Key Laboratory of Laboratory Medicine

Wenzhou, China

Key Laboratory of Laboratory Medicine

Wenzhou, China
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Zheng X.,Xi'an Jiaotong University | Zheng X.,Wenzhou University | Zheng X.,Key Laboratory of Laboratory Medicine | Gao Y.,First Peoples Hospital of Changde | And 8 more authors.
PLoS ONE | Year: 2015

Human cytomegalovirus (HCMV) interleukin-10 (hcmvIL-10), encoded by HCMV UL111A gene, is a homolog of human IL-10. It exerts immunomodulatory effects that allow HCMV to evade host defense mechanisms. However, the exact mechanism underlying the regulation of hcmvIL-10 expression is not well understood. The transcription factor acute myeloid leukemia 1 (AML-1) plays an important role in the regulation of various genes involved in the differentiation of hematopoietic lineages. A putative AML-1 binding site is present within the upstream regulatory region (URR) of UL111A gene. To provide evidence that AML-1 is involved in regulating UL111A gene expression, we examined the interaction of AML-1 with the URR of UL111A in HCMV-infected human monocytic THP-1 cells using a chromatin immunoprecipitation assay. HcmvIL-10 transcription was detected in differentiated THP-1 cells, but not in undifferentiated ones. Furthermore, the URR of UL111A showed a higher intensity of AML-1 binding, a higher level of histone H3 acetyl-K9, but a lower level of histone H3 dimethyl-K9 in differentiated THP-1 cells than undifferentiated cells. Down-regulation of AML1 by RNA interference decreased the expression of the UL111A gene. Our results suggest that AML-1 may contribute to the epigenetic regulation of UL111A gene via histone modification in HCMV-infected differentiated THP-1 cells. This finding could be useful for the development of new anti-viral therapies. © 2015 Zheng et al.

Liu Y.-H.,Tsinghua University | Liu Y.-H.,Key Laboratory of Laboratory Medicine | Jin J.-L.,Hebi Polytechnic | Wang Y.-Z.,Key Laboratory of Laboratory Medicine | And 9 more authors.
Acta Pharmacologica Sinica | Year: 2016

Aim:Recent evidence shows that localization of mRNAs and their protein products at cellular protrusions plays a decisive function in the metastasis of cancer cells. The aim of this study was to identify the variety of proteins encoded by protrusion-localized mRNAs and their roles in the metastasis and invasion of liver cancer cells.Methods:Highly metastatic hepatocellular carcinoma cell line HCCLM3 and non-metastatic hepatocellular carcinoma cell line SMMC-7721 were examined. Cell protrusions (Ps) were separated from cell bodies (CB) using a Boyden chamber assay; total mRNA population in CB and Ps fractions was analyzed using high-throughput direct RNA sequencing. The localization of STAT3 mRNA and protein at Ps was confirmed using RT-qPCR, RNA FISH, and immunofluorescence assays. Cell migration capacity and invasiveness of HCCLM3 cells were evaluated using MTT, wound healing migration and in vitro invasion assays. The interaction between Stat3 and growth factor receptors was explored with co-immunoprecipitation assays.Results:In HCCLM3 cells, 793 mRNAs were identified as being localized in the Ps fraction according to a cut-off value (Ps/CB ratio) >1.6. The Ps-localized mRNAs could be divided into 4 functional groups, and were all closely related to the invasive and metastatic properties. STAT3 mRNA accumulated in the Ps of HCCLM3 cells compared with non-metastatic SMMC-7721 cells. Treatment of HCCLM3 cells with siRNAs against STAT3 mRNA drastically decreased the cell migration and invasion. Moreover, Ps-localized Stat3 was found to interact with pseudopod-enriched platelet-derived growth factor receptor tyrosine kinase (PDGFRTK) in a growth factor-dependent manner.Conclusion:This study reveals STAT3 mRNA localization at the Ps of metastatic hepatocellular carcinoma HCCLM3 cells by combining application of genome-wide and gene specific description and functional analysis. © 2016 CPS and SIMM All rights reserved.

