MicroRNA-29a induces insulin resistance by targeting PPARδ in skeletal muscle cells.

Intrauterine growth retardation (IUGR) induces metabolic syndrome, which is often characterized by insulin resistance (IR), in adults. Previous research has shown that microRNAs (miRNAs or miRs) play a role in the target genes involved in this process, but the mechanisms remain unclear. In the present study, we examined miRNA profiles using samples of skeletal muscles from both IUGR and control rat offspring whose mothers were fed either a protein-restricted diet or a diet which involved normal amounts of protein during pregnancy, respectively. miR­29a was found to be upregulated in the skeletal muscles of IUGR offspring. The luciferase reporter assay confirmed the direct interaction between miR­29a and peroxisome proliferator­activated receptor δ (PPARδ). Overexpression of miR­29a in the skeletal muscle cell line C2C12 suppressed the expression of its target gene PPARδ, which, in turn, influenced the expression of its coactivator, peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α). Thus, PPARδ/PGC-1α­dependent signals together reduced insulin-dependent glucose uptake and adenosine triphosphate (ATP) production. Overexpression of miR­29a also caused a decrease in levels of glucose transporter 4 (GLUT4), the most important glucose transporter in skeletal muscle, which partially induced a decrease insulin­dependent glucose uptake. These findings provide evidence for a novel micro-RNA­mediated mechanism of PPARδ regulation, and we also noted the IR-promoting actions of miR-29a in skeletal muscles of IUGR.

The Roles of p38 MAPK and ERK1/2 in Coplanar Polychlorinated Biphenyls-Induced Apoptosis of Human Extravillous Cytotrophoblast-Derived Transformed Cells.

BACKGROUND/AIMS: The purpose of this study was to investigate the relationships among exposure to coplanar polychlorinated biphenyls (Co-PCBs), the expression of gC1qR and the underlying intracellular apoptotic signaling pathways of human extravillous cytotrophoblast (EVCT)-derived transformed cells (HTR-8/SVneo and HPT-8). METHODS: Apoptosis in HTR-8/SVneo and HPT-8 cells was assessed using flow cytometric analysis. gClqR expression was examined in the HTR-8/SVneo and HPT-8 cells using real-time qPCR and western blot analyses. The phosphorylations of p38 mitogen-activated protein kinase (p38 MAPK) (Thr180/Tyr182) and extracellular signal-regulated kinase (ERK) 1/2 (Thr202/Thr204) were detected using western blot analyses. RESULTS: The HTR-8/SVneo and HPT-8 cells treated with Co-PCBs exhibited significantly increased gClqR expression, p38 MAPK/ERK activation and an up-regulation of cellular apoptosis. These effects were abrogated by the application of gC1qR small interfering RNA (siRNA). Furthermore, apoptosis in HTR-8/SVneo and HPT-8 cells was observed upon treatment with Co-PCBs, and these effects were reversed by the p38 MAPK pathway inhibitor SB203580 or the ERK1/2 pathway inhibitor PD098059. CONCLUSION: These data support a mechanism wherein gC1qR plays a crucial p38 MAPK/ERK signaling pathway-dependent role in Co-PCBs-induced apoptosis of human EVCT-derived transformed cells.

Systematic gene microarray analysis of the lncRNA expression profiles in human uterine cervix carcinoma.

The human uterine cervix carcinoma is one of the most well-known malignancy reproductive system cancers, which threatens women health globally. However, the mechanisms of the oncogenesis and development process of cervix carcinoma are not yet fully understood. Long non-coding RNAs (lncRNAs) have been proved to play key roles in various biological processes, especially development of cancer. The function and mechanism of lncRNAs on cervix carcinoma is still rarely reported. We selected 3 cervix cancer and normal cervix tissues separately, then performed lncRNA microarray to detect the differentially expressed lncRNAs. Subsequently, we explored the potential function of these dysregulated lncRNAs through online bioinformatics databases. Finally, quantity real-time PCR was carried out to confirm the expression levels of these dysregulated lncRNAs in cervix cancer and normal tissues. We uncovered the profiles of differentially expressed lncRNAs between normal and cervix carcinoma tissues by using the microarray techniques, and found 1622 upregulated and 3026 downregulated lncRNAs (fold-change>2.0) in cervix carcinoma compared to the normal cervical tissue. Furthermore, we found HOXA11-AS might participate in cervix carcinogenesis by regulating HOXA11, which is involved in regulating biological processes of cervix cancer. This study afforded expression profiles of lncRNAs between cervix carcinoma tissue and normal cervical tissue, which could provide database for further research about the function and mechanism of key-lncRNAs in cervix carcinoma, and might be helpful to explore potential diagnosis factors and therapeutic targets for cervix carcinoma.

