| Multiple myeloma(MM)is a plasma cells malignant proliferation disease.The incidence of MM is the second in the malignant tumors of blood system.In recent years,the incidence of MM is increasing in China.The process of angiogenesis is very complex,recent studies have found that the biological behavior of hematological tumors can be affect by angiogenesis just like that of solid tumors,especially in the development of MM.In the process of angiogenesis,endothelial cells are in the core position.They are involved in the whole process of revascularization,neogenesis and stabilization,which are the key factors of neovascularization.It was found that as an important member of the rho(Ras homologous)family,RhoC plays an important role in the invasion and metastasis of multiple myeloma by regulating the cytoskeleton to participate in the formation of tumor blood vessels.Previous studies have found that RhoC was not only expressed in multiple myeloma cells,but also in multiple myeloma vascular endothelial cells(MVECs)and normal human umbilical vein endothelial cells(HUVECs).So we make a proposition that down regulating the expression of RhoC in MVECs will reduce the angiogenesis of myeloma,and RhoC can directly participate in the regulation of the morphology or function of MM vascular endothelial cells.The effect of down-regulation of RhoC in MM cells on angiogenesis in vivo and the effect of down-regulation of RhoC in MVECs how to affect the biological behavior of MM cells still need further study.This topic is divided into two parts.In the first part,MVECs and HUVECs were transfected by RhoC shRNA packaged with lentivirus,then the supernatant of endothelial cells in each group was used to culture RPMI8226 cells;the expression of RhoC mRNA and protein in RPMI8226 cells was detected by qRT-PCR and Western blot;The proliferative,invasive abilities and cell cycle changes of RPMI8226 cells were detected by CCK-8,Transwell invasion experiment and flow cytometry respectively;The expression of PI3K,CDK,CyclinDl,MMP2 and MMP9 proteins were detected by Western blot.Through the above experimental methods,to observe whether the down-regulation of RhoC expression in MVECs has an effect on the biological behavior of RPMI8226 cells,and to explore the possible mechanism of down-regulation of RhoC in MVECs affecting the proliferative,invasive abilities and cell cycle change of myeloma RPMI8226 cells.Angiogenesis of myeloma is a complex process involving many steps and affected by many factors.Previous studies have revealed that RhoC plays an important role in physiological activities of MVECs.However,the effect of down regulating RhoC expression in MM cells on angiogenesis in vivo is not clear.Therefore,in the second part,establishment of animal models of MM was constructed and the growth of MM transplantation tumor was recorded;Ki67 counts of MM transplantation tumor was detected by immunohistochemical staining;The microvascular density(MVD)in the MM transplantation tumor was counted;The number and length of angiogenesis induced by VEGF in Matrigel were counted.To investigate the effect of down-regulation of RhoC on MM cell proliferation,MM transplantation tumor growth and angiogenesis.In search of this study to provide a theoretical basis for the exploration of new methods of clinical treatment of multiple myeloma.Part I Down-regulates the expression of RhoC in MVEC and its effect on the biological behavior of MM cells1 Materials and methods1.1 Induction and identification of myeloma vascular endothelial cellsMyeloma cell supernatants was used as conditioned medium to induce HUVECs cells to obtain MVECs.Identification methods:(1)Cell proliferative activities was detected by CCK-8 assay;(2)Cell migrative and invasive abilities was detected by transwell migration test and transwell invasion test;(3)The expression of tumor vascular endothelial cell specific markers TEM1 and TEM8 were detected by qRT-PCR;(4)ELISA was used to detect the secretion of VEGFA in myeloma vascular endothelial cells.Whether the endothelial cells have the characteristics of tumor endothelial cells after induction is detected by the above method.1.2 Effect of MM vascular endothelial cells after RhoC shRNA transfection on the biological behavior of RPMI8226 cells(1)Group culture of cellsBased on