The Mechanisms Of AZD6244Inhibited Proliferation And Induced Apoptosis Of Lymphoma Raji And MOLT4Cell

Background and PurposeLymphoma is one of the most common hematological malignancies,of90%is non-Hodgkin lymphoma(NHL). The rate of morbidity and mortality of lymphoma has risen in recent years. Chemotherapy is always used as the main treatment of lymphoma, with some other ways, such as radiotherapy and transplantation. CHPO or RCHOP regimens is the first-line chemotherapy regimen of NHL. Althought its5-years disease-free survial rate is more than40-80%, there is still15-20%of patients accompanied by refractory, relapse or drug resistance do not respond to the treatment after first-line chemotherapy. Besides, traditional chemotherapy drugs show a lower selectivity and an obviously adverse reactions. So, a kind of newly efficacious drug of lymphoma is desiderated. In recent years, with the deepening understanding of the biological behavior of molecular signaling pathways regulated cell proliferation, molecular targeted drugs, by virtue of their higher specificity, pertinency and efficiency, well tolerance of patients, and lower toxicity reactions, have achieved great success in cancer therapy. More and more small molecular anti-cancer drugs are used clinically, and gradually become a hot topic in the field of tumor treatment at home and abroad.Mitogen activated protein kinase(MAPK) is a kind of serine/threonine protein kinase widely exists in cells, its abnormal signal pathway is closely related to the onset of tumor. Extracelluar signal regulated kinase(ERK) signal pathway is the earliest identified pathway in the MAPK superfamily. A large body of evidence has established that both T lymphoma and B lymphoma active MEK/ERK. Actived ERK1/2can phosphorylate a variety of substrates to promote cell proliferation, increase cell viability. AZD6244(Selumetinib,ARRY-142886), is a kind of small molecular anti-cancer drugs.As an ATP-uncompetitive inhibitor of mitogen-activated protein kinase1/2(MEK1/2), AZD6244has shown activity just in nanomolar concentrations against isolated MEK enzyme and several cancer cell lines. It has also shown activity in several tumor xenograft models of human tumor. Drugs with single-kinase target is the earliest and most successful example in clinical application of anti-tumor small molecule compound drugs. In clinical trials, certain types of cancer patients have shown responses to MEK inhibitor monotherapy. Whether AZD6244function in lymphoma cells is unknown.So, we aim to investigate the effects of AZD6244, a kind of MEK inhibitors, on the proliferation, apoptosis and cell cycle of lymphoma cells and its relevant mechanism, which can provide experimental and theoretical basis for further study.Methods一、Observation of AZD6244for lymphoma cells proliferation, apoptosis and cell cycle.Human Burkitt lymphoma cell line Raji and Human acute lymphoblastic leukemia cell line MOLT4(collectively known as lymphoma cell lines) were used in our experiments. Lympoma cell lines were treated with different concentrations(1、5、10、50uM) of AZD6244for48h, then the protein level of pERK was detected by Western blot, and the anti-proliferative effect, cell cycle profile and apoptosis level was estimated by CCK-8kit and Annexin V/PI Apoptosis Detection Kit, respectively.二、 Explore the mechanism of AZD6244for lymphoma cells proliferation, apoptosis and cell cycle.1. AZD6244down-regulates miR-92a expression through AP1transcriptional factor1.1Microarray to analyze the miRNAs that are modulated by AZD6244in Raji cellsmiRNAs were extracted from cells which treated with10uM AZD6244for48h. Speciments for gene microarray was sent into Kangcheng biological Ltd. Using chip scanner (Genepix4000B) to get the origina signal strength of chip image,and then using GenePix Pro6.0(Axon) software to analyze data.1.2quantitative real-time PCR(qRT-PCR)to validate the influence of AZD6244to miR-92aLympoma cells were treated with different concentrations of AZD6244for48hour. Total RNA were exacted using Tizols-assay, and then ultraviolet spec trophotometer and agarose gel electrophoresis was used to examine the concent ration and quality of RNA. cDNA was synthesized