The role of the globular heads of the C1q receptor in HPV-16 E2-induced human cervical squamous carcinoma cell apoptosis via a mitochondria-dependent pathway.

BACKGROUND: Human papillomavirus type-16 (HPV-16) E2 protein acts as a transcriptional modulator and plays a key role in regulating many biological responses. The purpose of this study was to investigate the relationship between HPV-16 E2, the receptor for the globular heads of human C1q (gC1qR) gene expression, mitochondrial dysfunction and apoptosis regulation in human cervical squamous carcinoma cells (C33a and SiHa). METHODS: HPV-16 E2 and gC1qR expression was examined using real-time PCR and western blot analysis. Apoptosis in C33a and SiHa cells was assessed by flow cytometry. Mitochondrial function was detected via ROS generation, the amount of cytosolic Ca2+, and changes in the mitochondrial membrane potential (Δψm). RESULTS: The expression of the HPV-16 E2 and gC1qR gene significantly decreased in human cervical squamous carcinoma samples relative to the non-cancerous cervix samples. C33a and SiHa cells that were transfected with a vector encoding HPV-16 E2 displayed significantly increased gC1qR gene expression and mitochondrial dysfunction as well as an up-regulation of cellular apoptosis, which was abrogated by the addition of gC1qR small-interfering RNA (siRNA). CONCLUSIONS: These data support a mechanism whereby gC1qR plays an important role in HPV-16 E2-induced human cervical squamous carcinoma cell apoptosis via a mitochondria-dependent pathway.

The globular heads of the C1q receptor regulate apoptosis in human extravillous cytotrophoblast-derived transformed cells via a mitochondria-dependent pathway.

PROBLEM: The receptor for the globular head of human C1q (gC1qR) predominantly localizes to the mitochondrial matrix. gC1qR mediates many biological responses, including growth perturbations, morphological abnormalities and the initiation of apoptosis. The purpose of this study was to investigate the relationship between gC1qR expression, mitochondrial dysfunction and the regulation of apoptosis in human extravillous cytotrophoblast (EVCT)-derived transformed cell lines (HTR-8/SVneo and HPT-8). METHOD OF STUDY: gC1qR expression was examined in human placental villi using real-time qPCR and Western blot analysis. The apoptotic death of HTR-8/SVneo and HPT-8 cells was assessed using flow cytometric analysis. Mitochondrial function was assessed via ROS generation, the amount of cytosolic Ca(2+) and changes in the mitochondrial membrane potential (Δψm). RESULTS: The expression of the gC1qR gene was significantly increased in spontaneous abortion samples relative to induced abortion samples. HTR-8/SVneo and HPT-8 cells transfected with a gC1qR vector showed upregulation of cellular apoptosis and mitochondrial dysfunction, interestingly, which were abrogated by the addition of metformin. Metformin may protect mitochondrial function. CONCLUSION: These data support a mechanism whereby gC1qR induces apoptosis through mitochondria-dependent pathways in human EVCT-derived transformed cells.

Neurotoxicity and biomarkers of lead exposure: a review.

