Aldo-keto reductase family member C3(AKR1C3)promotes hepatocellular carcinoma cell growth by producing prostaglandin F2α

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.

Analysis of the mechanism of aldo-keto reductase dependent cis-platin resistance in HepG2 cells based on transcriptomic and NADH metabolic state

Background:Aldo-keto oxidoreductase(AKR)inhibitors could reverse the resistance of several cancer cells to cis-platin,but their role in resistance remains unclear.Methods:We verified the difference of AKR1Cs expression by Western blot,RNA sequencing and qRT-PCR.The differences of AKR1Cs expression were analyzed and inferred.Use Assay of NADH and NAD^(+)content to verify the inference.The Docking experience was used to verify the affinity between MPA,MCFLA,MLS and AKR1C3.Results:Our RNA-seq results showed de novo NAD biosynthesis-related genes and NAD(P)H-dependent oxidoreductases were significantly upregulated in cis-platin-resistant HepG2 hepatic cancer cells(HepG2-RC cells)compared with HepG2 cells.At least 63 NAD(P)H-dependent reductase/oxidases were upregulated in HepG2-RC cells at least twofold.Knockdown of AKR1Cs could increase cis-platin sensitivity in HepG2-RC cells about two-fold.Interestingly,the AKR1C inhibitor meclofenamic acid could increase the cis-platin sensitivity of HepG2-RC cells about eight-fold,indicating that the knockdown of AKR1Cs only partially reversed the resistance.Meanwhile,the amount of total NAD and the ratio of NADH/NAD^(+)were increased in HepG2-RC cells compared with HepG2 cells.The ratio of NADH/NAD^(+)in HepG2-RC cells was almost seven-fold higher than in HepG2 or HL-7702 cells.Increased NADH expression could be explained as a directly operating antioxidant to scavenge cis-platin-induced radicals.Conclusion:We report here that NADH,which is produced by NAD(P)Hdependent oxidoreductases,plays a key role in the AKR-associated cis-platin resistance of HepG2 hepatic cancer cells.

Expression Analysis of Aldo-Keto Reductase 1 (AKR1) in Foxtail Millet (Setaria italica L.) Subjected to Abiotic Stresses

Foxtail millet (Setaria italica L.) is a drought-tolerant millet crop of arid and semi-arid regions. Aldo-keto reductases (AKRs) are significant part of plant defence mechanism, having an ability to confer multiple stress tolerance. In this study, AKR1 gene expression was studied in roots and leaves of foxtail millet subjected to different regimes of PEG- and NaCl-stress for seven days. The quantitative Real-time PCR expression analysis in both root and leaves showed upregulation of AKR1 gene during PEG and salt stress. A close correlation exits between expression of AKR1 gene and the rate of lipid peroxidation along with the retardation of growth. Tissue-specific differences were found in the AKR1 gene expression to the stress intensities studied. The reduction in root and shoot growth under both stress conditions were dependent on stress severity. The level of lipid peroxidation as indicated by MDA formation was significantly increased in roots and leaves along with increased stress levels. Finally, these findings support the early responsive nature of AKR1 gene and seem to be associated at least in part with its ability to contribute in antioxidant defence related pathways which could provide a better protection against oxidative stress under stress conditions.

Berberine inhibits androgen synthesis by interaction with aldo-keto reductase 1C3 in 22Rv1 prostate cancer cells

AIdo-keto reductase family 1 member C3 has recently been regarded as a potential therapeutic target in castrate-resistant prostate cancer. Herein, we investigated whether berberine delayed the progression of castrate-resistant prostate cancer by reducing androgen synthesis through the inhibition of Aldo-keto reductase family 1 member C3. Cell viability and cellular testosterone content were measured in prostate cancer cells. Aido-keto reductase family 1 member C3 mRNA and protein level were detected by RT-PCR and Western bolt analyses, respectively. Computer analysis with AutoDock Tools explored the molecular interaction of berberine with Aldo-keto reductase family 1 member C3. We found that berberine inhibited 22Rvl cells proliferation and decreased cellular testosterone formation in a dose-dependent manner. Berberine inhibited Aldo-keto reductase family I member C3 enzyme activity, rather than influenced mRNA and protein expressions. Molecular docking study demonstrated that berberine could enter the active center of Aldo-keto reductase family 1 member C3 and form π-π interaction with the amino-acid residue Phe306 and Phe311. In conclusion, the structural interaction of berberine with Aldo-keto reductase family 1 member C3 is attributed to the suppression of Aldo-keto reductase family I member C3 enzyme activity and the inhibition of 22Rvl prostate cancer cell growth by decreasing the intfacellular androgen synthesis. Our result provides the experimental basis for the design, research, and development of AKRlC3 inhibitors using berberine as the lead compound.

