NMN partially rescues cuproptosis by upregulating sirt2 to increase intracellular NADPH.

Cuproptosis is characterized by lipoylated protein aggregation and loss of iron-sulfur (Fe-S) proteins, which are crucial for a wide range of important cellular functions, including DNA replication and damage repair. Sirt2 and sirt4 are lipoamidases that remove the lipoyl moiety from lipoylated proteins using nicotinamide adenine dinucleotide (NAD+) as a cofactor. However, to date, it is not clear whether nicotinamide mononucleotide (NMN), a precursor of NAD+, affects cellular sensitivity to cuproptosis. Therefore, in the current study, cuproptosis was induced by the copper (Cu) ionophore elesclomol (Es) in HeLa cells. It was also found that Es/Cu treatment increased cellular DNA damage level. On the other hand, NMN treatment partially rescued cuproptosis in a dose-dependent manner, as well as reduced cellular DNA damage level. In addition, NMN upregulated the expression of Fe-S protein POLD1, without affecting the aggregation of lipoylated proteins. Mechanistic study revealed that NMN increased the expression of sirt2 and cellular reduced nicotinamide adenine dinucleotide phosphate (NADPH) level. Overexpression of sirt2 and sirt4 did not change the aggregation of lipoylated proteins, however, sirt2, but not sirt4, increased cellular NADPH levels and partially rescued cuproptosis. Inhibition of NAD+ kinase (NADK), which is responsible for generating NADPH, abolished the rescuing function of NMN and sirt2 for Es/Cu induced cell death. Taken together, our results suggested that DNA damage is a characteristic feature of cuproptosis. NMN can partially rescue cuproptosis by upregulating sirt2, increase intracellular NADPH content and maintain the level of Fe-S proteins, independent of the lipoamidase activity of sirt2.

Comparison of protective effects of nicotinamide mononucleotide and nicotinamide riboside on DNA damage induced by cisplatin in HeLa cells.

Background: Previous studies have shown that the nicotinamide adenine dinucleotide (NAD+) precursors, nicotinamide mononucleotide (NMN) and nicotinamide riboside (NR), protect against endogenously or exogenously induced DNA damage. However, whether the two compounds have the same or different efficacy against DNA damage is not clear. In the current study, we systematically compared the effects of NMN and NR on cisplatin-induced DNA damage in HeLa cells. Methods: To evaluate the protective effects of NMN or NR, HeLa cells were pretreated with different doses of NMN or NR followed with 10 µM of cisplatin treatment. Cell viability was examined by Trypan blue staining assay. For observing the DNA damage repair process, HeLa cells were treated with 10 µM of cisplatin for 12 h, followed with 10 mM NMN or NR treatment for another 8, 16, 24, or 32 h, DNA damage levels were assessed for each time point by immunofluorescent staining against phosphor-H2AX (γH2AX) and alkaline comet assay. The effects of NMN and NR on intracellular NAD+ and reactive oxygen species (ROS) levels were also determined. Results: NMN and NR treatment alone did not have any significant effects on cell viability, however, both can protect HeLa cells from cisplatin-induced decrease of cell viability with similar efficacy in a dose-dependent manner. On the other hand, while both can reduce the DNA damage levels in cisplatin-treated cells, NR exhibited better protective effect. However, both appeared to boost the DNA damage repair process with similar efficacy. NMN or NR treatment alone could increase cellular NAD+ levels, and both can reverse cisplatin-induced decrease of NAD+ levels. Finally, while neither NMN nor NR affected cellular ROS levels, both inhibited cisplatin-induced increase of ROS with no significant difference between them. Conclusion: NR have a better protective effect against cisplatin-induced DNA damage than NMN.

Protective effects of nicotinamide mononucleotide on ethanol-induced DNA damage in L02 cells / 预防医学