Appropriate selection and measurement of lead biomarkers of exposure are critically important for health care management purposes, public health decision making, and primary prevention synthesis. Lead is one of the neurotoxicants that seems to be involved in the etiology of psychologies. Biomarkers are generally classified into three groups: biomarkers of exposure, effect, and susceptibility.The main body compartments that store lead are the blood, soft tissues, and bone; the half-life of lead in these tissues is measured in weeks for blood, months for soft tissues, and years for bone. Within the brain, lead-induced damage in the prefrontal cerebral cortex, hippocampus, and cerebellum can lead to a variety of neurological disorders, such as brain damage, mental retardation, behavioral problems, nerve damage, and possibly Alzheimer's disease, Parkinsons disease, and schizophrenia. This paper presents an overview of biomarkers of lead exposure and discusses the neurotoxic effects of lead with regard to children and adults.

The role of globular heads of the C1q receptor in HPV 16 E2-induced human cervical squamous carcinoma cell apoptosis is associated with p38 MAPK/JNK activation.

BACKGROUND: Human papillomavirus type 16 (HPV 16) E2 protein is a multifunctional DNA-binding protein. HPV 16 E2 regulates many biological responses, including DNA replication, gene expression, and apoptosis. The purpose of this study was to investigate the relationship among the receptor for globular heads of the human C1q (gC1qR) gene expression, HPV 16 E2 transfection and apoptosis regulation in human cervical squamous carcinoma cells (C33a and SiHa). METHODS: gC1qR expression was examined in C33a and SiHa cells using real-time PCR and Western blot analysis. Apoptosis of C33a and SiHa cells was assessed by flow cytometry. C33a and SiHa cell viability, migration and proliferation were detected using the water-soluble tetrazolium salt (WST-1) assay, a transwell assay and 3H-thymidine incorporation into DNA (3H-TdR), respectively. RESULTS: C33a and SiHa cells that were transfected with a vector encoding HPV 16 E2 displayed significantly increased gC1qR gene expression and p38 mitogen-activated protein kinase (p38 MAPK)/c-jun N-terminal kinase (JNK) activation as well as up-regulation of cellular apoptosis, which was abrogated by the addition of gC1qR small interfering RNA (siRNA). Furthermore, the changes in C33a and SiHa cell viability, migration and proliferation that were observed upon HPV 16 E2 transfection were abrogated by SB203580 (a p38 MAPK inhibitor) or SP600125 (a JNK inhibitor) treatment. CONCLUSION: These data support a mechanism whereby HPV 16 E2 induces apoptosis by silencing the gC1qR gene or inhibiting p38 MAPK/JNK signalling in cervical squamous cell carcinoma.

Maternal protein restriction in rats leads to reduced PGC-1α expression via altered DNA methylation in skeletal muscle.

Intrauterine growth retardation (IUGR) is thought to program insulin resistance, type 2 diabetes and other metabolic diseases in later life. Skeletal muscle is an important tissue involved in regulating the metabolism. We therefore hypothesized that the regulation of glucose- and lipid-related genes in skeletal muscle may contribute to metabolic changes in rats with IUGR. In this study, IUGR rats were bred from pregnant rats fed a protein-restricted (PR) diet. Insulin resistance (IR)-related metabolic parameters and the expression of key regulatory IR genes such as peroxisome proliferator-activated receptor γ (PPARγ) coactivator-1α (PGC-1α) and glucose transporter 4 (GLUT4) were measured in skeletal muscle from 18-month-old female IUGR rats. The methylation status of promoters of PGC-1α and GLUT4 were assessed in the same tissues. During the aging process, IUGR rats exhibited catch-up growth and obesity. In old age, they showed impaired glucose tolerance demonstrated by high glucose and insulin AUC (area under the curve) values. The expression of glucose transporter 4 (GLUT4) and PGC-1α in skeletal muscle was significantly reduced in IUGR rats. Mean CpG island methylation in the PGC-1α promoter sequence was significantly increased. These results suggest that a PR diet during gestation may induce epigenetic changes, such as DNA methylation, in the promoters of specific genes. The changes may affect gene expression and account for the metabolic alterations in female rats with IUGR.

Effect of Pregnancy on the Levels of Blood Cadmium and Lead: analysis of 2006-2011 Nanjing Maternity and Child Health Care Hospital Survey Data.

