| Backgroud: Multiple myeloma(MM)is a kind of hyperplastic blood system tumor in plasma cells in the elderly malignant which can lead to bone damage,can also be invasion to other organs,such as the liver,spleen,lymph nodes,can be found in kidney,lung,heart,thyroid,alimentary canal,uterus,adrenal and subcutaneous tissue,and it also can cause the body metabolism and immune function abnormalities.The present treatment measures mainly include chemotherapy and stem cell transplantation.Because of tumor cell proliferation ratio is very low,the formation of multiple drug resistance and tumor cell invasive strong aspects of reasons,led to the current clinical treatment of multiple myeloma curative effect is not ideal.As is known to all,myeloma cell migration,invasion and spread of ability,at the same time,the study of the mechanism is less,but with the deepening of the research,we will have a more comprehensive understanding of the pathogenesis of MM and thorough treatment will be more effective and also have specificity,to reduce the patient marrow infiltration of outside,so as to fundamentally improve the prognosis of patients.Now it thinks that multiple myeloma have complex pathogenesis on development and marrow infiltration events related to the complex cell genetics.Some genetic abnormalities is often considered as multiple myeloma pathogenic factor.Study found that in recent years,in multiple myeloma cells and bone marrow microenvironment can generate many angiogenic factors,such as vascular endothelial growth factor(VEGF)and basic fibroblast growth factor(bFGF),interleukin-8,angiogenin-1,osteopontin(OPN),myeloma cells secrete growth factors and other matrix metalloproteinases(MMPs),especially the MMP-2 to promote angiogenesis is particularly important.At the sametime,some studies have demonstrated that HOXB7 genes can be induced bFGF specificity,GROa,VEGF,such as vascular growth factor expression level rises,the activation of PI3K/Akt signal pathway,inhibiting apoptosis of endothelial cells,and then further promote tumor angiogenesis,tumor growth and metastasis.HOXB7 genes promote tumor cell proliferation,invasion,metastasis and angiogenesis of tumor cells in a variety of solid tumors cells has been confirmed,and its role in the hematopoietic system and hematopoietic tumor cells is unclear,especially in the multiple myeloma research,reported at home and abroad is less.Therefore,we studies the role of the transcription factor HOXB7 in different clinical stages of patients with multiple myeloma,testing a series of related gene expression at the same time,further elaborated HOXB7 in occurrence,development and infiltration of multiple myeloma.By siRNA transfection multiple myeloma cells,make HOXB7 cut,it can be inspected for multiple myeloma cell proliferation,apoptosis,cell cycle,etc.In our previous study,we have confirmed the SHP-1 as tyrosine kinase 1gene in acute leukemia,may together other genes through phosphorylation off or go to methylation play the role of tumor suppressor genes.Domestic and foreign research in the field of multiple myeloma and is relatively small,in this study,we found that the SHP-1 gene in multiple myeloma has potential antiangiogenic and antitumor activities.By siRNA transfection multiple myeloma cells,make the SHP-1 gene,detect the expression of related genes,further illustrate the role of it in multiple myeloma,and provide strong theoretical basis for gene therapy for multiple myeloma.Study the following three parts:Part one The role of transcription factors HOXB7 in different clinicalstages of patients with multiple myelomaObjective:Detection from MM patients with bone marrow mononuclear cells were purified HOXB7 mRNA and its protein levels in the separation of multiple myeloma,and analyze the HOXB7 MM patients with multiplemyeloma,the relationship between outside marrow infiltration,clinical stage and explore its possible role in multiple myeloma.Methods : Collection of 49 patients with MM marrow mononuclear nuclear cells,and bone marrow pathology and bone marrow supernatant on at the same time,application of CD138 antibodies to purify the MM cells,at the same time and 20 patients with iron deficiency anemia bone marrow mononuclear cells,according to patients will Durie-Salmon,staging and whether with marrow infiltrates into groups,were analyzed.Real-time fluorescent quantitative PCR(FQ-PCR)method to detect the expression of HOXB7,Western blot method to measure its protein expression level,at the same time to bone marrow pathology immunohistochemistry and ElISA analysis of bone marrow supernatant.Results:1 In 49 patients with MM,including 35 patients with MM express HOXB7 mRNA and its expression level is(0.466 ± 0.148),the expression level of 20 cases of control group(0.179 ± 0.061),compared the two significant differences(P< 0.01).We applied Durie-Salmon in installment,the MM patients were divided into two groups.