Hepatitis B surface antigen inhibits MICA and MICB expression via induction of cellular miRNAs in hepatocellular carcinoma cells.

Hepatitis B surface antigen (HBsAg) seropositivity is an important risk factor for hepatocellular carcinoma (HCC), and HBsAg-transgenic mice have been reported to spontaneously develop HCC. The major histocompatibility complex class I-related molecules A and B (MICA and MICB) are NKG2D ligands that play important roles in tumor immune surveillance. In the present study, we found that HBsAg overexpression in HepG2 cells led to upregulation of 133 and downregulation of 9 microRNAs (miRNAs). Interestingly, several HBsAg-induced miRNAs repressed the expression of MICA and MICB via targeting their 3'-untranslated regions. In addition, the expression of MICA and MICB was significantly reduced upon HBsAg overexpression, which was partially restored by inhibiting the activities of HBsAg-induced miRNAs. Moreover, HBsAg-overexpressing HCC cells exhibited reduced sensitivity to natural killer cell-mediated cytolysis. Taken together, our data suggest that HBsAg supresses the expression of MICA and MICB via induction of cellular miRNAs, thereby preventing NKG2D-mediated elimination of HCC cells.

MicroRNA-581 promotes hepatitis B virus surface antigen expression by targeting Dicer and EDEM1.

Hepatitis B virus surface antigen (HBsAg) is an important risk factor for hepatocellular carcinoma (HCC) and is downregulated during hepatocarcinogenesis. MicroRNAs (miRNAs) are frequently deregulated in HCC tissues. However, whether the deregulation of certain miRNAs in HCC has an impact on HBsAg expression remains unclear. We found here that microRNA-581 (miR-581), which is deregulated during hepatocarcinogenesis, promoted HBsAg expression. Additionally, miR-581 targeted Dicer and endoplasmic reticulum degradation-enhancing alpha-mannosidase-like protein 1 (EDEM1) and repressed their expression. Although Dicer cannot process HBV transcripts, Dicer knockdown led to increased HBsAg secretion, most likely due to a reduction in the levels of Dicer-processed 7SL RNA fragments. Moreover, Dicer-processed 7SL RNA fragments partially inhibited the ability of miR-581 to stimulate HBsAg expression. Furthermore, we found that forced EDEM1 expression inhibited miR-581-mediated induction of HBsAg. Finally, transfection of miR-581 into HepG2.2.15 cells promoted cell proliferation and led to upregulation of genes involved in development, cell proliferation and protein secretion. Altogether, we conclude that miR-581 promotes HBsAg expression by targeting Dicer and EDEM1. Our findings suggest that downregulation of miR-581 during hepatocarcinogenesis may lead to a reduction in HBsAg expression and impede HCC development.

MicroRNA-34a inhibits migration and invasion of colon cancer cells via targeting to Fra-1.

MicroRNA-34a (miR-34a), a transcriptional target of p53, is a well-known tumor suppressor gene. Here, we identified Fra-1 as a new target of miR-34a and demonstrated that miR-34a inhibits Fra-1 expression at both protein and messenger RNA levels. In addition, we found that p53 indirectly regulates Fra-1 expression via a miR-34a-dependant manner in colon cancer cells. Overexpression of miR-34a strongly inhibited colon cancer cell migration and invasion, which can be partially rescued by forced expression of the Fra-1 transcript lacking the 3'-untranslated region. The expression of matrix metalloproteinase (MMP)-1 and MMP-9, two enzymes involved in cell migration and invasion, was decreased in miR-34a-transfected cells, and this can be rescued by Fra-1 overexpression. Moreover, we found that miR-34a was downregulated in 25 of 40 (62.5%) colon cancer tissues, as compared with the adjacent normal colon tissues and that the expression of miR-34a was correlated with the DNA-binding activity of p53. Unexpectedly, the DNA-binding activity of p53 was not inversely correlated with Fra-1 expression, and a significant statistical inverse correlation between miR-34a and Fra-1 expression was only observed in 14 of 40 (35%) colon cancer tissues. Taken together, our in vitro data suggest that p53 regulates Fra-1 expression, and eventually cell migration/invasion, via a miR-34a-dependent manner. However, in vivo data indicate that the p53-miR-34a pathway is not the major regulator of Fra-1 expression in human colon cancer tissues.

[Relationship between photosynthetic characteristics and environmental factors in leaves of Pueraria lobata].

OBJECTIVE: To study the relationship between photosynthetic characteristics and environmental factors in leaves of P. lobata. METHOD: Photosynthetic characteristics and environmental factors were measured by using CIRAS-2 portable photosynthesis system. RESULT: The apparent quantum yield in leaves was 0.0173 micromol CO2 x micromol(-1) photon. The dark respiration rate was 2.9333 micromol x m(-2) x s(-1). The light compensation point of photosynthesis was 180 micromol x m(-2) x s(-1). The light saturation point was 1600 micromol x m(-2) x s(-1). The carboxylation efficiency was 0.0338 micromol x m(-2) x s(-1). The light respiration rate was 2.5 micromol x m(-2) x s(-1). The CO2 compensation point was 100 micromol x mol(-1), The CO2 saturation point was 1 600 micromol x mol(-1). CONCLUSION: Photo flux density and air temperature are major environmental factors influencing diumal changes of net photosynthetic rate.