Li W.,Key Laboratory of Laboratory Medicine | Li W.,Zhejiang Provincial Key Laboratory of Medical Genetics | Li W.,Wenzhou University | Wen C.,Key Laboratory of Laboratory Medicine | And 8 more authors.
Molecular and Cellular Biochemistry | Year: 2015

Mitochondrial diabetes originates mainly from mutations located in maternally transmitted, mitochondrial tRNA-coding genes. In a genetic screening program of type 2 diabetes conducted with a Chinese Han population, we found one family with suggestive maternally transmitted diabetes. The proband’s mitochondrial genome was analyzed using DNA sequencing. Total 42 known nucleoside changes and 1 novel variant were identified, and the entire mitochondrial DNA sequence was assigned to haplogroup M11b. Phylogenetic analysis showed that a homoplasmic mutation, 10003T>C transition, occurred at the highly conserved site in the gene encoding tRNAGly. Using a transmitochondrial cybrid cell line harboring this mutation, we observed that the steady-state level of tRNAGly significantly affected and the amount of tRNAGly decreased by 97 %, production of reactive oxygen species was enhanced, and mitochondrial membrane potential, mtDNA copy number and cellular oxygen consumption rate were remarkably decreased compared with wild-type cybrid cells. The homoplasmic 10003T>C mutation in the mitochondrial tRNAGly gene suggested to be as a pathogenesis-related mutation which might contribute to the maternal inherited diabetes in the Han Chinese family. © 2015, Springer Science+Business Media New York.

Ye W.,Key Laboratory of Laboratory Medicine | Ye W.,Wenzhou University | Chen S.,Key Laboratory of Laboratory Medicine | Jin S.,Key Laboratory of Laboratory Medicine | And 2 more authors.
Molecular Medicine Reports | Year: 2013

Metabolic syndrome (MS) is a complex disorder characterized by a group of metabolic abnormalities. In the present study, the case of an 18-year-old male who presented with MS characteristics with central obesity (overweight and a waist circumference of 95 cm) and dyslipidemia (high TG, low HDL levels and low apoA-I/apoB-100) was reported. The patient's family has maternally inherited diabetes and a number of the patient's maternal relatives present MS features. For the investigation of the mitochondrial DNA variants in the patient and the patient's family, genomic DNA of all the family members were extracted from peripheral blood using routine methods. Amplification of mitochondrial DNA in 24 overlapping fragments by PCR, direct sequencing and denaturing high-performance liquid chromatography was utilized for genetic analysis. Sequences were compared to the reference sequence to identify variants. Bioinformatic methods and databases were used to analyze conservation of the variants and to predict the protein secondary structure. With the exception of the patient, five relatives were diagnosed with MS. Moreover, 5 of the 8 family members had been diagnosed with diabetes, hearing loss and mild kidney impairment according to serum biochemical analysis. Further molecular genetic analysis indicated that the MS-associated variant T16189C was detected in this family. Notably, a heteroplasmic mutation A8890G was detected in the patient in the mitochondrial ATP6 gene, which codes the ATP synthase subunit 6 (ATPase6). A8890G changed the highly conserved ATPase6 Lys122 into Glu122 in the mitochondrial inner membrane. However, this mutation was not detected in other family members. In conclusion, the mutation A8890G may affect the function of ATPase 6 and the production of ATP, thus contributing to juvenile-onset MS. It was not detected in other family members possibly due to the mitochondrial genetic segregation or production of a new germline mutation in the juvenile-onset patient.

Yang Y.,Wenzhou University | Meng H.,Wenzhou University | Peng Q.,Wenzhou University | Yang X.,Wenzhou University | And 7 more authors.
Cancer Gene Therapy | Year: 2015

Preliminary studies showed that miR-21 is overexpressed in some human cancers. However, the role of miR-21 in cancer is still unclear and even controversial. Our purpose was to investigate the biological effects of miR-21 on A549 non-small cell lung cancer (NSCLC) cells and the underlying mechanisms of those effects. The expression of miR-21 was quanti fied in serum samples from patients with NSCLC. A549 cells were transfected with miR-NC-sponge or miR-21-sponge only, or with miR-21-sponge plus PDCD4 small-interfering RNA (siRNA). The expression of miR-21 and PDCD4 mRNA in transfected cells was quantified by real-time polymerase chain reaction and the expression of PDCD4 protein by Western blot. Cell proliferation, apoptosis, migration, and invasion assays were performed to determine the biological effects of miR-21 expression and PDCD4 inhibition. miR-21 was overexpressed in serum from patients with NSCLC. Reduced miR-21 expression was observed following transfection with miR-21-sponge in A549 NSCLC cells. Co-transfection of miR-21-sponge with PDCD4 siRNA upregulated miR-21 expression in these cells. PDCD4 mRNA and protein levels were increased 2.14-fold and 2.16-fold, respectively, following inhibition of miR-21 expression. Inhibition of miR-21 expression following transfection of miR-21-sponge reduced cell proliferation, migration, and invasion of A549 cells. Depletion of PDCD4 by siRNA transfection reversed the reduction of cell proliferation, migration, and invasion induced by inhibition of miR-21 in A549 cells. It indicates that miR-21 is highly expressed in patients with NSCLC and inhibition of miR-21 expression reduces proliferation, migration, and invasion of A549 cells by upregulating PDCD4 expression. Modulation of miR-21 or PDCD4 expression may provide a potentially novel therapeutic approach for NSCLC. © 2015 Nature America, Inc.

