| Part1Study on the relation between the mRNA level of NGDN gene andthe clinical features in patients with acute myeloid leukemiaObjective To explore the relation between the mRNA level of NGDN gene andthe clinical features in patients with acute myeloid leukemia(AML).MethodsThe mRNA expression levels of NGDN gene in bone marrowmononuclear cells seperated from59patients with newly diagnosed AML and15healthy volunteers were detected by fluorescence quantitative RT-PCR. The mutationsof NGDN gene were detected by cDNAsequencing. The relationship between mRNAexpression levels of NGDN gene and clinical features in patients with AML wereanalyzed by the chi-square test for categorical variables.Results The average expression level of NGDN gene relative toreference genein bone marrow mononuclear cells seperated from15healthy volunteers was1.7.Thusthe59AML patients were divided into low NGDN expressiongroup (the mRNA expression level of NGDN <1.7) and high NGDN expression group (the mRNAexpressionlevel of NGDN>1.7). The results showed that there were no significant differencesbetween these two groups in age, gender, fever, anemia, bleeding, invasion outside thebone marrow, White Blood Count, PLT count, FAB genotyping, molecular and immunological phenotype (P>0.05). In high NGDN expression group, the bone marrow blastcell proportion of58.8%(20/34) patients was <70%, but in low NGDN expressiongroup only28%(7/25) patients <70%. The difference was statistically significant(P=0.019).The proportions of peripheral blood blast cells of16(47%) patients amonghigh NGDN expression group and6(24%) patients among in low NGDNexpressiongroup were less than30%. But the difference between these two groups inproportions of peripheral blood blast cells was not statistically significant (P=0.070)because of the limited patients number.In these59cases of patients,12kind of nonsense mutations of NGDN gene (7g>A,7g> R,9a> G,297g> A,297g> R,1560g> A,1560g> R,306c> A,306c> M,380t> Y,1306a> R,1183c> s) have been detected.One patient may have more than two kinds of mutations. In one patient, sense mutation of247a> M (A247C) wasdetected，resulting in the substitution of lysine at83site by glutamine.Conclusion The high NGDN mRNA expression level may be relative to the lowbone marrow blast cell proportion and less inducing chemotherapy courses to obtainCR；Mutations sites and types of NGDN gene in patients with AML were various, butthere wasseldom sense mutations.The clinical significances of mutations remains tobe further research.Part2Study of the effect of NGDN gene on the sensitivity of human myeloidleukemia cells to chemotherapeutic drugs in vitroObjectiveTo explore the relationship between the expression level of NGDNgene and chemosensitivity of human myeloid leukemia cells.MethodsTransfect the NGDN gene into human myeloid leukemia cell line K562and its multi-drug resistant subline K562/A02(P-glycoprotein positive) by lentivirusvector. Verify the transfection efficiency by the fluorescence microscope and flowcytometry assay. Detect the mRNA expression level of NGDN gene by RT-PCR.Detect the growth inhibition rates of K562, K562-CON (transfected with emptyvector), K562-NGDN, K562/A02，K562/A02-NGDN (transfected with NGDN gene)with the treatmentof VCR, VP-16, ADM and DNR by CCK-8method and analyze thedifferences between these cells. Detect the apoptosis rates of K562, K562-CON,K562-NGDN, K562/A02and K562/A02-NGDN with the treatmentof VCR, VP-16,ADM and DNR by the flow cytometry analysis.Results Human myeloid leukemia cell lines K562-NGDN and K562/A02-NGDNwith NGDN over-expression were successfully constructed. The GFP-positiverateswere greater than90%in K562-NGDN and K562/A02-NGDN cells. The mRNAexpression level of NGDN gene in K562-NGDN cells was8.2times compared withthat in K562cells and the expression level in K562/A02-NGDN cells compared withK562/A02cells was11.3times.After treatment with different concentration VCR, ADM, DNR and VP-16fordifferent time, the proliferation inhibition rates of K562-NGDN cells were Significant ly higher than those of K562and K562-vector cells (K562-CON)(P<0.05).For example,after0.5μM VCR treatment for36h, the proliferation inhibition rates of K562,K562-CON and K562-NGDN cells were57.50±2.1%,55.73±1.60%and72.26±3.83%respectively (K562-NGDN vs K562, P<0.05; K562-NGDN vsK562-CON,P <0.05). After20μMVP-16treatment for36h, the proliferation inhibition rates ofK562, K562-CON and K562-NGDN cells were43.06±2.67%,46.50±2.89%and65.10±3.65%respectively (K562-NGDN vs K562, P<0.05; K562-NGDN vsK562-CON, P <0.05).After8μMDNR treatment for36h, the proliferation inhibition ratesof K562, K562-vector and K562-NGDN cells were63.43±3.0%,65.20±1.49%and76.53±3.05%respectively (K562-NGDN vs K562, P<0.05; K562-NGDN vsK562-CON, P <0.05). After8μMADM treatment for36h, the proliferation inhibition ratesof K562, K562-CON and K562-NGDN cells were65.23±3.02%,63.40±3.0%and76.53±2.13%respectively(K562-NGDN vs K562, P<0.05; K562-NGDN vsK562-CON,P<0.05).In addition, after treatment with different concentration VCR, ADM and VP-16for different time, the proliferation inhibition