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Objective @#To investigate the effect of aluminum exposure on expression of miR-497-5p, wingless murine breast cancer virus integration site family member 3a (Wnt3a), β-catenin protein, glycogen synthase kinase-3β (GSK-3β) protein and tau protein in rat adrenal pheochromocytoma PC12 cells, so as to provide insight into unraveling the mechanisms underlying aluminum exposure-induced abnormal phosphorylation of tau protein.@* Methods@# PC12 cells were exposed to Al(mal)3 at concentrations of 0, 100, 200, 400 μmol/L for 24 h. The viability of PC12 cells was measured using cell counting kit-8 (CCK-8) assay. The relative expression of miR-497-5p and Wnt3a was detected using a real-time fluorescent quantitative PCR (RT-qPCR) assay, and the expression of Wnt3a, β-catenin, GSK-3β, P-GSK-3β (Ser9), tau and p-tau (Ser396) proteins were determined using Western blotting. @*Results @#The viability of PC12 cells appeared a tendency towards a decline with the increase of aluminum dose (Ftrend=323.473, P=0.001). RT-qPCR assay detected that the relative miR-497-5p expression appeared a tendency towards a rise with the increase of aluminum dose (Ftrend=14.888, P=0.031), and the relative Wnt3a expression appeared a tendency towards a decline with the increase of aluminum dose (Ftrend=165.934, P<0.001). The miR-497-5p expression negatively correlated with the relative Wnt3a expression (r=-0.693, P=0.012). The expression of Wnt3a (Ftrend=357.656, P=0.001), β-catenin (Ftrend=208.750, P=0.001) and p-GSK-3β (Ser9) proteins (Ftrend=512.583, P<0.001) appeared a tendency towards a decline with the increase of aluminum dose, and the expression of GSK-3β (Ftrend=39.965, P<0.001), tau (Ftrend=277.929, P=0.006) and p-tau (Ser396) proteins (Ftrend=96.247, P=0.002) appeared a tendency towards a rise with the increase of aluminum dose. @*Conclusion@# Up-regulation of miR-497-5p and GSK-3β expression and down-regulation of Wnt3a and β-catenin expression may be a mechanism underlying aluminum exposure-induced abnormal phosphorylation of tau protein.
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ObjectiveTo investigate the regulatory effect and molecular mechanism of berberine （BBR） on lipophagy in the prevention and treatment of atherosclerotic （AS） lesions in mice. MethodFifty apolipoprotein E-knockout （ApoE-/-） mice were randomly divided into an AS model group， an atorvastatin group （5 mg·kg-1）， and low-， medium-， and high-dose BBR groups （2.5， 5， 10 mg·kg-1）. Ten C57BL/6J mice were assigned to the control group. After 12 weeks， hematoxylin-eosin （HE） and oil red O staining were performed to assess the histopathological changes of AS plaques in the aorta. Biochemical analysis was used to measure serum lipid levels， and enzyme-linked immunosorbent assay （ELISA） was employed to measure the levels of inflammatory cytokines interleukin-6 （IL-6） and tumor necrosis factor-α （TNF-α）， oxidative stress marker reactive oxygen species （ROS）， and serum lipophagy marker Beclin1 and microtubule-associated protein 1 light chain 3 Ⅱ （LC3Ⅱ）. The xanthine oxidase method was used to measure serum superoxide dismutase （SOD） activity. Immunohistochemistry （IHC） was used to detect the distribution of wingless-type MMTV integration site family member 5a （Wnt5a） and Nieman Pick type C1 （NPC1） in the aorta， and Western blot was used to determine the protein expression of Wnt5a and NPC1 in the aorta. ResultCompared with the control group， the AS model group showed significant AS plaque formation， significantly elevated levels of serum total cholesterol （TC）， triglycerides （TG）， low-density lipoprotein cholesterol （LDL-C）， IL-6， TNF-α， and ROS， aortic Wnt5a distribution and protein expression （P<0.01）， and significantly reduced levels of serum high-density lipoprotein cholesterol （HDL-C）， SOD， Beclin1， LC3Ⅱ， and aortic NPC1 distribution and protein expression （P<0.01）. Compared with the AS model group， the atorvastatin group， and high- and medium-dose BBR groups showed a significant reduction in AS plaque area （P<0.05， P<0.01）， significantly decreased levels of serum TC， TG， LDL-C， IL-6， TNF-α， ROS， and aortic Wnt5a distribution and protein expression （P<0.05， P<0.01）， and significantly increased levels of serum HDL-C， SOD， Beclin1， LC3Ⅱ， and aortic NPC1 distribution and protein expression （P<0.05， P<0.01）. There was no statistically significant difference in the above indicators between the atorvastatin group and the medium-dose BBR group. ConclusionBBR can competitively bind to Wnt5a to activate NPC1 expression， upregulate lipophagy levels， reduce blood lipids， and inhibit the release of inflammatory mediators and oxidative stress damage， thereby exerting a preventive and therapeutic effect on AS.