Zhang Q.,Wenzhou University | Zhang Q.,Key Laboratory of Laboratory Medicine | Gao Y.,Wenzhou University | Gao Y.,Key Laboratory of Laboratory Medicine | And 11 more authors.
Virology Journal | Year: 2014

Background: This study investigated infection status and distribution of human cytomegalovirus (HCMV) serum markers in hospitalized children from the Wenzhou region. Methods. This survey was performed on 10,147 hospitalized children from birth to 14 years of age in Southeastern China (Wenzhou region) from March 2010 to March 2013. IgM and IgG antibodies to HCMV were quantitatively detected by chemiluminescence immunoassay (CLIA). HCMV IgM or IgG detection rates, concentration, and distribution in various age groups were retrospectively analyzed. Results: In this study of hospitalized children, the overall rates of HCMV IgM+ and IgG+ were 10.8% (1,099/10,147) and 83.0% (8,425/10,147), respectively. The lowest HCMV IgM + rate (1.0%, P < 0.001) was observed in the group of patients <28 days of age whereas the highest HCMV IgM+ rate (19.9%, P < 0.001) occurred in the 28 days ∼ 5 months old group. However, the concentrations of HCMV specific IgM in all age groups were not significantly different (P > 0.05). The HCMV IgG+ rate was highest in the <28 days group (98.1%, P < 0.001). The 28 days ∼ 5 months old group had the lowest HCMV specific IgG concentrations (median, 133.9 AU/mL, P < 0.001). Among 1,099 HCMV IgM+ children, 405 (36.9%) were diagnosed with respiratory infections which pneumonia accounted for 18.2% (200/1,099) of the total population. However, children with respiratory infections had the lowest HCMV IgG concentrations (median, 161.1 AU/mL, P < 0.05). Conclusions: HCMV specific antibody responses are very common in hospitalized children with respiratory infection in Wenzhou region. Protection against HCMV airway infection needs greater emphasis and further studies will be helpful to reveal the role of HCMV in children respiratory disease. © 2014 Zhang et al.; licensee BioMed Central Ltd.

Gan R.,Wenzhou University | Yang Y.,Wenzhou University | Yang X.,Wenzhou University | Zhao L.,Wenzhou University | And 4 more authors.
Cancer Gene Therapy | Year: 2014

Aberrantly expressed microRNAs (miRNAs) are involved in breast tumorigenesis. It is still unclear if and how miRNAs-221/222 are implicated in breast cancer and the resistance to estrogen receptor modulator tamoxifen. We investigated the roles and mechanisms of miR-221/222 in breast cancer cells, particularly in modulating response to tamoxifen therapy. MCF-7 and MDA-MB-231 breast cancer cells were transfected with antisense oligonucleotides AS-miR-221 and AS-miR-222 and their expression of miR-221 and miR-222 was assessed. The correlation of miR-221/222 with tissue inhibitor of metalloproteinase-3 (TIMP 3) expression was investigated by fluorescence quantitative PCR and western blotting analysis. The therapeutic sensitivity of these cells, transfected and untransfected, to tamoxifen was determined. Transfection of AS-miR-221 and AS-miR-222 dramatically inhibited expression of miR-221 and miR-222, respectively, in both MCF-7 and MDA-MB-231 cells (P<0.05-0.01). Downregulation of miR-221/222 significantly increased the expression of TIMP 3 compared with controls (P<0.05-0.01). The viability of estrogen receptor (ER)-positive MCF-7 cells transfected with AS-miR-221 or/and AS-miR-222 was significantly reduced by tamoxifen (P<0.05-0.01). We have demonstrated for the first time that suppression of miRNA-221/222 increases the sensitivity of ER-positive MCF-7 breast cancer cells to tamoxifen. This effect is mediated through upregulation of TIMP 3. These findings suggest that upregulation of TIMP 3 via inhibition of miRNA-221/222 could be a promising therapeutic approach for breast cancer. © 2014 Nature America, Inc.