previous research,lentivirus-packed RhoC shRNA-3 and RhoC shRNA-NC plasmids were used to transfect HUVECs and MVECs.The infection effect was evaluated by fluorescence microscopy.Western blot and qRT-PCR were used to detect RhoC expression before and after transfection.Serum-free DMEM medium was used to culture the HUVECs and MVECs of each group for 48 hours after transfection.Collected the supernatant and stored in the-80℃ refrigerator.The supernatants were used as a conditioned medium to induce RPMI8226 cells for 48 hours.RPMI8226 cells are divided as follows:M-S culture group:RPMI8226 cells were cultured in the supernatant of MVECs transfected with RhoC shRNA;M-NC culture group:RPMI8226 cells were cultured in the supernatant of MVECs transfected with RhoC shRNA-NC;H-S culture group:RPMI8226 cells were cultured in the supernatant of HUVECs transfected with RhoC shRNA;H-NC culture group:RPMI8226 cells were cultured in the supernatant of HUVECs transfected with RhoC shRNA-NC.(2)To observe the effect of down-regulating RhoC expression in MVECs on the biological behavior of MM cells and its possible mechanism.RhoC mRNA and protein expression in each group of RPMI8226 cells were detected by qRT-PCR and Western blot respectively;Cell cycle and invasive ability of RPMI8226 cells in each group were detected by Flow cytometry and transwell invasion experiments respectively;Western blot was used to detect the protein contents of PI3K,Akt,CDK,CyclinD1,MMP2,and MMP9 in RPMI8226 cells of each group.1.3 Statistical processingThe SPSS 21.0 statistical software package was used for data analysis.Each experiment is presented as mean±standard error(at least n=3).With p<0.05 considered to represent a statistically significant difference.2 Results2.1 Induction and identification of myeloma vascular endothelial cellsInduction results:(1)The results of CCK-8 assay showed that the proliferative activity of MVECs induced by the medium containing 20%and 50%of the supernatant was different from the control group(p<0.05),and the proliferation rate of MVECs induced by the medium containing 50%supernatant was the highest,the proliferation curve of MVECs induced by 50%supernatant medium was higher than HUVEC s;(2)The results of transwell migration and invasion experiments showed that the migration rate and invasion efficiency of MVECs were higher than the control group(p<0.05);(3)qRT-pCR results showed that the mRNA expressions of TEM1 and TEM8 in MVECs were not significantly different from those in the control group;(4)ELISA results showed that the expression levels of VEGFA in MVECs were different from those in the control group(p<0.05).2.2 Effect of MM vascular endothelial cells after RhoC shRNA transfection on the biological behavior of RPMI8226 myeloma cells(1)Transfection efficiency of endothelial cells in each group and culture of RPMI8226 cellsRhoC shRNA plasmids were used to transfect endothelial cells in each group,and the cells in each group were observed under a fluorescent microscope.The transfection efficiency of MVECs and HUVECs was(91.5 ± 4.5)%and(89.4 ±3.6)%respectively.Then,the transfected endothelial cell supernatants were collected and cultured in RPMI8226 cells to obtain M-S culture group;M-NC culture group;H-S culture group;H-NC culture group.(2)RhoC protein and mRNA expression in RPMI8226 cells of each group①The expression of RhoC protein in RPMI8226 cells of each group was detected by Western blot,results showed that MVEC-S group had a lower RhoC protein expression level than MVECs-NC group(p<0.05),and HUVECs-S group had a lower RhoC protein expression level than HUVECs-NC group(p<0.05).).②The expression of RhoC mRNA in RPMI8226 cells of each group was detected by qRT-PCR,the expression of RhoC mRNA of MVECs-S group was lower than MVECs-NC group(p<0.05).The expression of RhoC mRNA of HUVECs-S group was lower than HUVECs-NC group(p<0.05).(3)Proliferative and invasive abilities of RPMI8226 cells in each group①The cell cycle was detected by flow cytometry.Compared with the NC group,The proportive of MVECs-S and HUVECs-S groups in the G0/G1 phase increased,and the proportion of cells in the S phase decreased(p<0.05),showed that cell cycle arrest occurred in G0/G1 phase;②Transwell invasion test was used to detect the invasive ability,compared with the NC group,the invasive ability of cells in MVECs-S group and HUVECs-S group decreased(p<0.05).