Objective@#To investigate protective effects of nicotinamide mononucleotide (NMN) on ethanol-induced DNA damage in L02 cells, so as to provide the evidence for adjuvant therapy of NMN on alcoholic liver diseases.@*Methods@#L02 cells were pretreated with different concentrations of NMN (0, 1, 2, 4 and 8 mmol/L) for 6 h, and then were exposed to 0.4% ethanol for 12 h. The treated cells were divided into the control group, 0.4% ethanol group and different concentrations of NMN groups. Cell viability was analyzed using trypan blue staining for determining the concentration of NMN as a protective agent. The effects of NMN on ethanol-induced DNA damage in L02 cells were evaluated using immunofluorescence detection and reactive oxygen species (ROS) assay. L02 cells were exposed to 0.4% ethanol for 12 h, cultured in a medium containing a protective concentration of NMN, and divided into PBS group and NMN group. Cell viability was detected at 0, 2, 4, 8, 16 and 32 h, and the effects of NMN on repairing ethanol-induced DNA damage were evaluated by alkaline comet assay.@*Results@#The cell viability was lower in 0.4% ethanol group than than in the control group, and was higher in different concentrations of NMN groups than in 0.4% ethanol group (all P<0.05), with no significant difference in the cells viability between 4 mmol/L and higher concentrations of NMN groups and the control group (all P>0.05). Therefore, 4 mmol/L NMN was selected as a protective agent. The cell tail moments, relative immunofluorescence intensities of γH2AX and relative levels of ROS were higher in 0.4% ethanol group than in the control group, and lower in 4 mmol/L and higher concentrations of NMN groups than in 0.4% ethanol group (all P<0.05). The cell viability was increased and the cell tail moment was shortened with the increase of 4 mmol/L NMN intervention time; and the cell viability in 4 h and more of NMN groups were higher, and the cell tail moment were lower than that in PBS group (all P<0.05).@*Conclusions@#NMN attenuates DNA damage in a dose-dependent manner and promotes the repair of DNA damage in a time-dependent manner. NMN has a protective effect on ethanol-induced DNA damage in hepatocytes.

Effects of preoperative carbohydrate loading on recovery after elective surgery: A systematic review and Bayesian network meta-analysis of randomized controlled trials.

Background: Preoperative carbohydrate loading is an important element of the enhanced recovery after surgery (ERAS) paradigm in adult patients undergoing elective surgery. However, preoperative carbohydrate loading remains controversial in terms of improvement in postoperative outcomes and safety. We conducted a Bayesian network meta-analysis to evaluate the effects and safety of different doses of preoperative carbohydrates administrated in adult patients after elective surgery. Methods: MEDLINE (PubMed), Web of Science, EMBASE, EBSCO, the Cochrane Central Register of Controlled Trials, and China National Knowledge Infrastructure (CNKI) were searched to identify eligible trials until 16 September 2022. Outcomes included postoperative insulin resistance, residual gastric volume (RGV) during the surgery, insulin sensitivity, fasting plasma glucose (FPG), fasting serum insulin (Fin) level, the serum levels of C-reactive protein (CRP), postoperative scores of pain, patients' satisfaction, thirst, hunger, anxiety, nausea and vomit, fatigue, and weakness within the first 24 h after surgery and the occurrences of postoperative infection. The effect sizes were estimated using posterior mean difference (continuous variables) or odds ratios (dichotomous variables) and 95 credible intervals (CrIs) with the change from baseline in a Bayesian network meta-analysis with random effect. Results: Fifty-eight articles (N = 4936 patients) fulfilled the eligibility criteria and were included in the meta-analysis. Both preoperative oral low-dose carbohydrate loading (MD: -3.25, 95% CrI: -5.27 to -1.24) and oral high-dose carbohydrate loading (MD: -2.57, 95% CrI: -4.33 to -0.78) were associated with postoperative insulin resistance compared to placebo/water. When trials at high risk of bias were excluded, association with insulin resistance was found for oral low-dose carbohydrate loading compared with placebo/water (MD: -1.29, 95%CrI: -2.26 to -0.27) and overnight fasting (MD: -1.17, 95%CrI: -1.88 to -0.43). So, there was large uncertainty for all estimates vs. control groups. In terms of safety, oral low-dose carbohydrate administration was associated with the occurrences of postoperative infection compared with fasting by 0.42 (95%Crl: 0.20-0.81). In the other outcomes, there was no significant difference between the carbohydrate and control groups. Conclusion: Although preoperative carbohydrate loading was associated with postoperative insulin resistance and the occurrences of postoperative infection, there is no evidence that preoperative carbohydrate administration alleviates patients' discomfort. Systematic review registration: [https://www.crd.york.ac.uk/PROSPERO/], identifier [CRD42022312944].

Association Between ABCA1 Gene Polymorphisms and the Risk of Hypertension in the Chinese Han Population.

