are required to examine the role and target genes of miR-106b in RCC.

Atorvastatin Ameliorates Radiation-Induced Cardiac Fibrosis in Rats.

Radiation-induced heart injury is one of the major side effects of radiotherapy for thoracic malignancies. Previous studies have shown that radiotherapy induced myocardial fibrosis and intensified myocardial remodeling. In this study, we investigated whether atorvastatin could inhibit radiation-induced heart fibrosis in Sprague-Dawley rats, which were randomly divided into six groups: control; radiation only; and four treatment groups receiving atorvastatin plus radiation (E1, E2, E3 and E4). All rats, except the control group, received local heart irradiation in 7 daily fractions of 3 Gy for a total of 21 Gy. Rats in groups E1 (10 mg/kg/day) and E2 (20 mg/kg/day) received atorvastatin and radiation treatment until week 12 after exposure. Rats in groups E3 (10 mg/kg/day) and E4 (20 mg/kg/day) received atorvastatin treatment from 3 months before irradiation to week 12 after irradiation. The expressions of TGF-ß1, Smad2, Smad3, fibronectin, ROCK I and p-Akt in heart tissues were evaluated using real-time PCR or Western blot analyses. Atorvastatin significantly reduced the expression of TGF-ß1, Smad3/P-Smad3, ROCK I and p-Akt in rats of the E1-E4 groups and in a dose-dependent manner. Fibronectin exhibited a similar pattern of expression changes. In addition, echocardiography showed that atorvastatin treatment can inhibit the increase of left ventricular end-diastolic dimension, left ventricular end-systolic diameter and left ventricular posterior wall thickness, and prevent the decrease of ejection fraction and fraction shortening in E1-E4 groups compared with the radiation only group. This study demonstrated that radiation exposure increased the expression of fibronectin in cardiac fibroblasts and induced cardiac fibrosis through activation of the TGF-ß1/Smad3, RhoA/ROCK, and PI3K/AKT signaling pathways. Statins ameliorated radiation-induced cardiac fibrosis in Sprague-Dawley rats. Our results suggest that atorvastatin is effective for the treatment of radiation-induced cardiac fibrosis, especially with longer and higher dose atorvastatin treatment, as demonstrated in experimental group E4.

Induction of dedifferentiated male mouse adipose stromal vascular fraction cells to primordial germ cell-like cells.

The adipose stromal vascular fraction (SVF) contains abundant mesenchymal stem cell populations that have a limited ability to self-renew and differentiate. Male mouse adipose SVF cells were dedifferentiated by reprogramming factors (c-Myc, Oct4, Sox2, and Klf4) to form embryonic stem cell-like cells (ESCLCs), which upgraded their limited differentiation potential. The ESCLCs were induced to differentiate toward epiblast-like cells (EpiLCs) and primordial germ cell-like cells (PGCLCs) by culturing in media supplied with activin A and BMP-4, respectively. The derived ESCLCs possess embryonic stem cell features and can automatically form embryonic bodies. After culture in EpiLC induction medium for 2-3 days, ESCLCs formed flattened epithelial structures that were different from their original water drop-like colonies, and the expression of pluripotency-related genes decreased. When the cells that had been cultured in EpiLC induction medium for 2 days were isolated and cultured in PGCLC induction medium for 4-6 days, they formed typical water drop-like colonies again. Moreover, expression of the pluripotency-related genes and the primordial germ cell (PGC) specification-related genes increased. During progression from ESCLCs toward EpiLCs and PGCLCs, the levels of histone methylases H3K9me2 and H3K27me3 kept changing, which resembled those seen in PGC specification. The derived PGCLCs expressed SSEA-1, Blimp-1, and Stella. Furthermore, methylation of Igf2r and Snrpn was retained, but H19 and Kcnq1ot1 methylation levels were slightly reduced compared to non-PGCLCs, suggesting that the derived PGCLCs may have initiated the process of imprint erasure.

Mesenchymal stem cells derived from different origins have unique sensitivities to different chemotherapeutic agents.

