Hepatocellular carcinoma(HCC)is a leading cause of death worldwide.Current therapies are effective for HCC patients with early disease,but many patients suffer recurrence after surgery and have a poor response to chem...Hepatocellular carcinoma(HCC)is a leading cause of death worldwide.Current therapies are effective for HCC patients with early disease,but many patients suffer recurrence after surgery and have a poor response to chemotherapy.Therefore,new therapeutic targets are needed.We analyzed gene expression profiles between HCC tissues and normal adjacent tissues from public databases and found that the expression of genes involved in lipid metabolism was significantly different.The analysis showed that AKR1C3 was upregulated in tumors,and high AKR1C3 expression was associated with a poorer prognosis in HCC patients.In vitro,assays demonstrated that the knockdown of AKR1C3 or the addition of the AKR1C3 inhibitor indomethacin suppressed the growth and colony formation of HCC cell lines.Knockdown of AKR1C3 in Huh7 cells reduced tumor growth in vivo.To explore the mechanism,we performed pathway enrichment analysis,and the results linked the expression of AKR1C3 with prostaglandin F2 alpha(PGF2a)downstream target genes.Suppression of AKR1C3 activity reduced the production of PGF2a,and supplementation with PGF2a restored the growth of indomethacin-treated Huh7 cells.Knockdown of the PGF receptor(PTGFR)and treatment with a PTGFR inhibitor significantly reduced HCC growth.We showed that indomethacin potentiated the sensitivity of Huh7 cells to sorafenib.In summary,our results indicate that AKR1C3 upregulation may promote HCC growth by promoting the production of PGF2α,and suppression of PTGFR limited HCC growth.Therefore,targeting the AKR1C3-PGF2a-PTGFR axis may be a new strategy for the treatment of HCC.展开更多
Background:Cytochrome b5 reductase 2(CYB5R2) is a potential tumor suppressor that inhibits cell proliferation and motility in nasopharyngeal carcinoma(NPC).Inactivation of CYB5R2 is associated with lymph node metastas...Background:Cytochrome b5 reductase 2(CYB5R2) is a potential tumor suppressor that inhibits cell proliferation and motility in nasopharyngeal carcinoma(NPC).Inactivation of CYB5R2 is associated with lymph node metastasis in NPC.This study aimed to explore the mechanisms contributing to the anti-neoplastic effects of CYB5R2.Methods:Polymerase chain reaction(PCR) assays were used to analyze the transcription of 84 genes known to be involved in representative cancer pathways in the NPC cell line HONE1.NPC cell lines CNE2 and HONE1 were transiently transfected with CYB5R2,and data was validated by real-time PCR.A chick chorioallantoic membrane(CAM)embryo model was implanted with CYB5R2-expressing CNE2 and HONE1 cells to evaluate the effect of CYB5R2 on angiogenesis.An immunohistochemical assay of the CAM model was used to analyze the protein expression of vascular endothelial growth factor(VEGF).Results:In CYB5R2-transfected NPC cells,PCR assays revealed up-regulated mRNA levels of Fas cell surface death receptor(FAS),FBJ murine osteosarcoma viral oncogene homolog(FOS),phosphoinositide-3-kinase regulatory subunit 1(PIK3R1),integrin beta 3(ITGB3),metastasis suppressor 1(MTSSl),interferon beta 1(IFNB1),and cyclin-dependent kinase inhibitor 2A(CDKN2A) and down-regulated levels of integrin beta 5(ITGB5),insulin-like growth factor 1(IGF1),TEK tyrosine kinase(TEK),transforming growth factor beta receptor 1(TGFBRl),and VEGF.The angiogenesis in the CAM model implanted with CYB5R2-transfected NPC cells was inhibited.Down-regulation of VEGF by CYB5R2 in NPC cells was confirmed by immunohistochemical staining in the CAM model.Conclusion:CYB5R2 up-regulates the expression of genes that negatively modulate angiogenesis in NPC cells and down-regulates the expression of VEGF to reduce angiogenesis,thereby suppressing tumor formation.展开更多
Objective To investigate the association between total homocysteine(tHcy)level in plasma and methylenetetrahydrofblate reductase(MTHFR)C677T and A1298C genetic polymorphisms in a Chinese Han nationality population wit...Objective To investigate the association between total homocysteine(tHcy)level in plasma and methylenetetrahydrofblate reductase(MTHFR)C677T and A1298C genetic polymorphisms in a Chinese Han nationality population with type 2 diabetes mellitus(T2DM)accompanied by dyslipidemia.Methods This case-control study enrolled T2DM patients with dyslipidemia and without dyslipidemia respectively.Sanger dideoxy-mediated chain-termination method was used to detect the gene polymorphisms of MTHFR C677T and A1298C.Plasma tHcy and lipid levels were measured as well.The genotype frequency and allele frequency between the dyslipidemia and non-dyslipidemia groups were compared by using Chi-square test.Plasma tHcy level ofT2DM patients who carried the different genotypes was compared by Student's t test.Results Finally,82 T2DM patients with dyslipidemia and 94 ones without dyslipidemia were included in this study.There was a significant correlation between tHcy level and MTHFR C677T gene polymorphism inT2DM patients(t=2.27,P=0.02).Moreover,the plasma tHcy level in the dyslipidemia patients who carried MTHFR 677TT genotype was significantly higher than that in those with CT+CC genotype(13.62+6.97 vs.10.95+3.62pmol/L,t=2.2O,P=0.03);while for patients without dyslipidemia,comparison of the tHcy level between those who carried the above two alleles showed no significantly difference(13.34±6.03 vs.12.04±5.09μmol/L,t=1.08,P=0.29).Conclusion MTHFR 677TT genotype might associate with higher tHcy level in T2DM patients with dyslipidemia.展开更多