Quantitative Evaluation of Aldo-keto Reductase Expression in Hepatocellular Carcinoma (HCC) Cell Lines

The involvement of aldo-keto reductases （AKRs） in tumorigenesis is widely reported, but their roles in the pathological process are not generally recognized due to inconsistent measure- ments of their expression. To overcome this problem, we simultaneously employed real-time PCR to examine gene expression and multiple reaction monitoring （MRM） of mass spectrometry （MS） to examine the protein expression of AKRs in five different hepatic cell lines. These include one rela- tively normal hepatic cell line, L-02, and four hepatocellular carcinoma （HCC） cell lines, HepG2, HUH7, BEL7402 and SMMC7721. The results of real-time PCR showed that expression of genes encoding the AKR1C family members rather than AKR1A and AKR1B was associated with tumor, and most of genes encoding AKRs were highly expressed in HUH7. Similar observations were obtained through MRM. Different from HUH7, the protein abundance of AKR1A and AKR1B was relatively consistent among the other four hepatic cell lines, while protein expression of AKR1C varied significantly compared to L-02. Therefore, we conclude that the abundant distri- bution of AKR 1C proteins is likely to be associated with liver tumorigenesis, and the AKR expres- sion status in HuH7 is completely different from other liver cancer cell lines. This study, for the first time, provided both overall and quantitative information regarding the expression of AKRs at both mRNA and protein levels in hepatic cell lines. Our observations put the previous use of AKRs as a biomarker into question since it is only consistent with our data from HUH7. Furthermore, the data presented herein demonstrated that quantitative evaluation and comparisons within a protein fam- ily at both mRNA and protein levels were feasible using current techniques.

Study of Aldo-keto Reductase 1C3 Inhibitor with Novel Framework for Treating Leukaemia Based on Virtual Screening and In vitro Biological Activity Testing

Aldo-keto reductase 1C3(AKR1C3)is a potential target for the treatment of acute myeloid leukaemia and T-cell acute lymphoblastic leukaemia.In this study,pharmacophore models,molecular docking and virtual screening of target prediction were used to find a potential AKR1C3 inhibitor.Firstly,eight bacteriocin derivatives(Z1-Z8)were selected as training sets to construct 20 pharmacophore models.The best pharmacophore model MODEL_016 was obtained by Decoy test(the enrichment degree was 21.5117,and the fitting optimisation degree was 0.9668).Secondly,MODEL_016 was used for the virtual screening of ZINC database.Thirdly,the hit 83256 molecules were docked into the AKR1C3 protein.Compared to the total scores and interactions between compounds and protein,16532 candidate compounds with higher docking scores and interactions with important residues PHE306 and TRP227 were screened.Lastly,eight compounds(A1-A8)that had good absorption,distribution,metabolism,excretion and toxicity(ADMET)properties were obtained by target prediction.Compounds A3 and A7 with high total score and good target prediction results were selected for in vitro biological activity test,whose IC_(50) values were 268.3 and 88.94µmol/L,respectively.The results provide an important foundation for the discovery of novel AKR1C3 inhibitors.The research methods used in this study can also provide important references for the research and development of new drugs.

Astragalin attenuates diabetic cataracts via inhibiting aldose reductase activity in rats

AIM:To investigate the aldose reductase(AR)inhibition capacity of astragalin(AST)against streptozoticin-induced diabetic cataracts(DCs)in rats.METHODS:Ex vivo investigations were conducted by treating the lens of a goat placed for 72h in artificial aqueous humor(AAH)of pH 7.8 at room temperature with cataract-causing substance(55 mmol/L of galactose)and in vivo studies were performed on rats via induction with streptozotocin.AST was administered at different dose levels and scrutinize for DC activity.RESULTS:In diabetic rats,AST improved the body weight,blood insulin,and glucose as well as the levels of galactitol in a dose-dependent way,other biochemical parameters i.e.inflammatory mediators and cytokines,and also suppress AR activity.The level of the antioxidant parameters such as superoxide dismutase(SOD),catalase(CAT),and glutathione(GSH)activity were also altered on a diabetic lens after the administration of the AST.CONCLUSION:AST protects against lens opacification to avoid cataracts and polyols formation,indicating that it could be used as a potential therapeutic agent for diabetes.