BMSCs (bone-marrow-derived mesenchymal stem cells) and ADSCs (adipose tissue-derived mesenchymal stem cells) are virtually identical in cell surface marker profile and differentiation potential. These cell populations have promising characteristics for clinical application. We have investigated the sensitivity of these cell populations to various chemotherapeutic agents by testing the inhibition of cell proliferation, low molecular DNA bands formation, in situ apoptosis, apoptosis-related gene expression and cell senescence after treatment. BU (busulfan), methotrexate and doxorubicin treatment led to a marked and dose-dependent reduction in cell viability compared with 5-FU (5-fluorouracil) treatment. Different expression patterns of apoptosis-related genes were found in the BMSCs and ADSCs following treatment with the agents, but no low molecular mass DNA bands were detected. BMSCs had a higher percentage of apoptotic and senescent cells following treatment with chemotherapeutic agents compared with ADSCs. These findings suggest that these two cell populations respond differently to chemotherapy treatment. ADSCs are more resistant than BMSCs to chemotherapy-induced senescence and apoptosis, indicating that they might be more advantageous to use in the clinic than BMSCs.

Effect of all-trans-retinoic acid on the expression of primordial germ cell differentiation-associated genes in mESC-derived EBs.

atRA (all-trans-retinoic acid) is known to induce the differentiation of mESCs (mouse embryonic stem cells) into PGCs (primordial germ cells) in vitro. However, it is not clear as to what changes occur in PGC differentiation-associated genes or what mechanisms are involved when EBs (embryoid bodies) derived from mESCs are induced by atRA. EBs derived from mESCs were treated with 1, 2 or 5 µM atRA for 16 h, 2 days or 5 days. Real-time PCR and Western blot analysis were performed to detect the relative levels of PGC differentiation-associated genes (Lin28, Blimp1, Stra8 and Mvh) and the corresponding proteins respectively. Immunofluorescence was used to detect the protein location and distribution in EBs. The expression characteristics of genes could be divided into three categories: rapidly reached the peak value in 16 h and then decreased (Stra8, Lin28), initially low and then increased to reach the peak value in 5 days (Mvh) and relatively unchanged (Blimp1). A low level of Lin28 was expressed in EBs treated with atRA for 2 days or 5 days. The variation in the level of Lin28 mRNA did not influence the change in the level of Blimp1 mRNA. The changes in Stra8/Lin28 were consistent with the corresponding changes in the levels of their respective mRNAs, but the changes for Mvh/Blimp1 were not consistent with the corresponding changes in the levels of their respective mRNAs. Blimp1 expression may be independent of the effect of atRA on PGC differentiation. atRA may promote the start of a period in which there is a low level of Lin28 expression during PGC differentiation.

[Mouse-induced pluripotent stem cells have the potential to differentiate into induced primordial germ cells].

OBJECTIVE: To investigate whether mouse-induced pluripotent stem (iPS) cell line IP14D-1 has the potential to differentiate into induced primordial germ cells (iPGCs), and to explore the changes in the expression of iPGCs-differentiation associated genes and their possible mechanisms. METHODS: Undifferentiated IP14D-1 was cultured to proliferate and then differentiated to form 4-, 7- and 9-day-old induced embryoid bodies (iEBs) in vitro, respectively. RT-PCR and immunofluorescence were used to detect the expressions of Lin28, Blimpl, Stra8 and Mvh, as well as the localization of the corresponding protein in iEBs. RESULTS: The expression of Blimpl was higher than that of Lin28 in the undifferentiated IP14D-1 and mouse embryonic stem cells (mESCs). Mvh and Stra8 as well as mESCs and EBs were also expressed in IP14D-1 and iEBs, but with no significant differences. The expression of Lin28 was gradually increased in the IP14D-1-derived iEBs from 4 to 7 days, but decreased at 9 days, and the expression of Blimp1 was gradually reduced with the prolonged growing time of iEBs. CONCLUSION: A stable system was established for the culture and differentiation of IP14D-1 and IP14D-1-derived iEBs. The expressions of Lin28, Blimp1, Mvh and Stra8 were not significantly different between the undifferentiated IP14D-1 and mESCs, nor were the expressions of Mvh and Stra8 between iEBs and EBs. IP14D-1 and iEBs had the potential to differentiate into iPGCs, which increased in number in the 7-day-old iEBs, and the expression of iPGC-differentiation associated Lin28 became lower in the older iEBs.