Yang X.,Wenzhou University | Yang Y.,Wenzhou University | Gan R.,Wenzhou University | Zhao L.,Wenzhou University | And 12 more authors.
PLoS ONE | Year: 2014

Studies have shown that miR-221 and miR-222 are deregulated in many cancers, including prostate cancer. Nevertheless, the biological role and the underlying mechanisms of miR-221 and miR-222 in the pathogenesis of androgen-independent prostate cancer are still not clear. The proliferation, apoptosis, cell cycle distinction, and migration capacity of prostate cells were determined following transfection of miR-221 or miR-222 inhibitor. The biological impact and regulation of SIRT1 on prostate cancer cells were investigated. MiR-221 and miR-222 were highly expressed in PC-3 cells compared with in LNCap cells. After miR-221 or miR-222 expression was inhibited, the proliferation and migration rates of PC-3 cells decreased and the apoptosis rate increased. Moreover, SIRT1 protein was up-regulated in cells after they were transfected with miR-221 or miR-222 inhibitor. Cells transfected with siSIRT1 showed increased migration and a decreased apoptosis rate, but there was no significant effect on cell proliferation compared with the controls. There was a negative correlation between miR-221 or miR-222 and SIRT1, but no direct target relationship was identified. These data demonstrate that miR-221 and miR-222 are highly expressed in PC-3 cells. Their inhibition leads to reduced cell proliferation and migration and increased apoptosis in prostate cancer cells. These effects are potentially mediated by up-regulation of SIRT1. © 2014 Yang et al.

Peng Y.,Wenzhou University | Peng Y.,University of Missouri | Lai M.,Wenzhou University | Lai M.,Key Laboratory of Laboratory Medicine | And 8 more authors.
Immunology Letters | Year: 2016

Although human autologous B cells represent a promising alternative to dendritic cells (DCs) for easy large-scale preparation, the naive human B cells are always poor at antigen presentation. The safe and effective usage record of human adenovirus type 7 (HAdV7) live vaccines makes it attractive as a promising vaccine vector candidate. To investigate whether HAdV7 vector could be used to induce the human B cells cross-presentation, in the present study, we constructed the E3-defective recombinant HAdV7 vector encoding green fluorescent protein (GFP) and carcinoembryonic antigen (CEA). We demonstrated that naive human B cells can efficiently be transduced, and that the MAPKs/NF-κB pathway can be activated by recombinant HAdV7. We proved that cytokine TNF-α, IL-6 and IL-10, surface molecule MHC class I and the CD86, antigen-processing machinery (APM) compounds ERp57, TAP-1, and TAP-2. were upregulated in HAdV7 transduced human B cells. We also found that CEA-specific IFNγ expression, degranulation, and in vitro and ex vivo cytotoxicities are induced in autologous CD8+ T cells presensitized by HAd7CEA modified human B cells. Meanwhile, our evidences clearly show that Toll-like receptors 9 (TLR9) antagonist IRS 869 significantly eliminated most of the HAdV7 initiated B cell activation and CD8+ T cells response, supporting the role and contribution of TLR9 signaling in HAdV7 induced human B cell cross-presentation. Besides a better understanding of the interactions between recombinant HAdV7 and human naive B cells, to our knowledge, the present study provides the first evidence to support the use of HAdV7-modified B cells as a vehicle for vaccines and immunotherapy. © 2015 Elsevier B.V.

Bo J.,Wenzhou University | Xie S.,Wenzhou University | Guo Y.,Wenzhou University | Zhang C.,Wenzhou University | And 7 more authors.
Journal of Diabetes Research | Year: 2016

Methylglyoxal (MG) is a highly reactive glucose metabolic intermediate and a major precursor of advanced glycation end products. MG level is elevated in hyperglycemic disorders such as diabetes mellitus. Substantial evidence has shown that MG is involved in the pathogenesis of diabetes and diabetic complications. We investigated the impact of MG on insulin secretion by MIN6 and INS-1 cells and the potential mechanisms of this effect. Our study demonstrates that MG impaired insulin secretion by MIN6 or ISN-1 cells in a dose-dependent manner. It increased reactive oxygen species (ROS) production and apoptosis rate in MIN6 or ISN-1 cells and inhibited mitochondrial membrane potential (MMP) and ATP production. Furthermore, the expression of UCP2, JNK, and P38 as well as the phosphorylation JNK and P38 was increased by MG. These effects of MG were attenuated by MG scavenger N-acetyl cysteine. Collectively, these data indicate that MG impairs insulin secretion of pancreatic β-cells through increasing ROS production. High levels of ROS can damage β-cells directly via JNK/P38 upregulation and through activation of UCP2 resulting in reduced MMP and ATP production, leading to β-cell dysfunction and impairment of insulin production. © 2016 Jinshuang Bo et al.

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