Background: Hypertension is rising as a major public health burden around the world. This study explored the association between single-nucleotide polymorphisms (SNPs) in the adenosine triphosphate (ATP)-Binding Cassette Subfamily A1 (ABCA1) gene and hypertension among Chinese Han adults. Method: A total of 2,296 Han Chinese in southeast China were recruited for this study. We collected medical reports, lifestyle details, and blood samples from individuals. The polymerase chain reaction-ligase detection reaction (PCR-LDR) method was used to detect the genotypes of these SNPs in the ABCA1 gene. Results: After adjusting some covariates, the additive and recessive models of the rs2472510 and rs2515614 were significantly associated with hypertension. The haplotypes TCTA (rs2297406-rs2472433-rs2472510-rs2515614) were associated with high SBP, and the haplotypes CCTA, TCTA, and TTTA were associated with high diastolic blood pressure (DBP). Conclusion: The results of the relationship between the polymorphisms of rs2297406, rs2472433, rs2472510, and rs2515614 in ABCA1 and hypertension in southeastern China would provide a theoretical basis for genetic screening and disease prevention.

RTA and LANA Competitively Regulate let-7a/RBPJ Signal to Control KSHV Replication.

The recombination signal binding protein for immunoglobulin kappa J region (RBPJ) has a dual effect on Kaposi's sarcoma-associated herpesvirus (KSHV) replication. RBPJ interaction with replication and transcription activator (RTA) is essential for lytic replication, while the interaction with latency-associated nuclear antigen (LANA) facilitates latent infection. Furthermore, our previous study found that LANA decreased RBPJ through upregulating miRNA let-7a. However, it is unclear whether RTA regulates the expression of RBPJ. Here, we show RTA increases RBPJ by decreasing let-7a. During KSHV replication, the RBPJ expression level was positively correlated with the RTA expression level and negatively correlated with the LANA expression level. The let-7a expression level was inverse to RBPJ. Knockdown of RBPJ inhibited the self-activation of RTA promoter and LANA promoter and weakened LANA's inhibition of RTA promoter. Collectively, these findings indicate that RTA and LANA compete for let-7a/RBPJ signal to control the KSHV replication. Regulating the RBPJ expression level by RTA and LANA plays an important role during KSHV replication.

Arsenic on cholesterol efflux and the expression of ABCA1, ABCG1 and SRBI / 预防医学

Objective@#To explore the impact of arsenic on cholesterol efflux and the expression of ATP-binding cassette, sub-family A, member 1 ( ABCA1 ), ATP-binding cassette transporter G1 ( ABCG1 ), and scavenger receptor class B member I ( SRBI ) in macrophages, so as to provide the evidence for the mechanism of arsenic induced atherosclerosis.@*Methods@#The human myeloid leukemia mononuclear cells ( THP-1 ), induced by phorbol myristate acetate, and mouse primary macrophages were treated with 0, 0.625, 1.25, 2.5 and 5 μmol/L NaAsO2 for 48 hours. Then the cells treated with 2.5 μmol/L NaAsO2 were changed to arsenic free mediums for 48 hours and collected every 12 hours to analyze the time effect of arsenic. The expression levels of ABCA1, ABCG1 and SRBI were determined by quantitative polymerase chain reaction and western blotting. Cholesterol efflux rates were measured by 3H isotope tracer. @*Results@#Arsenic significantly down-regulated the expression levels of ABCA1 and ABCG1, and cholesterol efflux in a dose-dependent manner. The levels of ABCA1 mRNA decreased by 69% and 72%, the levels of ABCG1 mRNA decreased by 42% and 34%, and the rate of cholesterol efflux decreased by 55% and 59% in THP-1 and mouse primary macrophages cells treated with 5 μmol/L NaAsO2 ( all P<0.05 ). Arsenic had no significant effect on SRBI expression ( all P>0.05 ). Arsenic inhibited ABCA1 expression and cholesterol efflux in THP-1 in a time-dependent manner. Compared with cells before the exposure of arsenic, the level of ABCA1 mRNA and the rate of cholesterol efflux in THP-1 bottomed at 48 hours by 43% and 42%, and gradually recovered when arsenic was removed. @*Conclusions@#Arsenic inhibits cholesterol efflux by down-regulating the expression of ABCA1 and ABCG1 in macrophages.

Cigarette smoke-initiated autoimmunity facilitates sensitisation to elastin-induced COPD-like pathologies in mice.