Our previous studies showed that resveratrol could inhibit the proliferation of vascular smooth muscle cells (VSMCs) and repress mRNA and protein expression of quinone reductase 2 (NQO2). This study further explor...Our previous studies showed that resveratrol could inhibit the proliferation of vascular smooth muscle cells (VSMCs) and repress mRNA and protein expression of quinone reductase 2 (NQO2). This study further explored the potential mechanisms whereby resveratrol inhibits the proliferation of rat VSMCs. Lentiviral vectors that incorporated NQO2 small interfering RNA (siRNA) were constructed and transduced into rat VSMCs. The cell proliferation was detected using the bromodeoxyuridine (BrdU) assay. Cultured rat VSMCs were stimulated with angiotensin II and the level of reactive oxygen species (ROS) was measured using a ROS assay kit. A realtime quantitative PCR was used to detect NQO2 mRNA levels. Extracellular signal-regulated kinase (ERK1/2) and NQO2 protein expression were determined by Western blotting analysis. The inhibitory effect of resveratrol (10 and 50 μmol/L) on the proliferation of rat VSMCs in the NQO2 siRNA group was significantly weaker than that in the normal and scrambled siRNA group (P 〈 0.01). The ROS level in the NQO2 siRNA and resveratrol (50 μmol/L) treatment groups were lower than that in the normal and scrambled siRNA groups (P 〈 0.01 in both). Compared with the normal and scrambled siRNA group, the phosphorylation of ERK1/2 was significantly decreased in the NQO2 siRNA and resveratrol (50 μmol/L) treatment group (P 〈 0.01 in both). In conclusion, high concentration of resveratrol inhibits angiotensin II-induced ERK1/2 phosphorylation and subsequent proliferation by down-regulation of NQO2 in cultured rat VSMCs.展开更多
Objective Chronic cardiovascular diseases induced by long-term poor blood glucose control are the main cause of death in patients with type 2 diabetes mellitus(T2DM).Previous researches report that methylenetetrahydro...Objective Chronic cardiovascular diseases induced by long-term poor blood glucose control are the main cause of death in patients with type 2 diabetes mellitus(T2DM).Previous researches report that methylenetetrahydrofolate reductase gene(MTHFR)polymorphisms might influence the occurrence of coronary heart disease(CHD)in T2DM patients.The purpose of this study was to evaluate whether MTHFR C677T and A1298C mutations are associated with the risk of CHD inT2DM patients.Methods A total of 197 subjects with T2DM were studied,of which 95 patients with CHD.The genotypes of MTHFR C677T and A1298C were analyzed by using dideoxy chain-termination method,and compared between patients with CHD and those without CHD.Results We found that the frequency of the 677T allele was significantly higher in T2DM patients with CHD than those without CHD(P=0.011).However,there was no significant difference in any of the examined haplotypes between T2DM patients with and without CHD.Furthermore,the 677T allele was associated with a higher risk of CHD development in diabetic patients with lower homocysteine(Hey)levels(≤15μmol/L)(P=0.006),while no effect of MTHFR gene polymorphism on the incidence of CHD was found in patients with higher Hey levels(>15 μmol/L)(P=0.491).Conclusion The MTHFR C677T gene polymorphism is associated with the risk of CHD of diabetic patients and could be used as an effective marker for CHD in Chinese diabetic populations with normal Hey levels.展开更多
AIM:To investigate the roles of the ribonucleotide reductase M2 (RRM2) subunit in colorectal cancer (CRC) and ultraviolet (UV)-induced DNA damage repair. METHODS:Immunohistochemical staining of tissue microarray was p...AIM:To investigate the roles of the ribonucleotide reductase M2 (RRM2) subunit in colorectal cancer (CRC) and ultraviolet (UV)-induced DNA damage repair. METHODS:Immunohistochemical staining of tissue microarray was performed to detect the expression of RRM2. Seven CRC cell lines were cultured and three human colon cancer cell lines, i.e., HCT116, SW480 and SW620, were used. Reverse transcription polymerase chain reaction and Western blotting were performed to determine the mRNA and protein expression levels of RRM2, respectively. Cell proliferation assay, cell cycle analysis were performed. Cell apoptosis was evaluated by double staining with fluorescein isothiocyanate-conjugated Annexin Ⅴ and propidium iodide (PI) usingAnnexin Ⅴ/PI apoptosis kit. The motility and invasion of CRC cells were assessed by the Transwell chamber assay. Cells were irradiated with a 254 nm UV-C lamp to detect the UV sensitivity after RRM2 depletion. RESULTS:Immunohistochemical staining revealed elevated RRM2 levels in CRC tissues. RRM2 overexpression was positively correlated with invasion depth (P < 0.05), poorly differentiated type (P = 0.0051), and tumor node metastasis stage (P = 0.0015). The expression of RRM2 in HCT116 cells was downregulated after transfection, and HCT116 cell proliferation was obviously suppressed compared to control groups (P < 0.05). In the invasion test, the number of cells that passed through the chambers in the RRM2-siRNA group was 81 ± 3, which was lower than that in the negative control (289 ± 7) and blank control groups (301 ± 7.2). These differences were statistically significant (P < 0.01). Our data suggest that RRM2 overexpression may be associated with CRC progression. RRM2 silencing by siRNA may inhibit the hyperplasia and invasiveness of CRC cells, suggesting that RRM2 may play an important role in the infiltration and metastasis of CRC, which is a potential therapeutic strategy in CRC. In addition, RRM2 depletion increased UV sensitivity. CONCLUSION:These findings suggest that RRM2 may be a facilitating factor in colorectal tumorigenesis and UV-induced DNA damage repair.展开更多