BACKGROUND: Prenatal lead exposure could not only affect various organ systems of the mother, but also provide a plumbeous environment for the fetus and newborns, and may affect the fetus in a number of detrimental ways. The aim of this study was to adequately determine the interaction between these factors and risky behaviors such as smoking. METHODS: Data from Nanjing Maternity and Child Health Care Hospital survey during the years of 2006-2011 were used (n = 4400) to evaluate the effections of age, parity, body mass index (BMI), race/ethnicity, pregnancy, iron (Fe) storage status and smoking status on the consumption of the levels of blood cadmium (Cd) and lead (Pb) of females aged 16-35yr old. The blood samples were sent to determine blood lead / cadmium concentration by the Inductively Coupled Plasma Mass Spectrometry (ICP-MS). STATA 12.1 software (www.stata.com) was used to fit regression models for each of the two metals. RESULTS: For both of the two metals, age was positively while BMI was negatively associated with the levels of these metals in blood. Smokers showed statistically significantly higher levels of Cd and Pb (P=0.007), while irrespective of race/ethnicity and Fe storage status as compared to nonsmokers. CONCLUSION: Novel to this study, pregnancy was found to be associated with significantly lower levels of Cd and Pb, while irrespective of race/ethnicity and Fe storage status as compared to non-pregnant females. It is conceivable that pregnancy could thus accelerate clearance of these metals in the blood.

The globular heads of the C1q receptor regulate apoptosis in human cervical squamous carcinoma cells via a p53-dependent pathway.

BACKGROUND: The globular heads of the human C1q receptor (gC1qR) localize predominantly to the mitochondrial matrix. gC1qR mediates many biological responses, including growth perturbation, morphological abnormalities and the initiation of apoptosis. The purpose of this study was to investigate the relationship between mitochondrial dysfunction, p53 status and gC1qR expression and the regulation of apoptosis in human cervical squamous carcinoma cells (C33a and SiHa). METHODS: Here, gC1qR expression was examined in human cervical tissues using real-time PCR and Western blot analysis. Apoptotic death of C33a and SiHa cells was assessed by flow cytometric analysis that detected the subG1 population. Mitochondrial function was assessed via ROS generation, the content of cytosolic Ca2+, and the change in mitochondrial membrane potential (Δψm). The viability and migration of C33a and SiHa cells were detected via the water-soluble tetrazolium salt (WST-1) assay and the transwell assay, respectively. RESULTS: gC1qR expression was decreased in cervical squamous cell carcinoma tissues compared with normal tissues. C33a and SiHa cells transfected with a vector encoding gC1qR displayed mitochondrial dysfunction and apoptosis, which was abrogated by the addition of a mutant form of p53 or p53 small interference RNA (siRNA). Furthermore, upon overexpression of gC1qR, cell viability and migration were significantly enhanced, and the apoptosis of C33a and SiHa cells were decreased when cells were treated with mutant p53 or p53 siRNA. CONCLUSIONS: These data support a mechanism whereby gC1qR induces apoptosis through the mitochondrial and p53-dependent pathways in cervical squamous cell carcinoma.

The role of the globular heads of C1q receptor (gC1qR) gene in regulating apoptosis of human cervical squamous cell carcinoma.

BACKGROUND: The globular heads of the human C1q receptor (gC1qR) are multi-compartmental and multi-functional cellular proteins. The list of biological responses mediated by the gC1qR includes growth perturbation and morphological abnormalities, along with the initiation of apoptosis. However, the effects of the gC1qR on the apoptosis of cervical squamous carcinoma cells (C33a and SiHa) have not been demonstrated. METHODS: Here, human cervical tissues were examined for the expression of the gC1qR using real-time PCR and Western blot analysis. Apoptotic death of C33a and SiHa cells was assessed by flow cytometric analysis to detect the subG1 population. Viability, migration and proliferation of C33a and SiHa cells were detected via the water-soluble tetrazolium salt (WST-1) assay, the Transwell assay and the (3)H-thymidine incorporation into DNA assay ((3)H-TdR), respectively. RESULTS: These data showed that expression of the gC1qR protein was significantly decreased in human cervical squamous cell carcinoma tissues relative to normal cervix tissues. C33a and SiHa cells transfected with a GFP-gC1qR vector resulted in the up-regulation of cellular apoptosis and an apparent increase in the expression of the p38 mitogen-activated protein kinase (p38 MAPK). Further, the changes in C33a and SiHa cells viability, migration and proliferation observed upon overexpression of gC1qR could be abrogated using the p38 MAPK inhibitor SB202190. CONCLUSION: These data indicate that gC1qR inhibits viability, migration and proliferation of cervical squamous cells carcinoma via the p38 MAPK signalling pathway.