It is currently not understood whether cigarette smoke exposure facilitates sensitisation to self-antigens and whether ensuing auto-reactive T cells drive chronic obstructive pulmonary disease (COPD)-associated pathologies.To address this question, mice were exposed to cigarette smoke for 2 weeks. Following a 2-week period of rest, mice were challenged intratracheally with elastin for 3 days or 1 month. Rag1-/- , Mmp12-/- , and Il17a-/- mice and neutralising antibodies against active elastin fragments were used for mechanistic investigations. Human GVAPGVGVAPGV/HLA-A*02:01 tetramer was synthesised to assess the presence of elastin-specific T cells in patients with COPD.We observed that 2 weeks of cigarette smoke exposure induced an elastin-specific T cell response that led to neutrophilic airway inflammation and mucus hyperproduction following elastin recall challenge. Repeated elastin challenge for 1 month resulted in airway remodelling, lung function decline and airspace enlargement. Elastin-specific T cell recall responses were dose dependent and memory lasted for over 6 months. Adoptive T cell transfer and studies in T cells deficient Rag1-/- mice conclusively implicated T cells in these processes. Mechanistically, cigarette smoke exposure-induced elastin-specific T cell responses were matrix metalloproteinase (MMP)12-dependent, while the ensuing immune inflammatory processes were interleukin 17A-driven. Anti-elastin antibodies and T cells specific for elastin peptides were increased in patients with COPD.These data demonstrate that MMP12-generated elastin fragments serve as a self-antigen and drive the cigarette smoke-induced autoimmune processes in mice that result in a bronchitis-like phenotype and airspace enlargement. The study provides proof of concept of cigarette smoke-induced autoimmune processes and may serve as a novel mouse model of COPD.

Latency-associated nuclear antigen inhibits lytic replication of Kaposi's sarcoma-associated herpesvirus by regulating let-7a/RBPJ signaling.

Latency-associated nuclear antigen (LANA) is the key factor in the establishment and maintenance of latency of Kaposi's sarcoma-associated herpesvirus (KSHV). A cellular protein, recombination signal binding protein for immunoglobulin kappa J region (RBPJ), is essential for the lytic reactivation of KSHV. However, whether RBPJ expression is regulated by KSHV is not clear. Here, we show that LANA upregulates let-7a and its primary transcripts in parallel with its reduction of RBPJ expression. An increase in notch intracellular domain (NICD) and the downregulation of NF-κB and LIN28B contribute to the upregulation of let-7a by LANA. Let-7a represses RBPJ expression by directly binding the 3' untranslated region of RBPJ. Let-7a overexpression or RBPJ knockdown led to a dose- and time-dependent inhibition of lytic reactivation of KSHV. Collectively, these findings support a model wherein LANA inhibits the lytic replication of KSHV by regulating let-7a/RBPJ signaling.

ABCA1 Is Coordinated with ABCB1 in the Arsenic-Resistance of Human Cells.

Arsenic is one of the most widespread global environmental toxicants associated with endemic poisoning. ATP-binding cassette (ABC) proteins are transmembrane channels that transport and dispose of lipids and metabolic products across the plasma membrane. The majority of ABC family members (including ABCB1 and ABCC1) are reported to play a role in the development of arsenic and drug resistance in mammals. Previously, we established a human arsenic-resistant ECV-304 (AsRE) cell line and identified ABCA1 as a novel arsenic resistance gene. In the current study, we further investigated the potential contribution of ABCA1, ABCB1, and ABCC1 to arsenic resistance through measurement of survival rates and arsenic accumulation in AsRE cells with RNA interference. The arsenic resistance capacity of ABCC1 was the strongest among the three genes, while those of ABCA1 and ABCB1 were similar. Double or triple gene knockdown of ABCA1, ABCB1, and ABCC1 via RNA interference led to a decrease significant in arsenic resistance when ABCA1/ABCB1 or ABCB1/ABCC1 were simultaneously silenced. Interestingly, no differences were evident between cells with ABCA1/ABCC1 and ABCC1 only knockdown. Our findings suggest that ABCA1 and ABCB1 proteins display similar arsenic resistance capabilities and possibly coordinate to promote arsenic resistance in AsRE cells.

AKT/mTOR signaling pathway is involved in salvianolic acid B-induced autophagy and apoptosis in hepatocellular carcinoma cells.