according to the first chain retrovirus kit manual.Then miR-92a gene was amplificated according to the sp ecificity primer by two-step PCR.The expression of miR-92a was detected by ABI7900HT.1.3Promoter.2prediction server analyze the upstream sequence of miR-92a To explore the direct involvement of AZD6244on miR-92a expression, the upstream sequence of miR-92a was analyzed by the Promoter2.0Prediction Server.1.4measure miR-92a expression level by qRT-PCR after c-Jun gene be silencedAccording to the liposome transfection method, c-Jun siRNA was transfected into Raji and MOLT4cells by RNAiMAX for24h before10uM AZD6244treatment for48hs. Cells were harvested at the corresponding time, and the total RNA was exacted to synthesize cDNA.To test the efficiency of gene silencing, relative mRNA level of c-Jun gene was measured by qPCR,and miR-92a relative level with different treatment was checked by the same method.1.5qRT-PCR detect c-FOS mRNA level in lymphoma cell linesTotal RNA of Raji and MOLT4cell were extracted after usinglOuM AZD6244for48h, and then cDNA were synthesized before anaylze c-FOS mRNA level by qRT-PCR.2. Reintroduction of miR-92a reverses AZD6244induced growth arrest of Raji and MOLT4cellsAccording to the liposome transfection method, miR-92a mimics was transiently transfected into Raji and MOLT4cells for24h before10μM AZD6244for48h, and then cells viability was examined with CCK8kit. Overexpression of miR-92a was quantified by qRT-PCR. Apoptosis and cell cycle distribution change were detected by Flow cytometry.3. miR-92a directly inhibits Bim expression by targeting its3’UTR3.1Analyze the direct mRNA targets of miR-92aWe use the online bioinformatic prediction algorithm to analyze the direct mRNA targets of miR-92a.3.2Dual-luciferase reporter assay to verify Bim gene is the direct target of miR-92aThe3’UTR sequence of Bim was found by PubMed, firstly. Specific primers were designed and synthesized according to the miR-92a bingding site area sequence. The3’UTR sequence of Bim containing the putative miR-92a-binding site was amplified with PCR and cloned into the pSI-CHECK2vector, which was designated as wild-type3’UTR (wt3’UTR). Mutagenesis was done with a Quik-Change Site-Directed Mutagenesis Kit (Stratagene, USA), resulting in mutated3’UTR (mut3’UTR). Wt or mut3’UTR vector were co-transfected with miR-92a mimics or negative control into293T cells using Lipofectamine2000(Invitrogen). Luciferase activity was measured48h later using the Dual-Luciferase Reporter Assay System (Promega, USA). Renilla luciferase values were normalized to firefly, and the ratio of Renilla/firefly values is presented.33qRT-PCR and Western blot was used to measure Bim’s mRNA and protein level after transfected miR-92a mimics/AMO into lymphoma cell linesmiR-92a mimic,mimics NC,miR-92a AMO,AMO-NC were transfected into Raji and MOLT4cells using RNAiMAX for24h, after with10uM AZD6244treatment for48h.The total RNA and total protein were exacted from each group, and the mRNA and protein level was quatified by qRT-PCR and Western blot, respectively.4. Silencing of Bim reverses AZD6244mediated apoptosis and growth arrest of lymphoma cells4.1Western blot detect Bim protein level after AZD6244treat with Raji、 MOLT4cells Cells were treated with different concertations(1-50uM) of AZD6244for48h, then total protein were extracted to measure Bim expression in each group cells by Western blot.4.2silence Bim gene, CCK-8kit estimate viable cells and FCM analyze cell profile and apoptosis levelAccording to RNAi technology, Bim siRNA was transfected with RNiMAX into lymphoma cell lines, transfection efficiency was examed by Western blot. After silenced gene Bim of Raji and MOLT4cells, the rate of proliferation and apoptosis, and cell cycle were measured by CCK-8and FCM,respectively.Statistical MethodsThe date of experimental results was was represented as the mean±standard deviation and anaylized using SPSS13.0software package, P values less than0.05are considered statistically significant. One-sample t-test was performed to compare the different between two groups. Differences among groups were determined using One-way ANOVE.Results1. AZD6244inhibits proliferation, induces apoptosis and Gl-stage arrest of lymphoma cells.AZD6244(1-50uM) significantly blocke ERK phosphorylation in Raji and MOLT4cells, even in low concentration. AZD6244significantly inhibit proliferation of both Raji and MOLT4in a dose-dependent manner. IC50is13.29uM of Raji and16.45uM of MOLT4. By flow cytometry, consistent with the proliferation inhibition effect, AZD6244treatment increase rate of apoptosis and G1phase cells both of Raji and MOLT4in a dose-dependent manner compared to the vehicle(n=3, p<0.05). The rate of apoptosis is (9.10±0.61)%.