The Effects Of Hypoxia And Notch1on The Growth And Apoptosis Of The Human Multiplemyeloma Cells

Section oneTHE EFFECTS OF HYPOXIA ON THE GROWTH ANDAPOPTOSIS OF THE HUMAN MULTIPLE MYELOMA CELLSBackground:Multiple myeloma (MM) is a hematological malignancy, which is characterized bycloned proliferation of plasma cells (PCs) in the bone marrow (BM).Researches show thatthe abnormal growth and overgrowth of malignant cells create a state of hypoxia intumors. Consequently, hypoxia is thought to be one of the important environment factorsthat affects the growth of tumor,and the common features of solid tumors.By promotingthe transcription of target genes, hypoxia can lead to angiogenesis, glycolysis, andincreased erythropoiesis. Similarly,the BM microenvironment has been considered to behypoxia,which plays a crucial role to the growth of MM cells. Numerous studies haveexamined the state of hypoxia in the BM microenvironment of the mouse model ofMM and MM patients.However,the effects and the mechanism on cell growth,proliferation, apoptosis of myeloma is still not clear.Recently,related studies have shown that the malignant tumor contains a range ofoxygen pressures ranging from approximately5%O2in the well vascularized areas tocomplete anoxia (no oxygen) in the necrotic areas.The average oxygen pressure generallylied in the hypoxic range at approximately1%O2.What’s more,1%oxygen pressures wasapplied in the study of multiple myeloma.Now, we aim to simulate the hypoxic bonemarrow microenvironment in vitro. Investigate the effects of hypoxia on the growth andapoptosis of the human multiple myeloma cells and explore the potential molecular mechanisms involved in these processes, Looking forward to provide a new direction fortumor treatment.Objective:To investigate the effects of hypoxia on the growth and apoptosis of the humanmultiple myeloma cell line RPMI8226and explore the potential mechanisms involved inthese processes.Methods:1.Cell culture:Mutiple myeloma cell lines RPMI8226cells were cultured in vitro,andwere used in the experiment in logarithmic phase.RPMI8226cells were divided in thenormoxic cell culture incubator and the hypoxic cell culture incubator connecting to theoxycycler CO2and oxygen controller.There were two groups:hypoxia group (37°C,1%O2,5%CO2), normoxia group (37°C,21%O2,5%CO2).2.The effects of hypoxia on MM cells proliferation in the different time point wasassessed by CCK-8assay after72h of cultivation. RPMI8226cells were placed in thenormoxic and the hypoxic cell culture incubator respectively for12h,24h,48h or72h.WST-8solution was applied to the viability of MM cells under normoxic and hypoxicconditions.3.Flow cytometry was used to detect cell cycle regulation: RPMI8226cells wereplaced in the normoxic and the hypoxic cell culture incubator respectively for12h,24h.Then,cells were dyed with propidium iodide solution.DNA content wasanalyzed using flow cytometry instrument.4. Flow cytometry was used to detect apoptosis: RPMI8226cells were placed in thenormoxic and the hypoxic cell culture incubator respectively for24h. The apoptosisassay was performed using the Annexin V-FITC apoptosis detection kit.5.The mRNA levels of HIF-1a and p21, p27, CyclinD1cell cycle regulation genewere analyzed by RT-PCR: RPMI8226cells were placed under the normoxic and thehypoxic cell culture respectively for24h.Total RNA was extracted by Trizol according tothe manufacturer’s instructions.After determination of the concentration,cDNA wassynthesized with primescript RT reagent kit.RT-PCR was used to detect the expression ofHIF-1a, p21, p27, CyclinD1mRNA.6.HIF-1a protein levels were analyzed by the western blot assay: RPMI8226cellswere placed under the normoxic and the hypoxic cell culture incubator respectively for24h. Protein was extracted and its concentration was determinate.WB was used todetect the protein expression of HIF-1a. Results:1.Hypoxia inhibited RPMI8226cell proliferation, and the effect of the inhibition oncell proliferation was obvious in12h or24h. After24hours with the extension of time,the inhibitory effection was gradually weaken.2.Hypoxia induced cell cycle arrest at G0/G1phase. Cell apoptosis increasedobviously under the hypoxia group.3.The mRNA expression of HIF-1a and cell cycle regulation gene p21, p27wasnoticeably increased,along with decreased cyclinD1.4. High levels of HIF-1a expression was proved to exist in RPMI8226cells,and theexpression of HIF-1a a was noticeably increased under the hypoxic conditions.Conclusion:The hypoxic cell culture incubator connecting with the oxycycler CO2andoxygen controller can simulate the hypoxic bone marrow microenvironment inMM.Hypoxia can inhibit MM cells proliferation, and its molecular mechanisms areassociated with the change of p21, p27and CyclinD1expression. Section twoTHE EFFECTS OF DOWN-REGULATION OF NOTCH1MEDIATED BY SIRNA ON THE GROWTH AND APOPTOSISOF MM CELLS IN HYPOXIABackground:Notch signaling pathway is an important signal transduction system on cellproliferation and differentiation,which is closely associated with tumor development.Itparticipates in tumor’s proliferation, apoptosis and resistant process such as. Themammalian family of Notch receptors consists of Notch1through Notch4. Notch1downstream genes includs the hairy/enhancer-of-split (Hes) and Hey family ect.whichcontains basic helix-loop-helix (bHLH) structure domain.Studies have reported that theexpression of Notch1in multiple myeloma cells and gamma secretase inhibitors (GSI) caninhibit protease body activity in MM,which enhance antitumor effect with bortezomib.Atthe same time, specific siRNA of Notch signaling molecules or certain resistance genetargeting inhibition for cancer, can inhibit tumor cell proliferation and increase thesensitivity of chemotherapy drugs. Targeted siRNA mediated Notch1genes will probablybe important direction of treatment of MM.Increasing evidence suggests that Notch signalsand hypoxia exist close contact in different tumors. However, the effects and molecularmechanisms of Notch signaling pathway on the growth and apoptosis of multiplemyeloma cells in hypoxia still need further research.Objective:Clear the expression of Notch1and related downstream target genes in multiplemyeloma cells. Explore the change of Notch1expression quantity in hypoxia.Screening theeffective siRNA fragment which can down-regulatethe expression of Notch1. Toinvestigate the effects and molecular mechanisms of Notch signaling pathway on thegrowth and apoptosis of multiple myeloma cells in hypoxia. And provide theoretical basisfor targeting therapy of multiple myeloma.Methods:1.The mRNA levels of Notch1and the related downstream genes Hes1,Hey1wereanalyzed by RT-PCR: RPMI8226cells were placed under the normoxic and the hypoxicconditions respectively for24hours.Total RNA was extracted by Trizol according to themanufacturer’s instructions.After determination of the concentration,cDNA was synthesized with primescript RT reagent kit.RT-PCR was used to detect the expression ofNotch1, Hes1,Hey1mRNA.2.Notch1protein levels were analyzed by the western blot assay: RPMI8226cellswere placed under the normoxic and the hypoxic conditions respectively for24hours.Protein was extracted and its concentration was determinate. WB was used to detect theprotein expression of Notch1.3.Detecting cell activity and efficiency of transfection: X-ray tremeGENE siRNAtransfection reagent transfected fluorescent CY3-negative siRNA fragment to multiplemyeloma cells in the logarithmic phase for48hours.Cell activity detection: counting thecells by microscope after trypan blue staining.Cell activity=(the total number of cells-coloring cell number)/total number of cells by100%. Transfection efficiency measurement:transfection efficiency=the number of CY3fluorescent expression cells/total number ofcells by100%.4.Detect blocking efficiency of Notch1-siRNA fragment: Transfection reagenttransfected Notch1-siRNA fragment (concentration respectively:0,30,60,90nM) tomultiple myeloma cells in the logarithmic phase.48hours after siRNA transfection，cellswere used for RT-PCR,western blot for blocking efficiency detection.5.The effects of Notch1-siRNA on MM cells proliferation under normoxic andhypoxic conditions were assessed by CCK-8: siRNA transfection reagent transfectedNotch1-siRNA fragment to multiple myeloma RPMI8226cells in the logarithmic phase for48hours.Then, the cells were placed under the normoxic and the hypoxic conditionsrespectively for24h. WST-8solution was applied to the viability of MM cells.6.Notch1and p27protein levels were analyzed by the WB assay: siRNA transfectionreagent transfect Notch1-siRNA fragment to multiple myeloma RPMI8226cells in thelogarithmic phase for48hours.Then,RPMI8226cells were placed under the normoxic andthe hypoxic conditions respectively for24h. WB was used to detect the proteinexpression of Notch1and p27.7.Flow cytometry was used to detect apoptosis: siRNA transfection Reagent transfectfluorescent Notch1-siRNA fragment to multiple myeloma RPMI8226cells of thelogarithmic phase for48hours.Then,RPMI8226cells were placed in the normoxic and thehypoxic condition respectively for24h. The apoptosis assay was performed using theAnnexin V-FITC apoptosis detection kit. 1.High levels of Notch1and the related downstream genes Hes1,Hey1mRNAexpression was proved to exist in RPMI8226cells.The expression of Notch1wasnoticeably increased in hypoxic conditions. High levels of Notch1protein expression wasproved to exist in RPMI8226cells,which was noticeably increased under hypoxicconditions.2.Notch1-siRNA effectively down—regulated the expression level of Notch1inhuman multiple myeloma RPMI8226cells. After transfecfion of48hours，Notch1mRNAswere decreased by82％,and Notch1protein were decreased by80.1%.3.Down-regulation of Notchl expression by siRNA inhibited the proliferation ofhuman multiple myeloma RPMI8226cells. The effect of the inhibition on cell proliferationwas more obvious in the group of Notch1-siRNA under hypoxic conditions than the othergroups.4.Apoptosis protein p27in hypoxia has the higher expression than in normoxia.p27protein in transfection cells has the higher expression than in untransfectionones.And the group of Notch1-siRNA under hypoxic conditions has the highestexpression.5. Cell apoptosis of transfection cells showed a marked increase over theuntransfection ones. And the apoptosis rate of Notch1-siRNA cells under hypoxicconditions was higer than the other groups.Conclusion:Notch1protein and its downstream gene were expressed in Multiple myelomacells,whosegene expression increased obviously in hypoxia. Notch1-siRNA effectivelydown—regulated the expression level of Notchl in human multiple myeloma cells. Theeffect of the inhibition on cell proliferation was more obvious in the group ofNotch1-siRNA under hypoxic conditions than the other groups. Cell apoptosis oftransfection cells showed a marked increase over the untransfection ones, especially inhypoxia.