3β-Hydroxysteroid-△24 reductase (DHCR24) is a multifunctional enzyme that localizes to the endoplasmic reticulum and has neuroprotective and cholesterol-synthesizing activities. DHCR24 overexpression confers neuro...3β-Hydroxysteroid-△24 reductase (DHCR24) is a multifunctional enzyme that localizes to the endoplasmic reticulum and has neuroprotective and cholesterol-synthesizing activities. DHCR24 overexpression confers neuroprotection against apoptosis caused by amyloid β deposition. The present study aimed to construct two recombinant adenoviruses driving DHCR24 expression specifically in neurons. Two SYN1 promoter DNA fragments were obtained from human (h) and rat (r). Recombinant Ad-r(h)SYN1-DHCR24 was transfected into AD-293, N2A (mouse neuroblastoma), and MIN6 (mouse pancreatic carcinoma) cells. Western blot analysis showed DHCR24 was specially expressed in 293 and N2A cells, but no specific band was found in MIN6 cells. This demonstrates that the recombinant adenoviruses successfully express DHCR24, and no expression is observed in non-neuronal cells. TUNEL assay results showed apoptosis was inhibited in adenovirus-transfected neurons. Detecting reactive oxygen species by immunoflu- orescence, we found that adenovirus transfection inhibits apoptosis through scavenging excess reactive oxygen species. Our findings show that the recombinant DHCR24 adenoviruses induce neuron-specific DHCR24 expression, and thereby lay the foundation for further studies on DHCR24 gene therapy for Alzheimer's disease.展开更多
BACKGROUND Ribonucleotide reductase(RR)is a key enzyme in tumor proliferation,especially its subunit-RRM2.Although there are multiple therapeutics for tumors,they all have certain limitations.Given their advantages,tr...BACKGROUND Ribonucleotide reductase(RR)is a key enzyme in tumor proliferation,especially its subunit-RRM2.Although there are multiple therapeutics for tumors,they all have certain limitations.Given their advantages,traditional Chinese medicine(TCM)monomers have become an important source of anti-tumor drugs.Therefore,screening and analysis of TCM monomers with RRM2 inhibition can provide a reference for further anti-tumor drug development.AIM To screen and analyze potential anti-tumor TCM monomers with a good binding capacity to RRM2.METHODS The Gene Expression Profiling Interactive Analysis database was used to analyze the level of RRM2 gene expression in normal and tumor tissues as well as RRM2's effect on the overall survival rate of tumor patients.TCM monomers that potentially act on RRM2 were screened via literature mining.Using AutoDock software,the screened monomers were docked with the RRM2 protein.RESULTS The expression of RRM2 mRNA in multiple tumor tissues was significantly higher than that in normal tissues,and it was negatively correlated with the overall survival rate of patients with the majority of tumor types.Through literature mining,we discovered that berberine,ursolic acid,gambogic acid,cinobufagin,quercetin,daphnetin,and osalmide have inhibitory effects on RRM2.The results of molecular docking identified that the above TCM monomers have a strong binding capacity with RRM2 protein,which mainly interacted through hydrogen bonds and hydrophobic force.The main binding sites were Arg330,Tyr323,Ser263,and Met350.CONCLUSION RRM2 is an important tumor therapeutic target.The TCM monomers screened have a good binding capacity with the RRM2 protein.展开更多
Resveratrol is a dietary polyphenol espoused to have chemopreventive activity against a variety of human cancer types. We first reported that resveratrol significantly decreases the proliferation of both androgen-depe...Resveratrol is a dietary polyphenol espoused to have chemopreventive activity against a variety of human cancer types. We first reported that resveratrol significantly decreases the proliferation of both androgen-dependent and hormone-refractory prostate cancer cells. However, the effects of resveratrol in normal prostate epithelial and stromal cells, particularly with regard to its uptake, subcellular distribution and intracellular targets, have not been investigated. To advance the knowledge on accessibility and cellular disposition of resveratrol in prostate cells, [3H] resveratrol, fractionation of cell extracts into subcellular compartments, Western blot analysis, resveratrol affinity column chromatography and flow cytometry were used to study the uptake and intracellular distribution of resveratrol in normally cultured prostate stromal (PrSCs) and epithelial cells (PrECs). Pretreatment of both PrSCs and PrECs for 2 days with resveratrol modulated its uptake and selectively increased its distribution to the membrane and organelle compartments. Resveratrol affinity column chromatography studies showed differential expression of a previously identified resveratrol-targeting protein, quinone reductase 2 (QR2), in PrSCs and PrECs. Flow cytometric analysis comparing resveratrol-treated and untreated PrSCs showed a large decrease in G1-phase and a concomitant increase in S and G2/M-phases of the cell cycle. These results suggest that resveratrol suppresses PrSC proliferation by affecting cell cycle phase distribution, which may involve the participation by QR2.展开更多
Objective: To fractionate and identify polyphenols from Guazuma ulmifolia Lam. leaves, and to explore their antioxidant, 5-hydroxy-3-methylglutaryl-coenzyme A(HMG-Co A) reductase inhibitory, and Nrf2 modulatory activi...Objective: To fractionate and identify polyphenols from Guazuma ulmifolia Lam. leaves, and to explore their antioxidant, 5-hydroxy-3-methylglutaryl-coenzyme A(HMG-Co A) reductase inhibitory, and Nrf2 modulatory activities.Methods: The 1,1-diphenyl-2-picrylhydrazyl assay was used to evaluate the antioxidant activity of a polyphenolic fraction of the extract of Guazuma ulmifolia Lam. leaves. THP-1 gene reporter cell lines constructed with a transcriptional response element specific for Nrf2 and a minimal promoter for the firefly luciferase–green fluorescent protein transgene were used to determine the effect of the polyphenolic fraction on the Nrf2 signaling pathway. Furthermore, an assay of HMG-Co A reductase inhibitory activity was performed by using a commercial enzyme kit. Polyphenolic compounds were identified by liquid chromatographytandem mass spectrometry.Results: The polyphenolic fraction showed fairly strong antioxidant activity [IC50 =(14.90 ± 4.70) μg/m L] and inhibited HMG-Co A reductase activity by 69.10%, which was slightly lower than that by pravastatin(84.37%) and quercetin(84.25%). Additionally, the polyphenolic fraction activated the Nrf2 antioxidant signaling pathway at 500 μg/m L. Eleven subfractions resulting from the column chromatography separation of the polyphenolic fraction also showed relatively strong antioxidant activities(IC50: 17.46–217.14 μg/m L). The subfraction(F6) stimulated the Nrf2 signaling pathway and had HMG-Co A reductase inhibitory activity(65.43%). Moreover, the subfraction contained two main flavonoids: quercetin and quercimeritrin.Conclusions: The polyphenolic fraction of Guazuma ulmifolia could induce antioxidant genes via the Nrf2/antioxidant regulatory elements pathway, and is a promising candidate for an inhibitor of HMG-Co A reductase.展开更多
The changes in 5α-reductase (type 2 ) gene expression in the epi-didymis of puberty diabetic rats were studied by the Northern blot and Dotblot method. Rats were divided into 3 groups: the control group (C), thediabe...The changes in 5α-reductase (type 2 ) gene expression in the epi-didymis of puberty diabetic rats were studied by the Northern blot and Dotblot method. Rats were divided into 3 groups: the control group (C), thediabetic group (D), and the diabetic group with insulin treatment (DI).Results: The Northern blot intensity of the caput epididymis in Group D is展开更多
Paddy soils are an important source of atmospheric nitrous oxide(N_(2)O).However,numerous studies have focused on N_(2)O production during the soil tillage period,neglecting the N_(2)O production during the dry fallow...Paddy soils are an important source of atmospheric nitrous oxide(N_(2)O).However,numerous studies have focused on N_(2)O production during the soil tillage period,neglecting the N_(2)O production during the dry fallow period.In this study,we conducted an incubation experiment using the acetylene inhibition technique to investigate N_(2)O emission and reduction rates of paddy soil profiles(0-1 m)from Guangdong Province and Jinlin Province in China,with different heavy-metal pollution levels.The abundance and community structures of denitrifying bacteria were determined via quantitative-PCR and Illumina MiSeq sequencing of nosZ,nirK,and nirS genes.Our results showed that the potential N_(2)O emission rate,N_(2)O production rate,and denitrification rate have decreased with increasing soil vertical depth and heavy-metal pollution.More importantly,we found that the functional gene type of N_(2)O reductase switched with the tillage state of paddy soils,which cladeⅡnos Z genes were the dominant gene during the tillage period,while cladeⅠnos Z genes were the dominant gene during the dry fallow period.The heavy-metal pollution has less effect on the niche differentiation of the nos Z gene.The N_(2)O emission rate was significantly regulated by the genus Bradyhizobium,which contains both N_(2)O reductase and nitrite reductase genes.Our findings suggests that the nos Z gene of N_(2)O reductase can significantly impact the N_(2)O emission from paddy soils.展开更多
The present study enrolled 251 diabetic patients, including 101 with neuropathy and 150 without neuropathy. Of the 150 patients, 100 had no complications, such as retinopathy, nephropathy, or neuropathy. Polymerase ch...The present study enrolled 251 diabetic patients, including 101 with neuropathy and 150 without neuropathy. Of the 150 patients, 100 had no complications, such as retinopathy, nephropathy, or neuropathy. Polymerase chain reaction-restriction fragment length polymorphism analysis was used to identify methylenetetrahydrofolate reductase gene variants. Plasma homocysteine levels were also measured. Homocysteine levels and the frequency of hyperhomocysteinemia were significantly higher in patients with diabetic peripheral neuropathy compared with diabetic patients without neuropathy (P 〈 0.05). In logistic regression analysis with neuropathy as the dependent variable, the frequency of C677T in methylenetetrahydrofolate reductase was significantly higher in patients with diabetic peripheral neuropathy compared with patients without diabetic complications. Homocysteine levels were significantly higher in patients with diabetic peripheral neuropathy carrying the 677T allele and low folic acid levels. In conclusion, hyperhomocysteinemia is an independent risk factor for diabetic neuropathy in Chinese patients with diabetes. The C677T polymorphism in methylenetetrahydrofolate reductase and low folic acid levels may be risk factors for diabetic peripheral neuropathy in Chinese patients with diabetes.展开更多
Chloroplasts are essential for plant photosynthesis and growth.Many genes have been identified that regulate plant chloroplast development.However,it is not known at a molecular level how these genes regulate chloropl...Chloroplasts are essential for plant photosynthesis and growth.Many genes have been identified that regulate plant chloroplast development.However,it is not known at a molecular level how these genes regulate chloroplast biogenesis.In this study,we isolated a mutant ygl2(yellow-green leaf2)that exhibited a pigment-defective phenotype.YGL2 encodes a geranylgeranyl reductase,and in mutant ygl2,there was a single base change(T1361G)located in the third exon of YGL2 that resulted in a missense mutation(L454R)in the encoded product.Transmission electron microscopy revealed that chloroplast development was impaired in the ygl2 mutant.The expression levels of plastid-encoded genes were significantly altered in the ygl2 mutant.Furthermore,in a yeast two-hybrid assay,we found that YGL2 interacted with the RNA editing factor MORF8.展开更多
基金National Yang Ming Chiao Tung University Far Eastern Memorial Hospital Joint Research Programs(NYCU-FEMH 109DN03,110DN06,111DN04,112DN05).