Chinese medicines are emerging as an attractive new generation of anticancer drugs. Here, we explored the impact of salvianolic acid B (Sal B), the major water-soluble compounds of Danshen, on apoptosis and autophagy of human hepatocellular carcinoma cells (HCC). We also investigated the related molecular mechanisms. We found that Sal B exhibits potent ability to inhibit HCC cells viability in a concentration-dependent manner, and to induce apoptosis via the mitochondrial apoptosis pathway. Additionally, Sal B could also induce autophagy. Furthermore, pretreatment with the autophagy inhibitor chloroquine or 3-methyladenine showed the potential in attenuating the apoptosis rate induced by Sal B. Mechanistically, Sal B treatment inhibited the AKT/mTOR signaling cascade in vitro. Overexpression of AKT abolished the effects of Sal B on HCC cells, suggesting a critical role of the AKT/mTOR signaling pathway in Sal B-induced biological effects. Our results indicated that the mitochondrial pathway was involved in Sal B-induced apoptosis of HCC cells. Moreover, the AKT/mTOR signaling pathway was involved in Sal B-induced autophagy, which promoted apoptosis. This study may provide a promising strategy for using Sal B as a chemotherapeutic agent for patients with HCC.

Oncomirs miRNA-221/222 and Tumor Suppressors miRNA-199a/195 Are Crucial miRNAs in Liver Cancer: A Systematic Analysis.

BACKGROUND: The high mortality rate of hepatocellular carcinoma (HCC) is partly due to a lack of good diagnostic markers and treatment strategies. Recently, several microRNA (miRNA) profiling studies were conducted with HCC; however, their inconsistency means that their diagnostic or therapeutic value is debatable. AIMS: This study aims to systematically evaluate the consistency of miRNAs from multiple independent studies. METHODS: A systematic analysis of miRNAs from eligible publications was conducted, followed by real-time PCRs. The targets of highly consistent miRNAs were collected using online programs, followed by enrichment analyses for gene ontology terms and Kyoto encyclopedia of genes and genomes pathways. RESULTS: In total, 241 differentially expressed miRNAs were reported in 13 HCC profiling studies, of which 137 were upregulated and 104 downregulated. Among consistently upregulated miRNAs (cutoff > fourfold), miRNA-222, miRNA-21, miRNA-221, miRNA-210, and miRNA-224 were found increased in 8, 6, 6, 5, and 5 different studies, respectively. Among 137 downregulated miRNAs, miRNA-195, miRNA-199a, miRNA-125b, and miRNA-99a were reported in 8, 8, 5, and 5 studies, respectively. These results were confirmed by real-time PCR. Enrichment analyses demonstrated that programmed cell death and proliferation play important roles during the interplay of miRNA with HCC. CONCLUSIONS: miRNAs most consistently related to HCC are oncomirs miRNA-221/222 and tumor suppressors miRNA-199a/195.

Cellular MicroRNA Let-7a Suppresses KSHV Replication through Targeting MAP4K4 Signaling Pathways.

BACKGROUND: Kaposi's sarcoma (KS)-associated herpesvirus (KSHV) is the etiologic agent of KS, the most common AIDS-related malignancy. The majority of KS tumor cells harbor latent KSHV virus but only a small percentage undergoes spontaneous lytic replication. Viral reactivation from latency is crucial for the pathogenesis and development of KS, but the cellular mechanisms underlying the switch between viral latency and replication are not well understood. METHODS: The level of let-7 miRNAs and MAP4K4 in KSHV infected 293T cells were quantified by real-time PCRs. Let-7 expression was silenced by the miRNA sponge technique. In let-7a transfected 293T cells, the expression of MAP4K4 was measured by real-time PCR and western blot. Luciferease expression was employed to examine the effect of let-7a on the 3'-untranslated region (UTR) of the MAP4K4 gene in 293T cells. Real-time PCR was used to quantify the KSHV copy numbers in BC-3 cells in which the expression of let-7a and/or MAP4K4 were altered. Finally, ERK, JNK and p38 protein production and their phosphorylation status were detected by western blots in let-7a or MAP4K4 transfected BCBL-1 cells. RESULTS: The expression of microRNA let-7 was dramatically decreased in KSHV infected 293T cells, but that of MAP4K4 was increased significantly. Let-7a is physically associated with and targets the MAP4K4 3'UTR, and inhibits MAP4K4 expression at both mRNA and protein levels. MAP4K4 stimulates KSHV reactivation from latency, whereas let-7a inhibits the function of MAP4K4 by reversing the function of MAP4K4 on JNK, phospho-JNK and phospho-ERK1/2 levels. CONCLUSION: Our results establish that let-7a specifically suppresses MAP4K4 expression, and further inhibits KSHV reactivation by interfering with the function of MAP4K4 on the MAPK pathway, highlighting let-7a as a potential treatment for KS.