Rat bone marrow derived mesenchymal progenitor cells support mouse ES cell growth and germ-like cell differentiation.

Mouse embryonic fibroblasts (MEFs) have been used as feeder cells to support the growth of mouse embryonic stem cell (mESC) and primordial germ cells (PGC) in culture for many years. However, MEF preparation is a complex and tedious task. Recently, there are reports indicating that the microenvironment provided by bone marrow stromal cells could support the survival of embryonic-like stem cells in bone marrow. In this report, rat bone marrow derived mesenchymal progenitor cells (MPC) were used as feeder cells to culture mouse Oct4-GFP ES cell and ES cell derived germ cells. FACS results show that similar to MEF, rat MPC could efficiently support growth of the mouse Oct4-GFP ES cell line in culture (MPC 85.5 +/- 5.1% vs MEF 84.1 +/- 6.2%). ES cells could be subcultured for >15 passages without losing morphological characteristics. The cultured cells expressed stem cell marker alkaline phosphatase, Oct4, Sox2, and SSEA-1. Furthermore, rat MPC cells were able to support survival of germ cells isolated from mouse Oct4-GFP ES cell formed embryoid bodies (EB). After induction by retinoic acid for 7 days, some isolated cells differentiated to spermatogonial stem-like cells, expressing Mvh, Stra-8, Hsp90-a, integrinb1 and a6. Compared with traditional MEF culture systems, the rat MPC culture system is effective in supporting ES cell growth and is easy to prepare.

[Extracellular matrix regulates expressions of germ cell differentiation associated genes in mouse embryonic stem cells].

OBJECTIVE: Interactions of cells with the extracellular matrix (ECM) are essential for cell differentiation. The authors sought to determine the roles of different ECMs in the expressions of germ cell differentiation associated genes after mouse embryonic stem cells (mESCs) differentiated into embryoid bodies (EBs). METHODS: EBs derived from mESCs were maintained in suspension for 3 days and then cultured on the plates coated with various ECMs, including fibronectin (F), laminin (L), matrigel (M), collagen (C) and nonadhensive agarose (A), respectively, for 1, 2, 3 or 4 days, followed by evaluation of the expressions of the genes associated with germ cell differentiation by RT-PCR. RESULTS: The EBs of the F and L groups exhibited facilitated adherent differentiation. The expressions of the Blimp-1, Stella, Mvh and Stra8 genes were increased gradually in the F and L but not obviously in the M and C groups. The overall gene expressions were low in the A group, but high and then gradually decreased in the blank control group. Endogenous fibronectin, laminin and integrin beta1 were obviously expressed in the L and control groups. CONCLUSION: Laminin /integrin beta1 signaling may play a role in regulating the differentiation of mESCs into primordial germ cells (PGCs). Exogenous laminin can facilitate the differentiation of mESC-derived EBs into PGCs by acting on the integrin beta1 subunit, while exogenous fibronectin may be involved in the regulation of the differentiation through other integrin subunit. Endogenous laminin and fibronectin secreted by EBs may also facilitate cell differentiation in the absence of exogenous ECMs.

[Analysis of human metapneumovirus genotype in Hunan, China and its attachment protein G sequence character].

OBJECTIVE: To understand the genotypes of human metapneumovirus (hMPV) and the genetic character of hMPV attachment protein G sequence in Hunan, China. METHODS: 232 nasopharyngeal aspirates (NPA) samples from hospitalized children with acute respiratory infections were collected from Hunan, China in 2005. HMPV was detected. The full length of G glycoprotein genes were amplified and sequenced. Bioinformatics soft-wares were employed to analyze the sequences. RESULTS: 17/232 (7.3%) were showed hMPV positive. And co-infection rate with other viruses is 35%. The diagnoses of these hMPV positive cases are pneumonia, bronchiolitis and bronchopneumonia. Phylogenetic analysis for G genes from 13 hMPVs revealed the existence of four major subgroups: A1, A2, B1, B2 in Hunan, China in 2005. There are four types of sequence lengths of hMPV G glycoprotein, which are 711, 675, 660, 696nt. It is different in potential N-linked glycosylation sites and number of cysteine residues among these hMPVs of Hunan, China and Beijing, China. Also it is different from those in Japan and North America. CONCLUSION: The investigation of hMPV from Hunan, China in 2005 revealed the high speed of genetic variation and the marked character of geographic epidemic differences.