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[Abstract] Objective To investigate the effect of wingless-type MMTV integration site family member 5b(Wnt5b) gene overexpression mediated by recombinant adenovirus on the differentiation of mouse embryonic liver stem cells and repair of chronic liver injury in mice. Methods Recombinant adenoviruses expressing Wnt5b and green fluorescent protein (GFP) were applied respectively to infect mouse fetal liver stem cells HP14-19, and induced its differentiation and verified the expression of Wnt5b through Real-time PCR and Western blotting. It also applied indocyanine grean(ICG) uptake experiment and periodic acid-schiff(PAS) staining to detect the differentiation ability of HP14-19 into hepatocyte-like cells. The Real-time PCR was chosen to detect hepatocyte markers albumin (Alb) and cytokeratin 18 (CK18) expression. Forty-eight experimental male BALB/ c mice were randomly divided into control group, model group, stem cell treatment group and Wnt5b modified stem cell treatment group. The carbon tetrachloride(CCl
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Dickkopf-3 (DKK3) , as a critical inhibitor of the Wnt/p-catenin signaling pathway, may he involved in melanogenesis.In the current study, we investigated the effects of DKK3 on melanogenesis in melanocytes of alpaca.Overexpression of DKK3 in alpaca melanocytes, the expression of Wntl, Lefl , Myc and the major target genes termed microphthalmia-associated transcription factor (M1TF) and its downstream genes, including tyrosinase (TYR), tyrosinase-related protein 1 (TYRP1) and tyrosinase- related protein 2 (TYRP2) were significantly decreased at both mRNA and protein levels (P<0.05); total alkali melanin, pheomelanin and eumelanin were decreased by 80.30%, 72.17% and 64.60% (P <0.05), respectively.In contrast, in the melanocytes transfected with siRNA-DKK3 (a small interference RNA targeting DKK3) , the expression of Wntl, Lefl, Myc, MITF, TYR, TYRPl and TYRP2 were significantly increased at both mRNA and protein levels (P<0.05) ; total alkali melanin, pheomelanin and eumelanin were significantly increased by 1.65 folds, 1.25 folds and 1.21 folds (P< 0.05) , respectively.These results indicate that DKK3 regulates melanogenesis in alpaca melanocytes via the Wnt/p-catenin signaling pathway and down-regulates MITF.
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Objective To investigate the expression changes of provirus integration site 1 for moloney murine leukemia virus(Pim-1) gene in damaged neurons in vitro and related molecular basis of neurotrophic factors regulating Pim-1 expression and promoting the neurite regeneration of damaged neurons. Methods Neuro-2a(N-2a)cells were induced into neuron-like N-2a(N-2a-N) cells by retinoic acid,the proliferation of N-2a cells was inhibited by deferoxamine mesylate (DFO), and N-2a-N cells were injured by acrylamide. The N-2a-N cells were divided into normal control group, injury group, ciliary neurotrophic factors (CNTF) group and neuritin (Nrn1) group, with four samples in each group. The phenotype of N-2a cells and the expression of Pim-1 protein in N-2a cells were detected by immunofluorescence cytochemistry, and the expression of Pim-1 in each group was detected by Real-time PCR and Western blotting. Western blotting was used to detect the expression changes of relevant molecules involving in regulating activity of Pim-1, cells survival, apoptosis and axonal regeneration. Results Cell immunofluorescence showed that N-2a-N cells had neuronal phenotype to express β-Ⅲ tubulin and neurofilament-200, and Pim-1 protein was expressed in N-2a-N cells. N-2a cell proliferation was effectively inhibited by 50 μmol/ L DFO, and N-2a-N cell damage model was established by 1 mmol/ L acrylamide. Pim-1 gene expression showed a tendency of first decreasing, then increasing, and then decreasing after N-2aN cells were injured. Compared with the injury group, the proportion of the longest neurite in CNTF group and Nrn1 group increased significantly, the expressions of intracellular signal transducers extracellular regulated protein kinase 1/ 2 (ERK1/ 2), phosphorylated extracellular regulated protein kinase 1/ 2 (p-ERK1/ 2), signal transducers and activators of transcription 3(STAT3), phosphorylated signal transducers and activators of transcription 3 (p-STAT3), and Pim-1 were up-regulated, the expressions of apoptosis-related molecules cleaved Caspase-3 and Bax were down-regulated, the expression of anti-apoptosis-related molecule Bcl-2 was up-regulated, so the growth-associated protein 43 (GAP-43) protein involved neurite regeneration. Conclusion There is a need to repair damaged N-2a-N cells by overexpressing the Pim-1 gene. CNTF and Nrn1 can activate the ERK1/ 2 and STAT3 signaling pathways of damaged N-2a-N cells, and then up-regulate the expression of Pim-1 and GAP-43,and then promote cell neurite regeneration.