文摘Hepatocellular carcinoma(HCC)is a leading cause of death worldwide.Current therapies are effective for HCC patients with early disease,but many patients suffer recurrence after surgery and have a poor response to chemotherapy.Therefore,new therapeutic targets are needed.We analyzed gene expression profiles between HCC tissues and normal adjacent tissues from public databases and found that the expression of genes involved in lipid metabolism was significantly different.The analysis showed that AKR1C3 was upregulated in tumors,and high AKR1C3 expression was associated with a poorer prognosis in HCC patients.In vitro,assays demonstrated that the knockdown of AKR1C3 or the addition of the AKR1C3 inhibitor indomethacin suppressed the growth and colony formation of HCC cell lines.Knockdown of AKR1C3 in Huh7 cells reduced tumor growth in vivo.To explore the mechanism,we performed pathway enrichment analysis,and the results linked the expression of AKR1C3 with prostaglandin F2 alpha(PGF2a)downstream target genes.Suppression of AKR1C3 activity reduced the production of PGF2a,and supplementation with PGF2a restored the growth of indomethacin-treated Huh7 cells.Knockdown of the PGF receptor(PTGFR)and treatment with a PTGFR inhibitor significantly reduced HCC growth.We showed that indomethacin potentiated the sensitivity of Huh7 cells to sorafenib.In summary,our results indicate that AKR1C3 upregulation may promote HCC growth by promoting the production of PGF2α,and suppression of PTGFR limited HCC growth.Therefore,targeting the AKR1C3-PGF2a-PTGFR axis may be a new strategy for the treatment of HCC.
基金supported by Grants from the National Basic Research Program of China(No.2011CB504300)the Program for New Century Excellent Talents in University(No.NCET-12-0654)the Department of Education of the Guangxi Zhuang Autonomous Region(No.201203YB051)
文摘Background:Cytochrome b5 reductase 2(CYB5R2) is a potential tumor suppressor that inhibits cell proliferation and motility in nasopharyngeal carcinoma(NPC).Inactivation of CYB5R2 is associated with lymph node metastasis in NPC.This study aimed to explore the mechanisms contributing to the anti-neoplastic effects of CYB5R2.Methods:Polymerase chain reaction(PCR) assays were used to analyze the transcription of 84 genes known to be involved in representative cancer pathways in the NPC cell line HONE1.NPC cell lines CNE2 and HONE1 were transiently transfected with CYB5R2,and data was validated by real-time PCR.A chick chorioallantoic membrane(CAM)embryo model was implanted with CYB5R2-expressing CNE2 and HONE1 cells to evaluate the effect of CYB5R2 on angiogenesis.An immunohistochemical assay of the CAM model was used to analyze the protein expression of vascular endothelial growth factor(VEGF).Results:In CYB5R2-transfected NPC cells,PCR assays revealed up-regulated mRNA levels of Fas cell surface death receptor(FAS),FBJ murine osteosarcoma viral oncogene homolog(FOS),phosphoinositide-3-kinase regulatory subunit 1(PIK3R1),integrin beta 3(ITGB3),metastasis suppressor 1(MTSSl),interferon beta 1(IFNB1),and cyclin-dependent kinase inhibitor 2A(CDKN2A) and down-regulated levels of integrin beta 5(ITGB5),insulin-like growth factor 1(IGF1),TEK tyrosine kinase(TEK),transforming growth factor beta receptor 1(TGFBRl),and VEGF.The angiogenesis in the CAM model implanted with CYB5R2-transfected NPC cells was inhibited.Down-regulation of VEGF by CYB5R2 in NPC cells was confirmed by immunohistochemical staining in the CAM model.Conclusion:CYB5R2 up-regulates the expression of genes that negatively modulate angiogenesis in NPC cells and down-regulates the expression of VEGF to reduce angiogenesis,thereby suppressing tumor formation.
基金the National Key Development Plan for Precision Medicine Research(2017YFC0910004)Jinan Science Project(201602171),and Jinan Science and Technology Plan Project(201503009).
文摘Objective To investigate the association between total homocysteine(tHcy)level in plasma and methylenetetrahydrofblate reductase(MTHFR)C677T and A1298C genetic polymorphisms in a Chinese Han nationality population with type 2 diabetes mellitus(T2DM)accompanied by dyslipidemia.Methods This case-control study enrolled T2DM patients with dyslipidemia and without dyslipidemia respectively.Sanger dideoxy-mediated chain-termination method was used to detect the gene polymorphisms of MTHFR C677T and A1298C.Plasma tHcy and lipid levels were measured as well.The genotype frequency and allele frequency between the dyslipidemia and non-dyslipidemia groups were compared by using Chi-square test.Plasma tHcy level ofT2DM patients who carried the different genotypes was compared by Student's t test.Results Finally,82 T2DM patients with dyslipidemia and 94 ones without dyslipidemia were included in this study.There was a significant correlation between tHcy level and MTHFR C677T gene polymorphism inT2DM patients(t=2.27,P=0.02).Moreover,the plasma tHcy level in the dyslipidemia patients who carried MTHFR 677TT genotype was significantly higher than that in those with CT+CC genotype(13.62+6.97 vs.10.95+3.62pmol/L,t=2.2O,P=0.03);while for patients without dyslipidemia,comparison of the tHcy level between those who carried the above two alleles showed no significantly difference(13.34±6.03 vs.12.04±5.09μmol/L,t=1.08,P=0.29).Conclusion MTHFR 677TT genotype might associate with higher tHcy level in T2DM patients with dyslipidemia.