多枝赖草Glutathione Reductase基因克隆及胁迫表达分析

为了获得完整的谷胱甘肽还原酶基因序列,根据已克隆到的谷胱甘肽还原酶cDNA片段设计引物,利用RACE扩增获得了基因全长序列,应用Southern印迹杂交法分析基因存在状态,Northern印迹杂交法研究基因表达情况.结果表明,该基因全长1580 bp,含一个1 140 bp的开放阅读框架,编码380个氨基酸,与其它植物谷胱甘肽还原酶氨基酸序列的同源性在77%-92%之间;Southern杂交表明该基因有一个拷贝;Northern杂交表明在逆境胁迫下GR基因表达加强.

油菜素内酯合成酶(Steroid 5α-Reductase)基因的超量表达对毛白杨生长的影响

将棉花Steroid 5α-reductase基因(DET2)超量表达载体导入毛白杨中,观察其对毛白杨生长和芽休眠的影响。结果表明,超量表达DET2基因的毛白杨茎高度、直径生长和不定根的生长增强,芽的休眠破除提前。

Single nucleotide polymorphism C677T in the methylenetetrahydrofolate reductase gene might be a genetic risk factor for infertility for Chinese men with azoospermia or severe oligozoospermia

Aim： To analyze the distribution of the single nucleotide polymorphism （SNP） C677T in the methylenetetrahydrofolate reductase （MTHFR） gene in 355 infertile Chinese patients with idiopathic azoospermia or severe oligozoospermia and 252 fertile Chinese men as controls to explore the possible association of the SNP and male infertility. Methods： Using the polymerase chain reaction （PCR）-restriction fragment length polymorphism technique, the allele and genotype distribution of SNP C677T in the MTHFR gene were investigated in both patients and controls. Results： The frequencies of allele T （40.9% vs 30.4%, P = 0.002, odds ration [OR] = 1.58, 95% confidence interval [CI]： 1.24-2.02） and mutant homozygote （TT） （18.3% vs. 11.5%, P = 0.023, OR = 1.72, 95% CI： 1.07-2.76） as well as carrier with allele （TT ＋ CT） （63.4% vs. 49.2%, P = 0.0005, OR = 1.79, 95% CI： 1.29-2.48） in infertile patients were significantly higher than those in controls. After patient stratification, the significant differences in distribution of the SNP between each patient subgroup and control group still remained. Conclusion： Our findings indicate that there is an association of SNP C677T in the MTHFR gene with male infertility, suggesting that this polymorphism might be a genetic risk factor for male infertility in Chinese men.

Influences of Mo on Nitrate Reductase, Glutamine Synthetase and Nitrogen Accumulation and Utilization in Mo-Efficient and Mo-Inefficient Winter Wheat Cultivars

The objective is to study whether the accumulation and utilization of plant N are controlled by Mo status in winter wheat cultivars. Mo-efficient cultivar 97003 （eft） and Mo-inefficient cultivar 97014 （ineff） were grown in severely Mo-deficient acidic soil （Tamm-reagent-extractable Mo 0.112 mg kg^-1） with （＋Mo） and without （-Mo） the application of 0.13 mg kg^-1 Mo. The accumulation and use efficiency of plant total N were significantly higher in ＋Mo than that in -Mo and in eft than that in ineff under Mo deficiency. N use efficiency was remarkably higher in maturity but it was forwarded to jointing stage after Mo supply, thus indicating that Mo supply promoted the N use efficiency besides N uptake and eff was efficient in N uptake and utilization. The overall activity of nitrate reductase （NR, EC 1.6.6.1） was significantly higher in ＋Mo than in -Mo and ratio of ＋Mo/-Mo was even to 14.8 at filleting stage for ineff. Activity of glutamine synthetase （GS, EC 6.3.1.2） was significantly lower in ＋Mo than in -Mo. Concentration of nitrate and glutamate were also significantly lower in ＋Mo than in -Mo, thus provided evidences for enhancing N use efficiency by Mo supply. Activities of NR and GS were significantly higher and concentrations of nitrate and glutamate were significantly lower in eff than ineff under Mo deficiency, thus indicated eff was more efficient in N reduction and utilization. It is therefore concluded that Mo could promote N accumulation and utilization in winter wheat which was directly related to NR and feedback regulated by GS. Higher Mo status also results in higher accumulation and utilization of plant N in eft.