Decreased dicer expression enhances SRP-mediated protein targeting.

We have shown that Dicer processes 7SL RNA into different fragments ranging from ∼20 to more than 200 nucleotides. Here we addressed the molecular functions of these 7SL RNA fragments and found that some of them functioned as dominant-negative regulators of the full-length 7SL RNA, interfering with signal recognition particle (SRP) complex formation. Transfection of these 7SL RNA fragments inhibited the expression of cell surface glycoproteins, the targeting of a reporter protein to the endoplasmic reticulum, and the secretion of secreted alkaline phosphatase. These results suggest that some Dicer-processed 7SL RNA fragments interfered with SRP-mediated protein targeting. Moreover, we showed that Dicer knockdown enhanced SRP-mediated protein targeting and that transfection of a mixture of the 7SL RNA fragments partially restored this effect. Our data indicate that Dicer can fine-tune the efficiency of SRP-mediated protein targeting via processing a proportion of 7SL RNA into fragments of different lengths.

Dicer-dependent biogenesis of small RNAs derived from 7SL RNA.

It has been reported that decreased Dicer expression leads to Alu RNAs accumulation in human retinal pigmented epithelium cells, and Dicer may process the endogenous SINE/B1 RNAs (the rodent equivalent of the primate Alu RNAs) into small interfering RNAs (siRNAs). In this study, we aimed to address whether Dicer can process Alu RNAs and their common ancestor, 7SL RNA. Using Solexa sequencing technology, we showed that Alu-derived small RNAs accounted for 0.6% of the total cellular small RNAs in HepG2.2.15 cells, and the abundance decreased when Dicer was knocked down. However, Alu-derived small RNAs showed different characteristics from miRNAs and siRNAs, the classic Dicer-processed products. Interestingly, we found that small RNAs derived from 7SL RNA accounted for 3.1% of the total cellular small RNAs in the control cells, and the abundance dropped about 3.4 folds in Dicer knockdown cells. Dicer-dependent biogenesis of 7SL RNA-derived small RNAs was validated by northern blotting. In vitro cleavage assay using the recombinant human Dicer protein also showed that synthetic 7SL RNA was processed by Dicer into fragments of different lengths. Further functional analysis suggested that 7SL RNA-derived small RNAs do not function like miRNAs, neither do they regulate the expression of 7SL RNA. In conclusion, the current study demonstrated that Dicer can process 7SL RNA, however, the biological significance remains to be elucidated.

Monitoring of viral pathogens in pediatric intensive care unit and analysis of clinical significance / 中华儿科杂志

<p><b>OBJECTIVE</b>To study the characteristics of viral spectrum and clinical features of children in pediatric intensive care unit (PICU).</p><p><b>METHOD</b>Nasopharyngeal aspirate specimens (NPA) from 349 patients(1 from each) and 130 cerebrospinal fluids (CSF) specimens were collected from children who were admitted to the PICU of Second Affiliated Hospital of Shantou University Medical College. Additional 87 NPA specimens were collected from healthy children for routine examination on the physical examination center, and the clinical data were collected. Multiplex PCR was applied to detect 16 kinds of viruses from NPA and CSF. Fluorescence quantitative PCR was applied to detect 13 viruses from CSF and to analyze the clinical data of positive cases.</p><p><b>RESULT</b>There were 209 samples (59.9%) of the 349 NPA specimens were positive for viruses, which included 117 cases positive for human rhinovirus (HRV), 60 for respiratory syncytial virus (RSV), 20 for influenza virus A (Inf A), 10 for adenovirus (ADV), 6 for parainfluenza virus type 3(PIV-3), 6 for human Boca virus (HBoV), 5 for influenza virus C(Inf C), 4 for parainfluenza virus type 4(PIV-4), 4 for human coronavirus-HKU1/OC43, 3 for influenza virus B (Inf B), 3 for WU Polyomavirus (WUPyV), 2 parainfluenza virus type 1(PIV-1), 2 human metapneumovirus (HMPV) and 1 human coronavirus-NL63/229E. But none from 87 healthy controls were positive for any respiratory virus. Among the 130 CSF specimens, in 58 cases the diagnosis was viral encephalitis. There were 22 samples (37.9%) among the 58 CSF specimens positive for viruses, which included 14 enterovirus (EV), 3 human cytomegalovirus (HCMV), 2 mumps virus, 1 coxsackie virus A16 (Cox-A16), 1 herpes simplex virus (HSV) and 1 human rhinovirus (HRV). The total positive rate was 63.3% (221/349) . Co-infection by at least 2 viral pathogens under study was observed in 45 of the 349 patients (12.9% of the total number of cases, 20.4% of the positives cases). The commonest pathogens in co-infected samples were WUPyV (100%) and HMPV(100%). The positive rate of virus peaked in the first 6 months of life, the rate in boys were higher than in girls and the peak season was summer. The numbers of none serious cases in the virus positive group were less than those in the virus negative group while the numbers of extremely serious cases in the virus positive group were higher than in the virus negative group.</p><p><b>CONCLUSION</b>Viral pathogen is a major cause of infectious disease in pediatric critical illnesses and virus infection may lead to severe illness.</p>