基金supported by grants from the National Natural Science Foundation of China (No.30971255)
文摘Our previous studies showed that resveratrol could inhibit the proliferation of vascular smooth muscle cells (VSMCs) and repress mRNA and protein expression of quinone reductase 2 (NQO2). This study further explored the potential mechanisms whereby resveratrol inhibits the proliferation of rat VSMCs. Lentiviral vectors that incorporated NQO2 small interfering RNA (siRNA) were constructed and transduced into rat VSMCs. The cell proliferation was detected using the bromodeoxyuridine (BrdU) assay. Cultured rat VSMCs were stimulated with angiotensin II and the level of reactive oxygen species (ROS) was measured using a ROS assay kit. A realtime quantitative PCR was used to detect NQO2 mRNA levels. Extracellular signal-regulated kinase (ERK1/2) and NQO2 protein expression were determined by Western blotting analysis. The inhibitory effect of resveratrol (10 and 50 μmol/L) on the proliferation of rat VSMCs in the NQO2 siRNA group was significantly weaker than that in the normal and scrambled siRNA group (P 〈 0.01). The ROS level in the NQO2 siRNA and resveratrol (50 μmol/L) treatment groups were lower than that in the normal and scrambled siRNA groups (P 〈 0.01 in both). Compared with the normal and scrambled siRNA group, the phosphorylation of ERK1/2 was significantly decreased in the NQO2 siRNA and resveratrol (50 μmol/L) treatment group (P 〈 0.01 in both). In conclusion, high concentration of resveratrol inhibits angiotensin II-induced ERK1/2 phosphorylation and subsequent proliferation by down-regulation of NQO2 in cultured rat VSMCs.
基金the National Key Development Plan for Precision Medicine Research(project number:2017YFC0910004,running period:2018/03-2020/12)Jinan Science Project(project number:201602171,running period:2016/01-2018/12).
文摘Objective Chronic cardiovascular diseases induced by long-term poor blood glucose control are the main cause of death in patients with type 2 diabetes mellitus(T2DM).Previous researches report that methylenetetrahydrofolate reductase gene(MTHFR)polymorphisms might influence the occurrence of coronary heart disease(CHD)in T2DM patients.The purpose of this study was to evaluate whether MTHFR C677T and A1298C mutations are associated with the risk of CHD inT2DM patients.Methods A total of 197 subjects with T2DM were studied,of which 95 patients with CHD.The genotypes of MTHFR C677T and A1298C were analyzed by using dideoxy chain-termination method,and compared between patients with CHD and those without CHD.Results We found that the frequency of the 677T allele was significantly higher in T2DM patients with CHD than those without CHD(P=0.011).However,there was no significant difference in any of the examined haplotypes between T2DM patients with and without CHD.Furthermore,the 677T allele was associated with a higher risk of CHD development in diabetic patients with lower homocysteine(Hey)levels(≤15μmol/L)(P=0.006),while no effect of MTHFR gene polymorphism on the incidence of CHD was found in patients with higher Hey levels(>15 μmol/L)(P=0.491).Conclusion The MTHFR C677T gene polymorphism is associated with the risk of CHD of diabetic patients and could be used as an effective marker for CHD in Chinese diabetic populations with normal Hey levels.
文摘AIM:To investigate the roles of the ribonucleotide reductase M2 (RRM2) subunit in colorectal cancer (CRC) and ultraviolet (UV)-induced DNA damage repair. METHODS:Immunohistochemical staining of tissue microarray was performed to detect the expression of RRM2. Seven CRC cell lines were cultured and three human colon cancer cell lines, i.e., HCT116, SW480 and SW620, were used. Reverse transcription polymerase chain reaction and Western blotting were performed to determine the mRNA and protein expression levels of RRM2, respectively. Cell proliferation assay, cell cycle analysis were performed. Cell apoptosis was evaluated by double staining with fluorescein isothiocyanate-conjugated Annexin Ⅴ and propidium iodide (PI) usingAnnexin Ⅴ/PI apoptosis kit. The motility and invasion of CRC cells were assessed by the Transwell chamber assay. Cells were irradiated with a 254 nm UV-C lamp to detect the UV sensitivity after RRM2 depletion. RESULTS:Immunohistochemical staining revealed elevated RRM2 levels in CRC tissues. RRM2 overexpression was positively correlated with invasion depth (P < 0.05), poorly differentiated type (P = 0.0051), and tumor node metastasis stage (P = 0.0015). The expression of RRM2 in HCT116 cells was downregulated after transfection, and HCT116 cell proliferation was obviously suppressed compared to control groups (P < 0.05). In the invasion test, the number of cells that passed through the chambers in the RRM2-siRNA group was 81 ± 3, which was lower than that in the negative control (289 ± 7) and blank control groups (301 ± 7.2). These differences were statistically significant (P < 0.01). Our data suggest that RRM2 overexpression may be associated with CRC progression. RRM2 silencing by siRNA may inhibit the hyperplasia and invasiveness of CRC cells, suggesting that RRM2 may play an important role in the infiltration and metastasis of CRC, which is a potential therapeutic strategy in CRC. In addition, RRM2 depletion increased UV sensitivity. CONCLUSION:These findings suggest that RRM2 may be a facilitating factor in colorectal tumorigenesis and UV-induced DNA damage repair.