(30.10±0.98)%、(57.10±1.25)%、(69.9±1.32)%in Raji,and is (5.13±1.15)%、(36.70±1.49)%、(54.70±1.21)%、(71.80±1.55)%in MOLT4, with1-50uM AZD6244treatment, respectinvely. Differences are statistically significant(n=3, p<0.05).2. AZD6244down-regulates miR-92a expression through AP1transcriptional factorMiRNAs that modulated by AZD6244were screened in Raji cells by microarray. MiR-92a, a highly down-regulated miRNAs was of interest.The relative level of miR-92a in lymphoma cells was reduced along with the concentration of AZD6244increased, radually, the differences among groups were statistically significant. A potential transcriptional promoter and an API binding site respectively located on1.0kb and1.8kb upstream of pri-miR-92a-2were found by Promoter2.0Prediction Server. Compared with control group, miR-92a expression level notably decreased after silencing c-Jun gene by Qrt-PCR. c-FOS mRNA level in both cell lines which treated with10uM AZD6244for48h, was markly lower than control group, there was significant difference between two groups(n=3, p<0.05).3. Reintroduction of miR-92a reverses AZD6244induced growth arrest of Raji and MOLT4cellsCompared with the negative control,miR-92a mimic transfection markedly increases the viability of Raji and MOLT4cells after AZD6244treatment,the proliferation activity is61.83±4.50%�'�58.90±5.35%, respectively; the rate of apoptosis obviously reduced from40.31±0.35%to24.91±2.33%in Raji,and43.25±1.04%to43.25±1.04%in MOLT4; the percentage of G1-phase cells are significantly reduced from4.66±2.62%to60.13±1.37%in Raji,77.75±1.47%to 63.19±0.86%in MOLT4. Differences are statistically significant (n=3, p<0.05).4. miR-92a directly inhibits Bim expression by targeting its3’UTRWe use the online bioinformatic prediction algorithm www.microRNA.org to analyzed that Bim may be the direct mRNA targets of miR-92a. The dual-luciferase reporter assay shows that miR-92a mimics reduced by50%fluorescence activity of wt3’UTR compared with the negative control(p<0.01),whereas mut3’UTR showed an abrogated response to miR-92a. We measured Bim’s mRNA and protein expression levels after transfecting miR-92a mimics or AMO into Raji and MOLT4cells. Both levels can be down-regulated by miR-92a mimics and up-regulated by miR-92a AMO, compared with the negative control, there are significant different between groups(n=3, p<0.05).5. Silencing of Bim reverses AZD6244mediated apoptosis and growth arrest of lymphoma cellsAfter AZD6244treatment, Bim protein level in Raji and MOLT4cells increase in a dose-dependent manner. After treatment, silencing Bim gene markedly increases the viability of Raji and MOLT4cells compared with the negative control.The cell proliferation rate of Raji and MOLT4is63.27±6.10%and62.73±4.01%,respectively. Apoptosis decreased significantly, compared with control, the rate of apoptosis is reduced from44.20±1.76%to29.35±1.47%in Raji and from51.40±2.31%to2.80±1.56%in MOLT4. Consistent with role in the growth inhibition, the percentage of G1-stage cells reduced markly.There are significant different between groups(n=3, p<0.05). Conclusions1. AZD6244can inhibit the proliferation of lymphoma Raji, MOLT4cell lines by inducing apoptosis and G1stage arrest;2. AZD6244down-regulates miR-92a expression through AP1transcriptional factor;3. Reintroduction of miR-92a reverses AZD6244induced growth arrest of Raji and MOLT4cells;4. miR-92a directly inhibits Bim expression by targeting its3’UTR;5. Silencing of Bim reverses AZD6244mediated apoptosis and growth arrest of lymphoma cells;6. lymphoma cell proliferation, apoptosis and cell cycle are regulated by AZD6244through MEK/ERK/AP1/miR-92a/Bim pathway.