Endocrine disruptors, polychlorinated biphenyls-induced gC1qR-dependent apoptosis in human trophoblast cell line HTR-8/SVneo.

Although an association exists between exposure to polychlorinated biphenyls (PCBs) and spontaneous miscarriage, the mechanisms underlying this phenomenon remain unclear. In this study, PCBs content in plasma was detected by gas chromatography-mass spectrometry (GC-MS) and decidua tissues were examined for the expression of globular heads of C1q receptor (gC1qR) using Western blot in patients who underwent induced abortion and spontaneous abortion. Results showed increased PCBs content and gC1qR expression in patients who experienced spontaneous abortion. In vitro, Western blot analysis demonstrated significantly higher caspase 3 expression and apoptotic cell counts in green fluorescent protein (GFP)-gC1qR vector group. Additionally, gC1qR and caspase 3 showed decreased expression following PCBs plus gC1qR small interfering RNA (siRNA) treatment. The percentage of apoptotic cells increased in cells treated with PCBs alone or PCB plus negative siRNA. These data suggest that maternal exposure to PCBs is associated with adverse pregnancy outcomes and that upregulation of gC1qR is important for PCBs-mediated trophoblast cell apoptosis.

The role of gC1qR in regulating survival of human papillomavirus 16 oncogene-transfected cervical cancer cells.

Human papillomavirus 16 (HPV-16) is strongly associated with the development of 50% of cervical cancers. The E6 and E7 proteins encoded by high-risk HPV types are associated with the immune evasion of cervical cancer cells, but the mechanism is poorly understood. The purpose of this study was to investigate whether cells transfected with E6 and E7 expression constructs reduce the expression of the globular heads of the C1q receptor (gC1qR), a mitochondrial surface protein overexpressed in certain cancer cells. First, C-33A cells were transiently transfected with the HPV-16 E6 and E7 oncogenes which resulted in gC1qR inhibition and a reduction in apoptosis. Second, gC1qR overexpression in cells showed that caspase-3 activation and mitochondrial dysfunction were involved in gC1qR-induced apoptosis. Cells transfected with a GFP-gC1qR vector resulted in upregulated gC1qR protein and a gradual increase in the generation of reactive oxygen species (ROS). Additionally, ROS generation and increased Ca2+ influx in mitochondria resulted in the loss of the mitochondrial transmembrane potential. Interestingly, when gC1qR was overexpressed in C-33A cells, apoptosis was significantly inhibited when cells were treated with metformin, which may protect mitochondrial function. These data suggest that gC1qR could play an important role in HPV-16-induced cervical cancer immune evasion depending on its level of expression and subcellular localisation.

The role of eIF5A in epidermal growth factor-induced proliferation of corneal epithelial cell association with PI3-k/Akt activation.