基金financially supported by the National Natural Science Foundation of China(General Program),No.31271494Excellent Talent Support Program of Liaoning Province,No.LJQ2011004
文摘3β-Hydroxysteroid-△24 reductase (DHCR24) is a multifunctional enzyme that localizes to the endoplasmic reticulum and has neuroprotective and cholesterol-synthesizing activities. DHCR24 overexpression confers neuroprotection against apoptosis caused by amyloid β deposition. The present study aimed to construct two recombinant adenoviruses driving DHCR24 expression specifically in neurons. Two SYN1 promoter DNA fragments were obtained from human (h) and rat (r). Recombinant Ad-r(h)SYN1-DHCR24 was transfected into AD-293, N2A (mouse neuroblastoma), and MIN6 (mouse pancreatic carcinoma) cells. Western blot analysis showed DHCR24 was specially expressed in 293 and N2A cells, but no specific band was found in MIN6 cells. This demonstrates that the recombinant adenoviruses successfully express DHCR24, and no expression is observed in non-neuronal cells. TUNEL assay results showed apoptosis was inhibited in adenovirus-transfected neurons. Detecting reactive oxygen species by immunoflu- orescence, we found that adenovirus transfection inhibits apoptosis through scavenging excess reactive oxygen species. Our findings show that the recombinant DHCR24 adenoviruses induce neuron-specific DHCR24 expression, and thereby lay the foundation for further studies on DHCR24 gene therapy for Alzheimer's disease.
基金Supported by Nanchong City School’s Science and Technology Strategic Cooperation,China,No.20SXQT0304Research and Development Project Plan of Affiliated Hospital of North Sichuan Medical College,China,No.2020ZD003.
文摘BACKGROUND Ribonucleotide reductase(RR)is a key enzyme in tumor proliferation,especially its subunit-RRM2.Although there are multiple therapeutics for tumors,they all have certain limitations.Given their advantages,traditional Chinese medicine(TCM)monomers have become an important source of anti-tumor drugs.Therefore,screening and analysis of TCM monomers with RRM2 inhibition can provide a reference for further anti-tumor drug development.AIM To screen and analyze potential anti-tumor TCM monomers with a good binding capacity to RRM2.METHODS The Gene Expression Profiling Interactive Analysis database was used to analyze the level of RRM2 gene expression in normal and tumor tissues as well as RRM2's effect on the overall survival rate of tumor patients.TCM monomers that potentially act on RRM2 were screened via literature mining.Using AutoDock software,the screened monomers were docked with the RRM2 protein.RESULTS The expression of RRM2 mRNA in multiple tumor tissues was significantly higher than that in normal tissues,and it was negatively correlated with the overall survival rate of patients with the majority of tumor types.Through literature mining,we discovered that berberine,ursolic acid,gambogic acid,cinobufagin,quercetin,daphnetin,and osalmide have inhibitory effects on RRM2.The results of molecular docking identified that the above TCM monomers have a strong binding capacity with RRM2 protein,which mainly interacted through hydrogen bonds and hydrophobic force.The main binding sites were Arg330,Tyr323,Ser263,and Met350.CONCLUSION RRM2 is an important tumor therapeutic target.The TCM monomers screened have a good binding capacity with the RRM2 protein.
文摘Resveratrol is a dietary polyphenol espoused to have chemopreventive activity against a variety of human cancer types. We first reported that resveratrol significantly decreases the proliferation of both androgen-dependent and hormone-refractory prostate cancer cells. However, the effects of resveratrol in normal prostate epithelial and stromal cells, particularly with regard to its uptake, subcellular distribution and intracellular targets, have not been investigated. To advance the knowledge on accessibility and cellular disposition of resveratrol in prostate cells, [3H] resveratrol, fractionation of cell extracts into subcellular compartments, Western blot analysis, resveratrol affinity column chromatography and flow cytometry were used to study the uptake and intracellular distribution of resveratrol in normally cultured prostate stromal (PrSCs) and epithelial cells (PrECs). Pretreatment of both PrSCs and PrECs for 2 days with resveratrol modulated its uptake and selectively increased its distribution to the membrane and organelle compartments. Resveratrol affinity column chromatography studies showed differential expression of a previously identified resveratrol-targeting protein, quinone reductase 2 (QR2), in PrSCs and PrECs. Flow cytometric analysis comparing resveratrol-treated and untreated PrSCs showed a large decrease in G1-phase and a concomitant increase in S and G2/M-phases of the cell cycle. These results suggest that resveratrol suppresses PrSC proliferation by affecting cell cycle phase distribution, which may involve the participation by QR2.