rates of K562/A02-NGDN cells werealso significantly higher than those of K562/A02cells (P<0.05). After2μM VCRtreatment for36h, the proliferation inhibition rates of K562/A02and K562/A02-NGDN cells were24.75±2.08%and36.27±2.45%respectively (P<0.05). After90μMVP-16treatment for36h,the proliferation inhibition rates of K562/A02andK562/A02-NGDNcells were63.77±3.42%and76.66±2.25%respectively (P<0.05). After50μMADM treatment for36h, the proliferation inhibition rates of K562/A02andK562/A02-NGDN cells were45.73±1.93%and59.15±2.75%respectively(P<0.05).After treatment with different concentration VCR, ADM, DNR and VP-16fordifferent time, the apoptosis rates of K562-NGDN cells were significantly higher thanthose of K562and K562-vector cells (K562-CON)(P<0.05). After0.5μM VCRtreatment for36h, the apoptosis rates of K562, K562-CON and K562-NGDN cellswere34.17±2.37%,37.69±3.32%and52.75±5.51%respectively (K562-NG DNvs K562, P<0.05; K562-NGDN vsK562-CON, P<0.05). After20μM VP-16treatment for36h, the apoptosis rates of K562, K562-CON and K562-NGDN cellswere21.58±1.45%,22.35±1.07%and33.09±1.16%respectively (K562-NGDNvs K562, P<0.05; K562-NGDN vsK562-CON, P <0.05). After4μM DNR treatmentfor36h, the apoptosis rates of K562, K562-CON and K562-NGDN cells were40.03±3.52%,41.53±3.25%and55.84±2.61%respectively (K562-NGDN vs K562, P<0.05; K562-NGDN vsK562-CON, P <0.05).After8μM ADM treatment for36h,the apoptosis rates of K562, K562-CON and K562-NGDN cells were30.07±3.05%,28.92±2.51%and41.35±2.27%respectively (K562-NGDN vs K562, P<0.05;K562-NGDN vsK562-CON, P <0.05).Moreover, after treatment with different concentration VCR, ADM and VP-16for different time, the apoptosis ratesof K562/A02-NGDN cells were also Significantly higher than those of K562/A02cells (P<0.05).For example, After10μM VCRtreatment for24h, the apoptosis rates of K562/A02and K562/A02-NGDN cells were18.45±1.34%and32.8±4.73%respectively(P<0.05). After180μM VP-16treatmentfor24h, the apoptosis rates of K562/A02and K562/A02NGDN cells were19.3±1.69%and26.7±0.84%respectively (P<0.05). After200μM ADM treatment for24h, theapoptosisrates of K562/A02and K562/A02-NGDN cells were23.85±1.06%and41.9±3.25%respectively (P<0.05).Conclusions The over-expression of NGDN gene can increase the sensitivity ofhuman leukemia cells tofrequently-used chemotherapeutic drugs VCR, ADM, DNRand VP-16in vitro. The chemosensitization effect of NGDN is notinfluenced by theexpression of P-glycoprotein.Part3Study on the gene pathway of NGDN in human leukemiacellsObjectiveTo explore the possible action pathway of NGDN gene in leukemiacells.Methods To obtain NGDN gene knockdown model (K562/A02-KD) bylentivirus vector carryingshRNA-NGDN and negative control cells (K562/A02-NC)by lentivirus vector carrying unrelated gene from multidrug resistant cell line K562/A02. To detect the mRNA expression levels of genes involved in mTOR andmTOR related pathwaysin K562/A02-KD and K562/A02-NC cells by quantitativeRT-PCR chip.Results ThemRNA expression levels ofNGDN gene in the K562/A02-NCcells isabout4.7times compared with the K562/A02-KDcells (p=0.009). The results ofquantitative RT-PCR chip showed there were69genes with significantly differentmRNA expression levels between K562/A02-KD andK562/A02-NC cells (P<0.05).Otherwise,according the definition of least significancedifference (Log2(KD/NC)>0.585as up-regulation and Log2(KD/NC)<-0.585as down-regulation, P <0.05), there were40genes with differential expression betweenK562/A02-KD andK562/A02-NC cells.The genes up-regulated in K562/A02-KD cells include extracellular signals genes, signal transduction pathway genes, transcription factor genes，cellinvasion and metastasis-related genes, cytoskeletal genes and oncogenes. extracellularsignal genes include TNF, IGF1, etc. Signal transduction pathway genes involved inMAPK, NF-κB, JAK-STAT, Toll-like and mTORpathways.Transcription factor genesinclude NFAT, HSF1, E2F1, ELK1.Cell invasion andmetastasis-related genesinclude CTNN, FN1, VIM, ITGAM. Oncogenes include MYC, ras, FOSetc.Inaddition,there were some genes down-regulated in K562/A02-KD cellscompared K562/A02-NC, including the TP53, PDGFB, PECAM1, CD44,CYP19A1and ACTA2etc.Some PI3K-Akt-mTOR pathway core protein genes (PI3K, PDK1, Akt,mTOR)were significantly up-regulated in K562/A02-KD cells and some mTORpathway downstream genes (4EBP1, S6K) were also up-regulated. However S6Kdownstream gene rpS6was down-regulated in K562/A02-KD cells.ConclusionsInhibition of NGDN expression resulted in the activation of manysignaling transduction pathways and up-regulated expression of some transcriptionfactor genes and oncogenes, which may promote tumor proliferation, invasion andinhibittumor apoptosis. In addition, inhibition of NGDN expression caused up-regulation ofPI3K-Akt-mTOR-4EBP1/S6K pathway and down-regulationof downstreamrpS6gene. We inferred that NGDN located downstream of the PI3K-Akt-mTORpath way and upstream of rpS6. The functionof NGDN may be similar to4E-BP1/S6K.We believe NGDN could regulate the PI3K-Akt-mTOR pathway in a negative feedbackway. |
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