Ⅰ+Ⅱ period and Ⅲ period.21 cases of patients with stage Ⅰ+Ⅱ MM group,HOXB7 mRNA expression level(0.372 ± 0.131).Patients with stage Ⅲ MM group 28 cases,HOXB7 mRNA expression level(0.561 ±0.099),and the stageⅠ +Ⅱ and Ⅲ group of patients with MM,respectively,comparing with control group,according to the results have statistical significance(P < 0.01),while the stage Ⅰ+ Ⅱ group of patients with MM andⅢ group of patients with MM,after comparing results show significant difference(P = 0.018;P < 0.05).Among all MM patients specimen WNT5 A mRNA inspection,found that WNT5 A mRNA expression level is:(0.588±0.588),the control of expression levels for(0.192±0.011),stage Ⅰ + Ⅱ and Ⅲ MM expression level in the patient group,respectively(0.426±0.133),(0.751±0.176),after comparedboth show significant difference(P = 0.005;P < 0.01),and the comparison results show that after the control group(P < 0.01,significant difference was statistically significant.Expression level of VEGFA,MMP2,MMP9,b FGF,Cyclind D1 mRNA,compared with the control group(P < 0.05),the stage Ⅰ+Ⅱ group of patients with MM and Ⅲ group of patients with MM,after comparing results show significant difference(P < 0.01).2 In phase III MM patients,including 9 cases of patients with outside marrow infiltration,19 cases of patients without outside marrow infiltration,which accompanied by marrow infiltration of outside HOXB7 mRNA expression of the patients was(0.751±0.176),without marrow infiltration of outside HOXB7 mRNA patients expressed as(0.639 ± 0.051),through the statistical analysis showed significant difference(P < 0.05),WNT5 A mRNA in patients with outside marrow infiltration of expressed as(0.896±0.105),without outside marrow infiltration of patients expressed as(0.605 ±0.056).The two was statistically significant compared with control group,(P< 0.01).Compared to the statistical analysis of significant differences(P <0.05).3 According to NCCN guidelines in the treatment of multiple myeloma in the treatment of patients with multiple myeloma standard of reaction,divides into the multiple myeloma patients with newly diagnosed,s CR,CR,VGPR,PR,SD,progressive diseases,clinical relapse,and relapse from CR.The line in patients with MM Western blot detection.20 patients with Newly diagnosed early(treated)MM,5 patients with HsCR MM,3 patients with CR MM,VGPR MM patients in 1 case,2 patients with MM PR,SD MM patients in 5cases,a total of 16 cases of patients in stable condition,rogressive diseases,3cases of MM patients,clinical relapse in patients with MM,6 cases relapse from CR 4 cases of MM patients,a total of 13 patients with recurrence and progression of the MM.In 49 patients with MM were chosen Newly diagnosed,sCR and CR,VGPR,PR,SD)in patients with MM,(progressive diseases,clinical relapse,and relapse from CR)all the 6 patients with MM,10 cases of control group selection,including 10 cases of control group in average HOXB7 protein expression level for(0.075±0.026),18 cases of patients with MM HOXB7 protein expression level to an average of(0.268 ± 0.127),including Newly diagnosed group average HOXB7 protein expression level(0.278 ± 0.050),(sCR and CR,VGPR,PR,SD)group of MM patients with(progressive diseases,clinical relapse,and relapse from CR)a group of patients with MM HOXB7 protein expression level to an average of(0.199±0.039),respectively,(0.327±0.131).Early treatment of MM patients with recurrence,progression of MM patients,compared with the stable condition of patients with MM respectively,the result shows that there is significant difference(P< 0.01).And put at the beginning of MM patients with recurrence and progression of MM patients after compared,the results showed no statistical significance(P >0.05).A group of patients with early treatment,recurrence and progression of MM patients compared with controls,respectively,the results there were significant differences(P < 0.01).6 cases of patients with stage Ⅰ+Ⅱ MM group,HOXB7 average expression level was(0.216±0.047).Patients with stage Ⅲ MM group of 12 cases,HOXB7 average protein expression level for(0.320±0.114),and the stage Ⅰ+Ⅱ and Ⅲ group of patients with MM,respectively,comparing with control group,according to the results have statistical significance(P < 0.05),while the stage Ⅰ+Ⅱ group of patients with MM and Ⅲ group of patients with MM,after comparing the results