基金funded by International Collaboration Research Grant under the Ministry of Research, Technology and Higher Education of the Republic of Indonesia (No. 011/SP2H/LT/DRPM/VIII/2017)University of Missouri-Columbia, MO(USA)
文摘Objective: To fractionate and identify polyphenols from Guazuma ulmifolia Lam. leaves, and to explore their antioxidant, 5-hydroxy-3-methylglutaryl-coenzyme A(HMG-Co A) reductase inhibitory, and Nrf2 modulatory activities.Methods: The 1,1-diphenyl-2-picrylhydrazyl assay was used to evaluate the antioxidant activity of a polyphenolic fraction of the extract of Guazuma ulmifolia Lam. leaves. THP-1 gene reporter cell lines constructed with a transcriptional response element specific for Nrf2 and a minimal promoter for the firefly luciferase–green fluorescent protein transgene were used to determine the effect of the polyphenolic fraction on the Nrf2 signaling pathway. Furthermore, an assay of HMG-Co A reductase inhibitory activity was performed by using a commercial enzyme kit. Polyphenolic compounds were identified by liquid chromatographytandem mass spectrometry.Results: The polyphenolic fraction showed fairly strong antioxidant activity [IC50 =(14.90 ± 4.70) μg/m L] and inhibited HMG-Co A reductase activity by 69.10%, which was slightly lower than that by pravastatin(84.37%) and quercetin(84.25%). Additionally, the polyphenolic fraction activated the Nrf2 antioxidant signaling pathway at 500 μg/m L. Eleven subfractions resulting from the column chromatography separation of the polyphenolic fraction also showed relatively strong antioxidant activities(IC50: 17.46–217.14 μg/m L). The subfraction(F6) stimulated the Nrf2 signaling pathway and had HMG-Co A reductase inhibitory activity(65.43%). Moreover, the subfraction contained two main flavonoids: quercetin and quercimeritrin.Conclusions: The polyphenolic fraction of Guazuma ulmifolia could induce antioxidant genes via the Nrf2/antioxidant regulatory elements pathway, and is a promising candidate for an inhibitor of HMG-Co A reductase.
文摘The changes in 5α-reductase (type 2 ) gene expression in the epi-didymis of puberty diabetic rats were studied by the Northern blot and Dotblot method. Rats were divided into 3 groups: the control group (C), thediabetic group (D), and the diabetic group with insulin treatment (DI).Results: The Northern blot intensity of the caput epididymis in Group D is
基金supported by the National Natural Science Foundation of China (Nos.91851204 and 92251304)the Excellent Innovation Project of Research Center for EcoEnvironmental Sciences,Chinese Academy of Sciences (No.RCEES-EEI-2019-02)the Program of the Youth Innovation Promotion Association of Chinese Academy of Sciences。
文摘Paddy soils are an important source of atmospheric nitrous oxide(N_(2)O).However,numerous studies have focused on N_(2)O production during the soil tillage period,neglecting the N_(2)O production during the dry fallow period.In this study,we conducted an incubation experiment using the acetylene inhibition technique to investigate N_(2)O emission and reduction rates of paddy soil profiles(0-1 m)from Guangdong Province and Jinlin Province in China,with different heavy-metal pollution levels.The abundance and community structures of denitrifying bacteria were determined via quantitative-PCR and Illumina MiSeq sequencing of nosZ,nirK,and nirS genes.Our results showed that the potential N_(2)O emission rate,N_(2)O production rate,and denitrification rate have decreased with increasing soil vertical depth and heavy-metal pollution.More importantly,we found that the functional gene type of N_(2)O reductase switched with the tillage state of paddy soils,which cladeⅡnos Z genes were the dominant gene during the tillage period,while cladeⅠnos Z genes were the dominant gene during the dry fallow period.The heavy-metal pollution has less effect on the niche differentiation of the nos Z gene.The N_(2)O emission rate was significantly regulated by the genus Bradyhizobium,which contains both N_(2)O reductase and nitrite reductase genes.Our findings suggests that the nos Z gene of N_(2)O reductase can significantly impact the N_(2)O emission from paddy soils.
基金supported by the Natural Science Foundation of Beijing, No. 7102161
文摘The present study enrolled 251 diabetic patients, including 101 with neuropathy and 150 without neuropathy. Of the 150 patients, 100 had no complications, such as retinopathy, nephropathy, or neuropathy. Polymerase chain reaction-restriction fragment length polymorphism analysis was used to identify methylenetetrahydrofolate reductase gene variants. Plasma homocysteine levels were also measured. Homocysteine levels and the frequency of hyperhomocysteinemia were significantly higher in patients with diabetic peripheral neuropathy compared with diabetic patients without neuropathy (P 〈 0.05). In logistic regression analysis with neuropathy as the dependent variable, the frequency of C677T in methylenetetrahydrofolate reductase was significantly higher in patients with diabetic peripheral neuropathy compared with patients without diabetic complications. Homocysteine levels were significantly higher in patients with diabetic peripheral neuropathy carrying the 677T allele and low folic acid levels. In conclusion, hyperhomocysteinemia is an independent risk factor for diabetic neuropathy in Chinese patients with diabetes. The C677T polymorphism in methylenetetrahydrofolate reductase and low folic acid levels may be risk factors for diabetic peripheral neuropathy in Chinese patients with diabetes.
基金This work was supported by the National Natural Science Foundation of China(31900388)the Natural Science Foundation of Jiangsu Province,China(BK20191055)the Natural Science Foundation of Colleges of Jiangsu Province,China(19KJB180009).
文摘Chloroplasts are essential for plant photosynthesis and growth.Many genes have been identified that regulate plant chloroplast development.However,it is not known at a molecular level how these genes regulate chloroplast biogenesis.In this study,we isolated a mutant ygl2(yellow-green leaf2)that exhibited a pigment-defective phenotype.YGL2 encodes a geranylgeranyl reductase,and in mutant ygl2,there was a single base change(T1361G)located in the third exon of YGL2 that resulted in a missense mutation(L454R)in the encoded product.Transmission electron microscopy revealed that chloroplast development was impaired in the ygl2 mutant.The expression levels of plastid-encoded genes were significantly altered in the ygl2 mutant.Furthermore,in a yeast two-hybrid assay,we found that YGL2 interacted with the RNA editing factor MORF8.