Biological characteristics of human umbilical cord-derived mesenchymal stem cells and their differentiation into neurocyte-like cells / 中华儿科杂志

<p><b>OBJECTIVE</b>To investigate the isolation and expansion of mesenchymal stem cells (MSCs) from human umbilical cord Wharton's jelly and their biological identities, and explore the possibility of inducing human umbilical cord-derived MSCs to differentiate into neurocyte-like cells.</p><p><b>METHODS</b>The growth and proliferative abilities of human umbilical cord-derived MSCs were observed, and their immunophenotypes were determined by flow cytometry. Salvia miltiorrhiza and beta-sulfhydryl alcohol were adopted to induce the cells to differentiate. The differentiated and undifferentiated cells were identified with immunocytochemistry. The pleiotrophin and nestin genes were measured by RT-PCR.</p><p><b>RESULTS</b>A population of human umbilical cord-derived MSCs were isolated from human umbilical Wharton's jelly; they were processed to obtain a fibroblast-like population of cells and could be maintained in vitro for extended periods with stable population doubling, and they were expanded as undifferentiated cells in culture for more than 10 passages, indicating their proliferative capacity. The human umbilical cord-derived MSCs were positive for CD(29), CD(44), CD(59), CD(105), but negative or weakly expressed the markers of hematopoietic cells such as CD(14), CD(33), CD(34), CD(28), CD(45) and CD(117). The important GVHD correlation markers were negative or weakly expressed, including CD(80) (B7-1), CD(86) (B7-2), CD(40) and CD(40L). Salvia miltiorrhiza beta-sulfhydryl alcohol could induce the MSCs to express nestin, a marker of neuronal precursor stem cells at early stage of differentiation. Later, they exhibited neural phenotypes, expressing beta-tubulin III and neurofilament (NF) and glial fibrillary acidic protein (GFAP). It was confirmed by RT-PCR that the MSCs could express pleiotrophin either before or after the induction of salvia miltiorrhiza, furthermore, after the induction the expression was markedly enhanced and the nestin gene was also expressed.</p><p><b>CONCLUSION</b>The human MSCs could be isolated from human umbilical cord Wharton's jelly, and it was easy to propagate these MSCs. The negative GVHD correlated markers might result from the fact that MSCs had no HLA barrier, which may suggest potential clinical significance. The MSCs are capable of differentiating into neurocyte-like cells and they may represent an alternative stem cell source for CNS cells transplantation.</p>

Human umbilical cord Wharton's Jelly-derived mesenchymal stem cells differentiation into nerve-like cells / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>The two most basic properties of mesenchymal stem cells (MSCs) are the capacities to self-renew indefinitely and differentiate into multiple cells and tissue types. The cells from human umbilical cord Wharton's Jelly have properties of MSCs and represent a rich source of primitive cells. This study was conducted to explore the possibility of inducing human umbilical cord Wharton's Jelly-derived MSCs to differentiate into nerve-like cells.</p><p><b>METHODS</b>MSCs were cultured from the Wharton's Jelly taken from human umbilical cord of babies delivered after full-term normal labor. Salvia miltiorrhiza and beta-mercaptoethanol were used to induce the human umbilical cord-derived MSCs to differentiate. The expression of neural protein markers was shown by immunocytochemistry. The induction process was monitored by phase contrast microscopy, electron microscopy (EM), and laser scanning confocal microscopy (LSCM). The pleiotrophin and nestin genes were measured by reverse transcription-polymerase chain reaction (RT-PCR).</p><p><b>RESULTS</b>MSCs in the Wharton's Jelly were easily attainable and could be maintained and expanded in culture. They were positive for markers of MSCs, but negative for markers of hematopoietic cells and graft-versus-host disease (GVHD)-related cells. Treatment with Salvia miltiorrhiza caused Wharton's Jelly cells to undergo profound morphological changes. The induced MSCs developed rounded cell bodies with multiple neurite-like extensions. Eventually they developed processes that formed networks reminiscent of primary cultures of neurons. Salvia miltiorrhiza and beta-mercaptoethanol also induced MSCs to express nestin, beta-tubulinIII, neurofilament (NF) and glial fibrillary acidic protein (GFAP). It was confirmed by RT-PCR that MSCs could express pleiotrophin both before and after induction by Salvia miltiorrhiza. The expression was markedly enhanced after induction and the nestin gene was also expressed.</p><p><b>CONCLUSIONS</b>MSCs could be isolated from human umbilical cord Wharton's Jelly. They were capable of differentiating into nerve-like cells using Salvia miltiorrhiza or beta-mercaptoethanol. The induced MSCs not only underwent morphologic changes, but also expressed the neuron-related genes and neuronal cell markers. They may represent an alternative source of stem cells for central nervous system cell transplantation.</p>