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The study aims to use CRISPR/Cas9 introducing foreign gene targeted knock-in into chicken EAV-HP genome. First, specific primers were designed for amplification of EAV-HP left, right homologous arms and enhanced green fluorescent protein (eGFP) expression cassette. PCR products of homologous arms were ligated to both sides of eGFP by overlap extension PCR, resulting in full-length donor DNA fragment designated as LER. Then LER fragments were cloned into pMD19-T to obtain donor vector pMDT-LER. Subsequently, the donor vector pMDT-LER was transfected into HEK293T cells to verify the expression of eGFP gene. Furthermore, co-transfection of CRISPR/Cas9 expression vector and pMDT-LER into chicken DF-1 cells was performed to achieve eGFP transgenic cells. Meanwhile, eGFP expression was observed in cells, and the event of eGFP integration into EAV-HP genome was detectable by amplification of target DNA. Finally, the transgenic DF-1 cells were passaged seven times, and the stable integration and expression of eGFP was checked by PCR and Western blotting. These results demonstrated that eGFP gene was knocked into the EAV-HP genome successfully, which provides a new integration site for research of transgenic chicken.
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Animals , Humans , CRISPR-Cas Systems , Chickens , Clustered Regularly Interspaced Short Palindromic Repeats , Gene Knock-In Techniques , Genome , HEK293 CellsABSTRACT
Objective To investigate the regulation mechanism of microRNA - 9(miR - 9)by ecotropic viral integration site1(EVI1)its impact on proliferation of AML cells and its role in the pathogenesis of myelogenous leuke-mia. Methods EVI1 was forced to express in Uocm1 cell lines by murine stem cell virus - EVI1(MSCV - EVI1) plasmid infection. EVI1 overexpressed Uocm1 cells were then treated with 0. 1 μmol/ L 5 - aza - 2′ - deoxycytidine (5 - AZA)dissolved in dimethyl sulfoxide (DMSO). The methylation level of miR - 9 promoter was tested by DNA bi-sulfite sequencing technology. The cell cycle was observed by flow cytometry (FCM). The proliferation ability of the cells was detected by the colony forming assay in semi - solid Methylcellulose medium culture. Results EVI1 level was dramatically increased after being infected by MSCV - EVI1 plasmid. Forced expression of EVI1 in Uocm1 signifi-cantly downregulated miR - 9 by inducing hypermethylation of miR - 9 promoter. Relative expression level of miR - 9 was lower in EVI1 overexpressed group(0. 004 ± 0. 000)than that of the control group(0. 006 ± 0. 001)(t = 4. 09,P <0. 05). When EVI1 was overexpressed in Uocm1,the rate of G0 / G1 cells decreased markedly(P < 0. 05),while rates of S phage and G2 phage increased significantly(all P < 0. 05). Seven days after 500 cells plated in semi - solid medium, EVI1 overexpressed Uocm1 cells gave rise to more colony (122. 3 ± 7. 8)than Uocm1 cells infected with vector (45. 7 ± 6. 1)(t = - 13. 44,P < 0. 01). 0. 1 μmol/ L 5 - AZA recovered miR - 9 expression(P < 0. 01)by decreasing EVI1 induced hypermethylation of miR - 9 promoter. G0 / G1 phase cell proportion was(48. 25 ± 2. 19)％ in control group,while (65. 90 ± 2. 90)％ in 5 - AZA group (t = - 6. 85,P < 0. 05). 5 - AZA group formed less colony (51. 00 ± 10. 01)than the control group (123. 40 ± 8. 12)(t = 9. 59,P < 0. 01),which indicated that 5 - AZA inhibi-ted cell proliferation by G0 / G1 cell cycle retardation in EVI1 overexpressed uocm1 cells. Conclusions EVI1 may en-hance proliferation ability of myeloid leukemia cells by downregulating miR - 9 through inducing hypermethylation of miR - 9 promotor,which plays a crucial role in the pathogenesis of AML. 5 - AZA may be an effective hypomethylating agent in the therapy of EVI1 high acute myeloid leukemia.