PURPOSE: After excimer laser surgery, epidermal growth factor (EGF) plays an important role in injured corneal epithelial cell on myofibroblastic cell formation in corneal stroma. The purpose of the study is to investigate the precise mechanism of EGF on corneal wound healing, particularly on epithelial proliferation and migration. METHODS: In this study we applied small interference RNA (siRNA) to knock down the expression of eukaryotic translation initiation factor 5A (eIF5A) in corneal epithelial cells. The relative mRNA and protein expression of matrix metallopeptidase 9 (MMP9) and proliferating cell nuclear antigen (PCNA) was determined via real-time PCR and western blot analysis. The proliferative potential of EGF was evaluated via a proliferation assay using the measurement of (3)H-thymidine incorporation ((3)H-TdR). HCEpiC apoptosis was subjected to flow cytometric analysis. RESULTS: The results showed eIF5A expression was enhanced and there was a statistically significant increase in EGF treatment compared to the control group. Real-time PCR, western blot analysis, and the proliferation assay demonstrated significantly lower MMP9 and PCNA expression and proliferation cell counts in eIF5A siRNA-treated groups versus significantly higher levels in EGF plus eIF5A siRNA-treated groups. The data analysis showed that eIF5A, MMP9, and PCNA expression decreased as a result of the inhibitor LY294002. Apoptotic cells were increased in the EGF plus eIF5A siRNA, EGF plus LY294002, and EGF plus eIF5A siRNA plus LY294002 groups as compared with the EGF siRNA group. CONCLUSIONS: These results indicate that EGF-induced upregulation of eIF5A stimulates corneal epithelial cell proliferation in vitro. EGF stimulation of corneal epithelial proliferation was through the phosphatidylinositol 3-kinase (PI3-k)/protein kinase B (Akt) signaling pathway.

Role of decay-accelerating factor in regulating survival of human cervical cancer cells.

BACKGROUND: Decay-accelerating factor (DAF) is one of the key molecules involved in cell protection against autologous complement, which restricts the action of complement at critical stages of the cascade reaction. The effect of DAF on the survival of human cervical cancer cell (ME180) has not been demonstrated. METHODS: In this study we applied, for the first time, small interference RNA (siRNA) to knock down the expression of the DAF with the aim of exploiting complement more effectively for tumor cell damage. Meanwhile, we investigated the effects of DAF on the viability and migration, moreover the proliferation of ME180 cell. RESULTS: The results showed that the expression of DAF was significantly increased in human cervical cancer tissues. SiRNA inhibition of DAF expression enhanced complement-dependent cytolysis up to 32% in ME180 cells, which contributed to the control of C3 activation and increased the cells viability, migration and augment the number of ME180 cells. CONCLUSION: These data indicated that DAF siRNA described in this study may offer an additional alternative to improve the efficacy of antibody- and complement-based cancer immunotherapy.

Cooperation of decay-accelerating factor and membrane cofactor protein in regulating survival of human cervical cancer cells.

BACKGROUND: Decay-accelerating factor (DAF) and membrane cofactor protein (MCP) are the key molecules involved in cell protection against autologus complement, which restricts the action of complement at critical stages of the cascade reaction. The cooperative effect of DAF and MCP on the survival of human cervical cancer cell (ME180) has not been demonstrated. METHODS: In this study we applied, for the first time, short hairpin RNA (shRNA) to knock down the expression of the DAF and MCP with the aim of exploiting complement more effectively for tumor cell damage. Meanwhile, we investigated the cooperative effects of DAF and MCP on the viability and migration, moreover the proliferation of ME180 cell. RESULTS: The results showed that shRNA inhibition of DAF and MCP expression enhanced complement-dependent cytolysis (CDC) up to 39% for MCP and up to 36% for DAF, and the combined inhibition of both regulators yielded further additive effects in ME180 cells. Thus, the activities of DAF and MCP, when present together, are greater than the sum of the two protein individually. CONCLUSION: These data indicated that combined DAF and MCP shRNA described in this study may offer an additional alternative to improve the efficacy of antibody-and complement-based cancer immunotherapy.

Over-expression of COX-2 induces human ovarian cancer cells (CAOV-3) viability, migration and proliferation in association with PI3-k/Akt activation.

COX-2 is involved in several pathological processes. The action of COX-2 on human ovarian cancer cell line (CAOV-3) and relative signal pathway has not been demonstrated. We explored the effects of COX-2 on the CAOV-3 viability and migration, moreover the proliferation of CAOV-3, and then ascertained the roles of PI3-k/Akt in these processes. The results showed that COX-2 increased the cells viability, migration and augment the number of CAOV-3. LY294002 could reduce COX-2 induce cells viability, migration and proliferation. These data indicate that COX-2 induce cells viability and migration, moreover promote the proliferation of CAOV-3 via PI3-k/Akt signal pathway.