Phospholipase A2-like activity of human bocavirus VP1 unique region.

Human bocavirus (HBoV) is a new parvovirus first discovered in 2005, which is associated with acute respiratory infection. Analysis of sequence homology has revealed that a putative phospholipase A2 (PLA2) motif exists in the VP1 unique region of HBoV. However, little is known about whether the VP1 unique region of HBoV has PLA2 enzymatic activity and how these critical residues contribute to its PLA2 activity. To address these issues, the VP1 unique region protein and four of its mutants, were expressed in Eschericha coli. The purified VP1 unique protein (VP1U) showed a typical Ca2+-dependent secreted PLA2-like (sPLA2) activity, which was inhibited by sPLA2-specific inhibitors in a time-dependent manner. Mutation of one of the amino acids (21Pro, 41His, 42Asp or 63Asp) in VP1U almost eliminated the sPLA2 activity of HBoV VP1U. These data indicate that VP1U of HBoV has sPLA2-like enzymatic activity, and these residues are crucial for its sPLA2-like activity. Potentially, VP1U may be a target for the development of anti-viral drugs for HBoV.

[Genome cloning and phylogenetic analysis of human bocavirus capsid gene].

The full-length genome of one human bocavirus (HBoV) and the VP1 sequences of nine HBoV were amplified from patients' samples by PCR, cloned into pGEM-T vector separately, and sequenced. In this study, the one full length gemome and nine VP1 sequences of HBoV were aligened with 14 sequences of Parvoviruses which were canonical exemplars in Parvovirinae. Phylogenetic analysis showed that HBoV capsid sequences positioned closely to B19 parvovirus, although they positioned far in phylogenetic tree based on full length genome. Many similarities were found between HBoV and B19 in capsid by alignment on secondary structural elements. Because both B19 and HBoV are the only Parvoviruses that infect mankind, so study on HBoV may be used for reference to B19 which had been studied for about 30 years. By analysis of mutational sites, HBoV capsid protein showed a highly conserved secondary structural elements, but highly active in VP1-U, leading end of VP2 and insertions between the strands of the betaG-H. This cued that HBoV inclined to immune evasion and infectant adaptive faculty.

[Clinical characteristics of bocavirus infection among children].

OBJECTIVE: To study the clinical characteristics of human bocavirus (HBoV) among children and to understand the association of HBoV with human diseases. METHOD: Totally 148 nasopharyngeal aspirate (NPA) samples were collect from hospitalized children with acute respiratory infection during Oct. 2005 to Feb. 2006. Two serum samples were obtained from HBoV positive patients. PCR was used to assay all these samples and PCR products were sequenced. RESULT: HBoV was positive in 11 of 148 NPA samples. The positive rate was 7.4 percent. The serum samples of HBoV infected patients showed that serum contained HBoV by PCR assay. All these HBoV positive patients had the clinical symptoms of bronchitis, bronchopneumonia and pneumonia. Some patients had diarrhea. CONCLUSION: All patients infected with HBoV had upper and lower respiratory tract infections. HBoV is a probable important pathogen of upper and lower respiratory tract infection. The HBoV could cause viremia. In addition, some HBoV patients had diarrhea. HBoV infection probably could also result in intestinal disease and other related symptoms.

Human bocavirus infection, People's Republic of China.

A newly identified parvovirus, human bocavirus (HBoV), was found in 21 (8.3%) of 252 nasopharyngeal aspirates from hospitalized children with lower respiratory tract infection in Hunan Province, People's Republic of China. Viral loads were 10(4) to 10(10) copies/mL. Phylogenetic analysis of the VP1 gene showed a single genetic lineage of HBoV worldwide.