Methylenetetrahydrofolate reductase C677T and A1298C polymorphisms and gastric cancer susceptibility

AIM: To identify the association between methylenetetrahydrofolate reductase(MTHFR) polymorphisms and gastric cancer(GC) susceptibility.METHODS: Systematic searches were performed on the electronic databases PubMed, ISI, Web of knowledge, CNKI and Wanfang, as well as manual searching of the references of the identified articles. A total of 26 papers were included in this meta-analysis. Overall and subgroup analyses were performed. Odds ratio(OR) and 95%CI were used to evaluate the associations between MTHFR polymorphisms and GC risk. The I2 statistics were used to evaluate between-study heterogeneity. Sensitivity analysis was also performed.RESULTS: Increased risk was found for the MTHFRC677T polymorphism under four genetic models(TT + CT vs CC: OR = 1.23, P = 0.002; T vs C: OR = 1.15, P = 0.001; TT vs CC: OR = 1.37, P = 0.0005; TT vs CT + CC: OR = 1.17, P = 0.0008). Subgroup analysis by ethnicity suggested that C677 T polymorphism conferred a risk of GC in eastern but not in western populations. Stratification by tumor site showed an association between the C677 T polymorphism and gastric cardia cancer and non-cardia GC in the worldwide population and in eastern populations. Regardless of comparisons with controls or diffuse-type GC, a positive association was found for the C677 T polymorphism and an increased risk of intestinal-type GC in the whole population and in western populations. With regard to the A1298 C polymorphism, we found that genotype CC was significantly decreased and conferred protection against GC in eastern populations(CC vs AA: OR = 0.44, P = 0.03; CC vs AC + AA: OR = 0.46, P = 0.04). CONCLUSION: MTHFR C677 T polymorphism is a risk factor for GC, and the A1298 C polymorphism may be a protective factor against GC in eastern populations.

Inhibitory effect of two Indian medicinal plants on aldose reductase of rat lens in vitro

Objective:To assesse the inhibitor) effect of alcoholic extract of two Indian medicinal plants namely Ceasalpinia digyna Rottler and.Alangium lamarckii Thwaits on aldose reductase（AR） of rat lens.Methods:Rats lens were enucleated through posterior approach and their homogenate was prepared and centrifuged to obtain a clear supernatant for the determination of AR activity and protein content.Results:The alcoholic extract of Ceasalpirda digyna and Alangium lamarckii had a potent inhibitory effect on the lens AR enzyme.The IC50 values of alcoholic extract of the selected plants were calculated and were（46.29±11.17） and（106.00±5.11）μg/mL,respectively. Quercetin was used as a positive control and its IC50 value was（2.95±1.53）μg/mL.Conclusions:Thus,it is concluded that alcoholic extracts of the selected plant exhibit significant inhibitory effects on AR in the rat lens in vitro.

Changes of Nitrate Reductase Activity in Cucumber Seedlings in Response to Nitrate Stress

In China, nitrogen fertilizer application rates in intensive agricultural systems have increased dramatically in recent years, especially in protected vegetable production systems. This excessive use of nitrogen fertilizer has resulted in soil secondary salinization, which has become a significant environmental stress for crops such as cucumber, in the protected farmland of China. So it is necessary to illuminate how crops respond to nitrate stress. The objective of this work was to examine the effects of increased nitrate concentration [14 （CK） and 140 mmol L^-1 （T）] on NO3- concentration, and in vitro and in vivo nitrate reductase activities in the roots and leaves of cucumber （Cucumis sativus L. cv. Xintaimici） seedlings with hydroponic culture. The results showed that the NO3- concentration in the roots and leaves of T seedlings significantly increased over treatment course, and at 12 d increased by 1.08 and 1.72 times with respect to CK seedlings, respectively; in vitro nitrate reductase activity of T was increased dramatically to 1.74 times of CK in the roots at 2 d and 1.56 times of CK in the leaves at 6 d, and then decreased. At 12 d, in vitro activity was still 24.3% higher in the roots and only 9.9% lower in the leaves than CK. Compared with in vitro nitrate reductase activity, in vivo activity responded differently to the increase of treatment time. At the beginning, in vivo nitrate reductase activity in the roots and leaves of T had no significant difference from CK, whereas with the increase of treatment duration, the activity decreased. At 12 d, in vivo activity in the roots and leaves of T lowered by 20.1 and 52.8% with respect to CK, respectively. This evidence suggests that posttranslational activation of nitrate reductase in cucumber seedlings may be seriously inhibited by nitrate stress.