showed significant difference(P <0.01).WNT5A protein in patients with stage Ⅰ+Ⅱ MM average expression level was(0.398 ± 0.136),patients with stage Ⅲ MM group average expression level was(0.549 ± 0.142),the expression level and the control group had significant differences P < 0.05,both are statistically significant(P< 0.01.4 Newly diagnosed,(sCR and CR,VGPR,PR,SD)MM patients,clinical relapse and clinical relapse each line 6 patients with MM WNT5 a,VEGFA,MMP-2,β-catenin,MMP-9,Cyclin D1,survivin protein detection,including MMP-2,β-catenin,VEGFA,MMP-9 protein in Newly diagnosed and(progressive diseases,clinical relapse,and relapse from CR)in patients with MM average expression level was significantly higher(s CR and CR,VGPR,PR,SD)average expression level in patients with MM,after statistics analysis,significant difference(P< 0.01),compared with control group,with statistical significance(P< 0.05).5 Whether the patients with multiple myeloma with marrow infiltration,all of the patients with multiple myeloma can be divided into two groups.In 4of the 12 patients with stage III MM marrow infiltration of MM patients and 8patients with marrow infiltration of the MM outside testing HOXB7 protein level,average expression level respectively(0.265 ± 0.085)and(0.389 ±0.051),respectively,compared with control group,significant difference,there is statistical significance,the comparison between them,there are significant differences(P< 0.05).WNT5 A protein in the absence of marrow infiltration in patients with MM outside the average protein levels was(0.441±0.075),with marrow infiltration of MM patients with average WNT5 A protein expression level(0.673±0.185),respectively,compared with control group,significant difference,there is statistical significance,the comparison between them,there are significant differences(P< 0.01).After statistical analysis showed that both(P < 0.01)and significant difference.6 Bone marrow pathology results are as follows:(1)the expression of HOXB7 in 18 cases of bone marrow biopsy specimens is positive,positive rate was 36.7%,and in 20 cases in the control group,there are 1 case expression is positive,positive rate was 5%,positive rate of the two groups are obvious difference(P<0.05),the intensity is significantly lower than the control group expression MM group(P<0.05);In the different stages in the MM group,with the increase of pathological stagingHOXB7 positive expression rate and expression intensity were increased(P<0.05);In MM different classification group,HOXB7 expression of no significant difference(P>0.05).(2)the expression of VEGFA : 49 cases of bone marrow biopsy specimens of 25 patients with MM positive,positive rate was 51%,and 2 cases in 20 cases in the control group expressed positive,positive rate was 10%,positive rate of the two groups are obvious difference(P<0.05),the intensity is significantly lower than the control group expression MM group(P<0.05);In the different stages in the MM group,with the increase of the pathological staging of VEGFA positive expression rate and expression intensity were increased(P<0.05);In MM different classification group,the expression of VEGFA has no obvious difference(P>0.05).(3)the expression of βcatenin expression: 49 cases of MM patients with bone marrow biopsy specimens of 10 cases were positive,positive rate was20.4%,and no expression in 20 cases in the control group,two groups were more obvious difference(P<0.05),in the different stages in the MM group,with the increase of pathological staging HOXB7 positive expression rate and expression intensity were increased(P< 0.05);In MM different classification group,HOXB7 expression of no significant difference(P>0.05).(4)the expression of WNT5 a : 49 cases of MM patients with bone marrow biopsy specimen of 32 cases positive,positive rate was 65.3%,and 2 cases in20 cases in the control group expressed positive,positive rate was 10%,positive rate of the two groups are obvious difference(P< 0.05),the intensity is significantly lower than the control group expression MM group(P<0.05);In the different stages in the MM group,with the increase of the pathological staging of WNT5 a positive expression rate and expression intensity were increased(P< 0.05);In MM different classification group,the expression of WNT5 a has no obvious difference(P> 0.05).7 The results of Bone marrow on the ELISA :(1)MM patients with bone marrow IL-6 protein content is higher thanthe control group,both compared statistically significant(P<0.05).Initial progress IL-6 MM patients with recurrence protein content was significantly higher in a stable condition in patients with MM,both statistically significant(P< 0.05).