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Objective To investigate the levels of serum Wnt5a and Sfrp5 in elderly male patients with type 2 diabetes mellitus (T2DM), and identify associations between their levels and glycemic control. Methods A total of 67 elderly male T2DM patients and 65 nondiabetic subjects were studied. Participants were divided into four groups:normal control (NC group), T2DM patients were categorized by HbA1C quartile(Group Ⅰ: HbA1C<7％, Group Ⅱ:7％≤HbA1C < 9％, Group Ⅲ: HbA1C ≥9％). The serum Wnt5a and Sfrp5 concentrations were measured through ELISA. Influencing factors for Wnt5a and Sfrp5 were analyzed. Results Compared with the NC group, Wnt5a levels of elderly T2DM were decreased in groups Ⅱ and Ⅲ, in contrast, Sfrp5 levels were elevated in groups Ⅱ and Ⅲ than NC group(all P<0.05). Spearman correlation analysis suggested that Wnt5a levels were negatively correlated with HbA1C , GA, FPG, and 2hPG(r were -0.277, -0.298, -0.185, and -0.254 respectively, all P<0.05);Sfrp5 levels were positively correlated with HbA1C , GA, and FPG(r were 0.311, 0.247, and 0.200 respectively, all P<0.05) while negatively correlated with BMI and LDL-C( r were - 0.193 and - 0.190, both P< 0.05). Multivariate linear regression analysis showed that HbA1C was an independent association factor for Wnt5a, and FPG was an independent association factor for Sfrp5. Conclusions In the elderly male T2DM with worse glycemic control, Wnt5a levels were more decreased, and in contrast, Sfrp5 levels were elevated. This result indicated that Wnt5a and Sfrp5 may be associated with the level of glycemic control in elderly male T2DM patients.
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As an proto-oncogene,ecotropic viral integration site 1 (EVI1) gene is over-expressed in acute myeloid leukemia (AML) because of chromosomal translocation or other genetic abnormalities,finally cause poor prognosis.In recent years,an increasing research of the expression of EVI1 in acute lymphoblastic leukemia (ALL),especially in pediatric ALL.The overexpression of EVI1 gene also suggest a poor prognosis,which is closely relate to the age.But the relationship between the expression of EVI1 gene and mixed lineage leukemia (MLL) and BCR-ABL gene rearrangement needs to be further studied.Researching the expression of EVI1 gene in ALL and exploring targeted therapeutic agents are important research directions in the future.
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As an proto-oncogene,ecotropic viral integration site 1 (EVI1) gene is over-expressed in acute myeloid leukemia (AML) because of chromosomal translocation or other genetic abnormalities,finally cause poor prognosis.In recent years,an increasing research of the expression of EVI1 in acute lymphoblastic leukemia (ALL),especially in pediatric ALL.The overexpression of EVI1 gene also suggest a poor prognosis,which is closely relate to the age.But the relationship between the expression of EVI1 gene and mixed lineage leukemia (MLL) and BCR-ABL gene rearrangement needs to be further studied.Researching the expression of EVI1 gene in ALL and exploring targeted therapeutic agents are important research directions in the future.