Thioredoxin and glutaredoxin-mediated redox regulation of ribonucleotide reductase

Ribonucleotide reductase(RNR), the rate-limitingenzyme in DNA synthesis, catalyzes reduction of thedifferent ribonucleotides to their corresponding deoxyri-bonucleotides. The crucial role of RNR in DNA synthesishas made it an important target for the development ofantiviral and anticancer drugs. Taking account of the re-cent developments in this field of research, this reviewfocuses on the role of thioredoxin and glutaredoxin sys-tems in the redox reactions of the RNR catalysis.

Cytochrome b5 reductase 2 suppresses tumor formation in nasopharyngeal carcinoma by attenuating angiogenesis

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.

Evaluation of in vitro aldose reductase inhibitory potential of different fraction of Hybanthus enneaspermus Linn F.Muell

Objective:To evaluate the aldose reductase inhibitory(ARI)activity of different fractions of Hybanthus enneaspermus for potential use in diabetic cataract.Methods:Total phenol and flavonoid content of different fractions was determined.ARI activity of different fractions in rat lens was investigated in vitro.Results:The results showed significant level of phenolic and flavonoid content in ethyl acetate fraction[total phenol(212.15±0.79 mg/g),total flavonoid(39.11±2.27mg/g)]and aqueous fraction[total phenol(140.62±0.57mg/g),total flavonoid(26.07±1.49 mg/g)]as compared with the chloroform fraction[total phenol(68.56±0.51mg/g),total flavonoid(13.41±0.82mg/g)]and petrolium ether fraction[total phenol(36.68±0.43mg/g),total flavonoid(11.55±1.06mg/g)].There was a significant difference in the ARI activity of each fraction,and it was found to be the highest in ethyl acetate fraction[IC_(50)(49.26±1.76μg/mL)]followed by aqueous extract[IC_(50)(70.83±2.82μg/mL)]and it was least in the petroleum ether fraction[IC_(50)(118.89±0.71μg/mL)].Chloroform fraction showed moderate activity[IC_(50)(98.52±1.80μg/mL)].Conclusions:Different fractions showed significanct amount of ARI activity,where in ethyl acetate fraction it was found to be maximum which may be due to its high phenolic and flavonoid content.The extract after further evaluation may be used in the treatment of diabetic cataract.

Polymorphisms of the oxidant enzymes glutathione S-transferase and glutathione reductase and their association with resistance of Plasmodium falciparum isolates to antimalarial drugs

Objective:To investigate the association between amplification of the two regulatory genes controlling glutathione(GSH) levels,glutathione reductase(PfGR) and glutathione S-transferase (PfGST) genes and sensitivity of Plasmodium falciparum(P.falciparum) isolates collected from different malaria endemic areas of Thailand to standard antimalarial drugs.Methods:A total of 70 P.falciparum isolates were collected from endemic areas of multi-drug resistance (Tak,Chantaburi and Ranong Provinces) during the year 2008-2009.The in vitro assessment of antimalarial activity of P.falciparum clones(K1- and Dd2 chloroquine resistant and 3D7- chloroquine sensitive) and isolates to chloroquine,quinine,mefloquine and arteusnate was performed based on SYBR Green modified assay.Results:68(97.14%),11(15.71%) and 28(40%) isolates respectively were classified as chloroquine-,quinine- and mefloquine-resistant isolates. With this limited number of P.falciparum isolates included in the analysis,no significant association between amplification of PfGST gene and sensitivity of the parasite to chloroquine, quinine,mefloquine and quinine was found.Based on PCR analysis,Dd2,Kl and 3D7 clones all contained only one copy of the PfGST gene.All isolates(70) also carried only one copy number of PfGST gene.There appears to be an association between amplification of PfGR gene and chloroquine resistance.The 3D7 and Dd2 clones were found to carry only one PfGR gene copy, whereas the K1 clone carried two gene copies.Conclusions:Chloroquine resistance is likely to be a consequence of multi-factors and enzymes in the GSH system may be partly involved. Larger number of parasite isolates are required to increase power of the hypothesis testing in order to confirm the involvement of both genes as well as other genes implicated in glutathione metabolism in conferring chloroquine resistance.

Association Between Homocysteine Level and Methylenetetrahydrofolate Reductase Gene Polymorphisms in Type 2 Diabetes Accompanied by Dyslipidemia

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.

Emerging roles of the ribonucleotide reductase M2 in colorectal cancer and ultraviolet-induced DNA damage repair

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.