(2)MM patients on bone marrow VEGF protein content is higher than the control group,both compared statistically significant(P<0.05).Early cure and recurrence progress in MM patients with VEGF protein is significantly higher than in a stable condition in patients with MM,both statistically significant(P< 0.05).(3)MM patients with bone marrow MMP-2 protein content is higher than the control group,both compared statistically significant(P< 0.05).Initial progress MMP-2 MM patients with recurrence protein content was significantly higher in a stable condition in patients with MM,both statistically significant(P< 0.05).Conclusions:HOXB7mRNA at different stages in patients with multiple myeloma stage there are differences between the expression level,with the increase of installment,HOXB7 mRNA expression increased.Treat patients with MM,early recurrence and disease progression in patients with MM there are differences in the expression level of comparison with patients with stable condition,was statistically significant(P<0.01).Initial MM patients with recurrence and disease progression in patients with the protein expression level of no statistical significance.Patients with MM HOXB7 mRNA expression level may be related to the MM patients with disease progression and marrow infiltration are closely related.Part two: the effect of proliferation and apoptosis on multiple myelomacells with transcription factor HOXB7 and after HOXB7suppression on multiple myeloma cellObjective : With HOXB7-siRNA decreased HOXB7 gene expression,discusses HOXB7 gene of RPMI8226 cell proliferation,cell cycle and the influence of the aggressivity,and discusses the molecular mechanisms.Methods:With mice HOXB7 sequence specificity of small interfering RNA(small interfering RNA,siRNA)transfection RPMI 8226 cells,block the expression of endogenous HOXB7.Determined by MTT test cell growth curve;Flow cytometry instrument to detect the cell cycle distribution;Transwell Chambers experiment test cell activity;Fluorescence quantitative PCR(FQ-PCR)to detect mRNA level change,Western Blot detection protein levels change.Results:1 Detect HOXB7 mRNA and protein detection to determine HOXB7-siRNA transfection efficiency.Results show that the HOXB7-siRNA transfection RPMI8226 cell 48 h,HOXB7 mRNA expression level in NS-siRNA transfection group was 1.128±0.315,HOXB7-siRNA transfection group was 0.142 ± 0.045,compared two groups exist significant difference(P<0.01).HOXB7 protein expression level in NS-siRNA transfection group was 0.698 ± 0.128,HOXB7-siRNA transfection group was 0.083 ± 0.021,compared the differences statistically significant(P<0.01)International working group on multiple myeloma according to NCCN treatment of multiple myeloma treatment guidelines in the treatment of multiple myeloma in the reaction of the standard,divides into the multiple myeloma newly diagnosed,sCR,CR,VGPR,PR,SD,progressive diseases,clinical relapse,and relapse from CR.2 Determined by MTT,according to the results of transfection HOXB7-siRNA 3 d,the cell survival rate(132.55 ± 7.24)%,NS-siRNA transfection group and HOXB7-siRNA transfection group OD 490 compared with statistical significance(P< 0.01)3 Cell cycle analysis showed that HOXB7-siRNA transfection RPMI8226 cells after 48 h,compared with not transfection cells,cells in G0 /G1 phase ratio is reduced,with an increase in G2 / M phase and S phase cells,apoptosis peak showed an increased proportion of cell apoptosis.4 The expression of mRNA : HOXB7-siRNA transfection RPMI8226 cells after 48 h,not the transfection group,NS-siRNA,HOXB7 siRNA group,HOXB7 relative mRNA expression levels were 1.058 ± 0.341;1.128 ±0.315;0.142 ± 0.045,compared with NS-siRNA and HOXB7-siRNA group was statistically significant(P< 0.01).MMP-2 mRNA expression level relatively were 2.606±0.521;1.858 ±0.186;0.882 ± 0.353(P< 0.01).MMP-9 mRNA expression level relatively were 1.896 ± 0.653;1.025 ± 0.276;0.457 ± 0.068;(P<0.05).WNT5 a mRNA expression level relatively are: 1.016±0.195;1.002 ±0.206;0.507 ±0.026(P<0.05).β-catenin mRNA expression level relatively were 1.012 ±0.205;1.006±0.179;0.682±0.351(P<0.01),NS-siRNA and HOXB7-siRNA compared two groups,with significant difference statistically significant(P<0.01).5 The protein level expression: HOXB7-si RNA transfection RPMI8226 cells after 48 h,not transfection group,NS-siRNA group,HOXB7 siRNA,HOXB7 proteins relative expression levels were 0.858±0.204,0.619 ±0.118 and 0.118 ± 0.021,NS-siRNA and HOXB7-siRNA group compared with significant difference(P< 0.01)MMP-2 protein expression level relatively were 1.018±0.216;0.734 ±0.159;0.283±0.114(P< 0.01).MMP-9 protein relative expression levels were1.236±0.253;1.022±0.259;0.453±0.098;(P< 0.05).Relative WNT5 a protein expression level are: 1.016 ± 0.129;0.947 ± 0.221;0.207 ± 0.028(P<0.028).