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Objective To analyze the functional changes and the clinical significance of B cell specific mono-clonal murine leukemia virus integration site -1 (Bmi -1 )and Th1 /Th2 cells in children with newly diagnosed im-mune thrombocytopenia(ITP)by testing the mRNA expressions of Bmi -1,helper T cell -related cytokine interferon (IFN)-γand interleukin(IL)-4 in children with newly diagnosed ITP.Methods Thirty -six cases of patients with newly diagnosed ITP in the experimental group came from the inpatient and outpatient children admitted to the Depart-ment of Pediatrics of the First Affiliated Hospital of Xinxiang Medical University from April to December 201 3.In the control group,26 cases of children requiring selective operation were admitted to the Department of Pediatric Surgery during the same period.The mRNA expressions of Bmi -1,IFN -γand IL -4 in the peripheral blood lymphocytes were detected by means of the reverse transcription -polymerase chain reaction(RT -PCR)method,and were analyzed and compared by t test and linear correlation analysis.Results (1 )The mRNA expressions of Bmi -1,IFN -γand IL -4 in peripheral blood lymphocytes in the experimental group were 2.63 ±0.54,3.84 ±0.43 and 1 .44 ±0.39,respec-tively;while the mRNA expressions of Bmi -1,IFN -γand IL -4 in the peripheral blood lymphocytes in the control group were 3.91 ±0.92,2.88 ±0.57 and 1 .87 ±0.34,respectively.The levels of IFN -γof the experimental group were significantly higher than those of the control group (P 0.05).Conclusions Bmi -1 may be involved in the pathogenesis of ITP by regulating Th cell, and Th cell dysfunction may occur in the children with ITP,and the disproportion between Th1 and Th2 may be due to the advantages of Th1 .
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<p><b>OBJECTIVE</b>To observe the expression of wingless-type MMTV integration site family, member 5A (Wnt5A)/receptor tyrosine kinase-like orphan receptor 2 (Ror2) signal in the dental follicle cells during the normal eruption of the teeth as well as to explore the relationship between the expression of dental follicle cells and the formation of mature osteoclasts and eruption of the teeth.</p><p><b>METHODS</b>The mandibulars of 1-13 d old SD rats were separated to observe the growth and develop-ment of the teeth and alveolar bone through hematoxylin-eosin(HE) staining. Ror2 and Wnt5A expressions in rat dental follicle were also observed through immunohistochemistry. Dental follicle cells from the lower first intact molar germs of 5-6-day old SD rats were separated and cultured.</p><p><b>RESULTS</b>On the second day after birth, the dental follicle began to differentiate into periodontal tissues, but no obvious changes were observed in the alveolar bone one to three days after birth. On the fourth day, the number of osteoclasts increased significantly. The results of immunohistochemistry showed that Wnt5A was not significantly expressed in rat dental follicle tissues before the fourth day, but positive expression was expressed in the next day and continued to express to thirteenth days. Ror2 was expressed in the rat dental follicle at postnatal days 1-3, but weak expression was found in days 4-13.</p><p><b>CONCLUSIONS</b>Wnt5A and Ror2 expressions in the process of tooth eruption have specific time distributions, suggesting that these expressions may participate in the regulation of the eruption of the teeth.</p>
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Animals , Rats , Dental Sac , Molar , Osteoclasts , Periodontium , Rats, Sprague-Dawley , Receptor Tyrosine Kinase-like Orphan Receptors , Tooth Eruption , Wnt Proteins , Wnt-5a ProteinABSTRACT
EVI1 gene mainly controls embryo development.3q rearrangement,MLL translocations,monosomy 7,which react with other genes,can lead to EVI1 aberrant expresion or fusion gene.Recent studies have shown aberrant expression of EVI1 gene in parts of acute lymphatic leukemia,acute myeloid leukemia and chronic granulocytic Leukemia and correlation with poor prognosis.Leukemogemc mechanism include epigenetic modifications,transcriptional control,regulation of signaling pathways,upregulation of cell adhesion,proliferation,and colony formation and antiapoptosis.Drugs of genetic therapy include epigenetic agents,mTOR inhibitor,human ITGA6/ITGB4 complex antibody and CD52 monoclonal antibody.
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Objective To study the integration site and arrangement of SfII and SfX prophages in Shigella flexneri serotype 2b strains. Methods A series of primers were designed based on potential integration site of SfII and SfX prophages in Shigella flexneri serotype 2b strains, and PCR were performed for 50 serotype 2b strains to amplify special genes located in host and prophages. PCR products were sequenced to identify integration sites and arrangement of SfII and SfX. Results In all the serotype 2b strains, prophage SfII and SfX were adjacent to each other, and integrated into the thrW tRNA gene of the host, which were located between genes proA and yaiC of host. Prophage SfX was located immediately upstream of prophage SfII in all the detected 50 serotype 2b strains exception for strain 51251. Conclusion This was the first report on the integration site and arrangement of serotype-converting prophages SfII and SfX in Shigella flexneri 2b strains.