Study Of Effect And Mechanisms Of Augmenter Of Liver Regeneration On Human Multiple Myeloma Cell Line

Multiple myeloma（MM） is a malignant tumor derived from plasmacells. The incidence of MM is in the second place of blood systemtumor[1].Now the pathogenesis of MM is unclear.The relationship betweenmyeloma cells and cytokines in the bone marrow microvironment is ahotspot of research.A large number of studies have found interleukin-6,interleukin-10, interleukin-21,tumor necrosis factor(TNF),hepatocytegrowth factor(HGF),vascular endothelial growth factor(VEGF) and someother cytokines plays a very important role in anti-apoptosis andproliferation of myeloma[2-5],these cytokines led to myeloma cellproliferation bypass JAK/STAT3, PI-3kinase/AKT, Ras/MAPK,NF-κB,β-catenin pathways.There’s an accidentally found that when our team product themonoclonal antibodies of augmenter of liver regeneration(ALR) using thehybridoma cells which fused by myeloma cells of BALB/c mice SP2/0andB lymphocyte of BALB/c mice,ascitic fluid with anti-ALR in two or threeof five mice vaccinated by hybridoma cells disappeared itself and there’s no any trace of hybridoma cells when we open up the abdomen andcheck.Thus we speculated that myeloma cells may produce and secrete largeamounts of ALR and strongly rely on ALR to sustain its malignantgrowth.When the ALR of myeloma neutralized by monoclonal antibodiesof ALR secreted by B lymphocyte of BALB/c mice,the ascitic fluiddisappeared for lack of stimulate signal which can maintain theproliferation of myeloma.Augmenter of liver regeneration is a thermostable cytokines cloned inthe early1990s which can promote the repair of the liver cells. Themolecular weight of ALR is about15KD and the molecular structure andamino acid sequences are different from hepatocyte growthfactor(HGF).ALR is expressed in various tissues and organs but the researchof its biological function mainly confined to the liver[6]. ALR is highexpressed in hepatoma tissue and it can stimulate the proliferation ofhepatoma cells in dose dependent manner, but didn’t expressed in normalliver tissue and have no effect on liver primary cells[7-10].All of these suggestthat ALR played an important role in development of liver cancer.Our teamhad proved that the proliferation of hepatoma cells and the growth oftransplantation tumor can be significantly inhibited by prevent thecombination of ALR and receptor with monoclonal antibody of ALR,orblock the expression of ALR in transcription using siRNA[11、12].There is no any research reports about the expression of ALR in MM and its effect on the proliferation of MM. Therefore,we aimed at ALR mayparticipate the development of MM,expound the mechanism,and offers anew reference target and experimental basis for the treatment of MM.This reseach can be divided into three parts:Part1:Study of expression of ALR in human multiple myeloma cellline U266.Objective: To prove the high expression of ALR in multiple myelomacells by detect the mRNA level and protein level and lay the foundation forinvestigate the function of ALR in MM.Methods: Detect the mRNA expression in human multiple myelomacell line U266, mouse myeloma cell line SP2/0and normal humanperipheral blood mononuclear cell line PMBC by Realtime PCR.Detect theprotein expression in these cell lines by Western blot.Results: Realtime PCR results showed that the mRNAexpression ofALR in human multiple myeloma cell line U266and in mouse myelomacell line SP2/0were significantly higher than that in normal humanperipheral blood mononuclear cell line PMBC.Western blot results showedthat their were high level protein expression of ALR in U266and SP2/0,buttheir was no expression could be detected in PMBC.Conclusion: Both on protein level and mRNA level ALR were highexpressed in human multiple myeloma cell line U266. Part2: Study of the effection of ALR in human multiplemyeloma cell line U266proliferation and apoptosis.Objective: Detect the effection of exogenous ALR and monoclonalantibody of ALR on the proliferation of U266.Construct lentivirus whichexpress interference gene of ALR detect proliferation and apoptosis ofU266cells after endogenous ALR gene was silenced.Methods: Detect the proliferation of U266cells after addingexogenous ALR and monoclonal antibody of ALR by trpan blue stain andMTS. Construct lentivirus with ALR shRNA gene and observe theinfection efficiency under the fluorescence microscope by greenfluorescent protein (GFP). Detect the mRNA expression and the proteinexpression in U266cell line by Real-time PCR and western blot. Detect theU266cells proliferation infected by lentivirus with ALR shRNA gene bytrpan blue stain,MTS and BrdU incorporation. Finally, detect U266cellsapoptosis by flow cytometry and detect apoptosis related cytokinesP53,Bax, Bcl-2and survivin of U266cells by Realtime PCR.Results:1.Trypan blue stain and MTS experimental results showed thatexogenous ALR can promote U266cells proliferation,and this promotion has a certain range of concentration-effect relationship， exogenousmonoclonal antibody of ALR can inhibit U266cells proliferation.2. Successfully construct lentivirus with ALR shRNA gene. Thefluorescence microscope showed when the multiplicity of infection（MOI）was1,the lentivirus can infect U266cells and the efficiency of infectionwas reached80%.3. Realtime PCR results showed that after72h the mRNAexpression ofALR in U266cells infected by the lentivirus was significantlydecreased,it’s about1/4-1/5of the control group. Western blot resultsshowed that the protein expression of ALR in U266cells infected by thelentivirus after72h was significantly decreased too.4. Trpan blue stain,MTS and BrdU incorporation showed that after72hthe proliferation of U266cells infected by the lentivirus was inhibited andthe difference was statistically significant(P<0.05).5.Flow cytometry showed after72h the apoptosis of U266cellsinfected by the lentivirus increased and Realtime PCR showed thatapoptosis related cytokines Bax raised obviously, and the Bcl-2lowered,the difference was statistically significant(P<0.05).Conclusion:1. Exogenous ALR can promote proliferation of human multiplemyeloma cell line U266.2. Exogenous monoclonal antibody of ALR can inhibit proliferation of human multiple myeloma cell line U266by neutralize the autocrine ALR.3. The lentivirus with ALR shRNA gene can endogenous silence theexpression of ALR.4. Endogenous silence the expression of ALR can inhibit proliferationof U266and regulate apoptosis of U266cells by affect apoptosis relatedfactors Bax and Bcl–2. Part3：Study of the changes of multiple family cytokines in U266cells after infected by lentivirus with ALR shRNA gene.Objective: We confirmed the effect on proliferation and apoptosis ofU266cell lines in the first two parts.In this part,we detected multiplefamily cytokines which associated with the proliferation and apoptosis ofU266cells infected by lentivirus. Selected cytokines which associated withALR protein expression and lay the foundation for investigate themechanism of ALR in MM.Methods: Detect cytokines IL-6,IL-10,VEGF and TNF-αin U266cellsinfected by lentivirus by ELISA method.Results: ELISA results showed the IL-6level decreased obviouslycompared with control group and the difference was statisticallysignificant(P<0.05). But the level of IL-10,VEGF and TNF-αshowed nostatistical difference compared with controls(P>0.05). Conclusion: Endogenous silent ALR gene express in U266cells canobviously down-regulate cytokine IL-6level but had no effection oncytokines IL-10,VEGF and TNF-α.