β-catenin protein expression level relatively 1.154 ± 0.205respectively;1.006±0.179;0.682±0.351(P< 0.682)NS-siRNA and HOXB7-siRNA compared two groups,with significant difference(P< 0.01).Conclusions : Transfection HOXB7 interference RNA can abate RPMI8226 cell proliferation,and increase the cell apoptosis,cell cycle in G0 /G1 phase detection results indicate cell percentage decline,G2 / M and S phase cells increased,cells invade its ability.May be related to HOXB7 expression level is reduced,and further cause apoptosis related gene and WNT signaling transduction pathway signal molecules mRNA and proteinexpression level of abnormalities.Part three Ginseng saponin Rg3(Rg3)on the impact of human multiplemyeloma cell proliferation and apoptosisObjective:Study ginseng saponin Rg3 affect human multiple myeloma cell proliferation and apoptosis,and reveals the underlying molecular mechanism..Methods : Collection of 49 patients with MM marrow mononuclear nuclear cells,and bone marrow pathology and bone marrow supernatant on at the same time,application of CD138 antibodies to purify the MM cells,at the same time and 20 patients with iron deficiency anemia bone marrow mononuclear cells,according to patients will Durie-Salmon,staging and whether with marrow infiltrates into groups,were analyzed.Real-time fluorescent quantitative PCR(FQ-PCR)method to detect the expression of HOXB7,Western blot method to measure its protein expression level,at the same time to bone marrow pathology immunohistochemistry and ElISA analysis of bone marrow supernatant.Results:1 Effects of Rg3 on the viability of multiple myeloma cell lines.The uncontrolled cell proliferation of cancer cells is vital in the progression of cancer,therefore,the present study evaluated the effects of Rg3 on the viability of multiple myeloma cells.Treatment with Rg3 inhibited the viability of the U266 cells in a time-and dose-dependent manner.The inhibition of cell viability in the U266 cells following treatment with Rg3 for 48 h reached the maximal level at 80 μg/ml,and the half maximal inhibitory concentration(IC50)value was 47.5 mg/l.In addition to U266,two other multiple myeloma cell lines,RPMI8226,were also included in the present study to examine these effects.Rg3 had a more potent effect in inhibiting the viability of the RPMI8226(IC50=36.8 μg/ml)compared with the U266 cells.2 Arrests the cell cycle of multiple myeloma cells.Flow cytometric cell cycle analysis was performed using PI staining to determine the effect of Rg3 on cell cycle progression.Treat-ment of the U266 cells with 20,40 and 80μg/ml Rg3 increased the percentage of the cell population in the G1 phase and decreased the percentage in the S phase.However,Rg3 had no effect on the G2/M phase.3 Similar results were observed in the RPMI8226.Bcl-2 and Bax are anti-apoptotic and pro-apoptotic proteins,and the ratio of Bcl-2/Bax appears to be a determinant of cell survival and death.To understand the mechanism by which Rg3 induces cell apoptosis,the expression levels of Bcl-2 and Bax,and the ratio of Bcl-2 to Bax were measured.The results of the western blot analysis showed that Rg3 treatment markedly decreased the protein expression of Bcl-2 and increased the protein expression of Bax in the U266 cells,resulting in a further decrease in the Bcl-2/Bax ratio.As expected,Rg3 treatment caused a significant decrease in the protein expression of cytochrome C in the mitochondria,and an increase in the cytoplasm.Cytochrome C release sequentially activates downstream apoptosis associated proteins,including caspases.Western blot analysis demon-strated that Rg3 increased the protein expression levels of cleaved caspase-9,caspase-8 and caspase-3.Collectively,these data suggested that mitochondrial dysfunction may underlie,at least partially,the enhanced effect of Rg3 on myeloma cell apoptosis.Conclusions:Ginseng saponin Rg3 U266,RPMI8226 cell activity,have the time-dose-dependent,and by regulating the cell cycle protein-dependent kinase pathway leading to cell cycle arrest in G1 phase.Moreover,Rg3 apoptosis induction of multiple myeloma cells,involved in regulating B cell lymphoma-2(Bcl2)/Bcl2 protein balance,regulation of caspase activity and the release of cytochrome C from mitochondria in the cytoplasm.Rg3 effectively suppress multiple myeloma cell proliferation and induce cell apoptosis. |
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