ATP-binding cassette transporter A1 (ABCA1) promotes arsenic tolerance in human cells by reducing cellular arsenic accumulation.

Arsenic is a toxic element widely distributed in nature, such as water and soil. To survive this metalloid in the environment, nearly all organisms develop strategies to tolerate arsenic toxicity to some degree. Some arsenic-resistance genes have been identified in bacteria and yeast, but for mammals, especially humans, these genes are largely unknown. The aim of the present study was to identify these genes and benefit our intervention of arsenic resistance. We first established a human arsenic-resistant ECV-304 (AsRE) cell line and then used suppression subtractive hybridization and microarray analysis to identify arsenic-resistant genes in these cells. Of the significantly upregulated genes, three ATP-binding cassette (ABC) subfamily members, namely ABCA1, ABCE1 and ABCF1, were chosen for further study with RNA interference and overexpression analyses. The 3-(4,5-dimethyl-2 thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide assay was used to determine the cell survival rate and the IC50 , whereas atomic fluorescence spectrophotometry was used to determine intracellular arsenic levels. We found that among the three ABC genes, only when ABCA1 gene expression was silenced did cells obviously lose their arsenic tolerance. The arsenic accumulation in ABCA1 deficiency AsRE cells was greater than that in wild type AsRE cells. Overexpression of ABCA1 in HeLa cells decreased arsenic accumulation in the cells and the cells were more resistant to As(III) than control cells transfected with empty vector. These results suggest a new functional role for ABCA1 in the development of arsenic resistance in human cells.

Distribution of CCR5-Delta32, CCR5m303A, CCR2-64I and SDF1-3'A in HIV-1 infected and uninfected high-risk Uighurs in Xinjiang, China.

BACKGROUND: Genetic variants of the genes encoding HIV-1 co-receptors and their ligands, CCR5-Delta32, CCR5m303A, CCR2-64I and SDF1-3'A, are implicated in the susceptibility to HIV-1 infection, and the prevalence of these mutations varies by ethnicity. However, little is known about their distribution in Uighurs. OBJECTIVES: This study aimed at characterizing the frequency of these HIV-related gene variants in a high-risk Uighur population. STUDY DESIGNS: A total of 251 HIV-1 seropositive and 238 seronegative high-risk Uighurs were recruited and their genotypes of CCR5-Delta32, CCR5m303A, CCR2-64I and SDF1-3'A were analyzed by PCR and PCR-ligase detection reaction (PCR-LDR). RESULTS: The allelic frequency of CCR5-Delta32, CCR5m303A, CCR2-64I and SDF1-3'A was 4.40%, 2.66%, 25.66% and 57.36%, respectively, in this population. Apparently, the Uighur population has low frequency of CCR5-Delta32 and CCR5m303A, but high frequency of CCR2-64I and SDF1-3'A. While there was no significant difference in the frequency of CCR5-Delta32, CCR2-64I and SDF1-3' A between HIV-1 seropositive and seronegative groups the frequency of CCR5m303A in HIV-1 seropositive group was significantly higher than that in seronegative group (P=0.006, OR=3.982 and 95%CI 1.514-10.476). CONCLUSIONS: Our data suggest that the CCR5-Delta32, CCR2-64I and SDF1-3'A variants may have limited effect on protecting from HIV-1 infection in Uighurs. Rather, the CCR5m303A may be associated with the risk for HIV-1 infection in high-risk Uighurs.

[Construction of subtractive cDNA library of apoptosis-related genes in NB4 cells treated by arsenic trioxide].

OBJECTIVE: Construct the gene library of apoptosis related genes in acute promyelocytic leukemia (APL) cell line NB4 cells treated by arsenic trioxide to clarify the apoptotic mechanism of NB4 cells. METHOD: APL cell line NB4 cells treated with or without arsenic trioxide for 24 hours. Total RNA was extracted and suppress subtractive hybridization (SSH) was conducted according to the manual. With the cDNA of the apoptosis cells as the tester and that of control cells as the driver, forward and reverse hybridization was performed. Differentially expressed genes were linked with pGEM-Teasy cloning vector and transformed into E. coli DH5alpha. The positive clones were screened by blue and white spot. PCR were used to amplify these genes. RESULT: The subtractive cDNA libraries related with apoptosis of NB4 cells were successfully constructed. CONCLUSION: The constructed subtractive libraries are suitable for further study on the functional genes associated with apoptosis ofNB4 cells induced by arsenic trioxide.