(4)The expression content of PI3K,Akt,CDK,CyclinDl,MMP2,MMP9 proteins in RPMI8226 cells of each groupthe expression of PI3K,Akt,CDK,CyclinD1,MMP2,and MMP9 proteins in each groupof cells was detected by Western blot.Compared with the NC group,the expression of PI3K,Akt,CDK,CyclinDl,MMP2,and MMP9 proteins in MVECs-S and HUVECs-S groups were reduced.(p<0.05).Part Ⅱ Effect of down-regulating RhoC expression in MM cells on angiogenesis of MM transplantation tumors1 Materials and methods1.1 Effect of RhoC shRNA on proliferation and angiogenesis of MM transplanted tumor cells in vivo(1)Group transfection of myeloma cellsRPMI8226 cells were transfected with RhoC shRNA and RhoC shRNA-NC lentiviral vectors.The method of transfection was similar with Part Ⅰ 2.2,(1).The cell groups were as follows:1)RPMI8226-S group:RPMI8226 cells transfected with lentiviral packaging RhoC shRNA plasmid;2)RPMI8226-NC group:RPMI8226 cells transfected with lentiviral packaging RhoC shRNA-NC plasmid.(2)Construction of MM transplantation modelsTen healthy 5-week-old BALB/c mice(12-18g)with SPF level.The RPMI8226 cells cultured in(1)were subcutaneously injected into the underarm of the left forelimb of nude mice.When the transplanted tumor in nude mice grows to a size of 1 cm3,500 μl of Matrigel mixed with VEGF(100 ng/ml)was injected into the right forelimb of the nude mice as an angiogenesis attractant.Nude mice are grouped as follows:S Group:each nude mouse injected with 100 μl of RPMI8226-S cell suspension(1.0×07/ml);NC group:each nude mouse injected with100 μl of RPMI8226-NC cell suspension(1.0×107/ml).(3)To observe the growth of MM transplantation tumor in each groupFrom the day of inoculation,observe the activity and tumor growth of nude mice every two days.After tumor formation,measure and record tumors twice a week(accuracy:0.1mm).Using vernier caliper measure the longest diameter a and shortest diameter b of the tumor.When the volum of tumor≥100mm3 and does not fade the next day,it can be judged that the tumor was successfully loaded,that is,the long diameter is about 6-7mm.Since the tumor was successfully loaded,tumors were measured twice a week and tumor growth and nude mice survival were observed.At the fourth week,nude mice were sacrificed by neck removal,and the tumors of the left forelimb were completely dissected out.Length(a)and width(b)of tumors were measured,calculate tumor volume(V)=ab2/2 according to the following formula,take the average of each group,and draw the tumor growth curve of nude mice.(4)To detect the expression of RhoC mRNA and protein in MM transplanted tumors of each groupTransplantion tumor tissues of each group were extracted with protein and mRNA on ice.the expression of RhoC protein in each group was detected by Western blot.The expression of RhoC mRNA in each group was detected by qRT-PCR.(5)To detect angiogenesis ability of MM transplanted tumors in each group① Nude mice were sacrificed at the fourth week,and Matrigel under the armpit of the right forelimbs was removed.Count the number of angiogenesis in the microscope(200×),count the length of new blood vessels(μm)in the image,randomly obtain five images per group,and take the average;② Myeloma transplanted tumors were prepared as paraffin sections,and CD31-labeled vascular endothelial cells were detected by immunohistochemistry.MVD values of the transplanted tumor tissues in each group in five non-repeated fields were counted under a high-power microscope(400 ×)under an inverted microscope.Counting standard:Single endothelial cells or endothelial cell clusters with positive staining,or blood vessels with an inner diameter of less than 8 red blood cells and no muscle layer in the tumor tissue were selected.Counting microvessels in 5 fields of view that do not overlap.(6)To detect cell proliferation of MM transplantion tumors in each groupThe expression of Ki-67 in transplanted tumors of each group was detected by immunohistochemical staining.Ki-67 positive cells were counted at high magnification(400 ×).Ki-67 index aim to count the proportion of Ki-67 positive cells.Ten fields were randomly selected to count the average percentage of positive labeled cells in the total number of cells.1.2 Statistical processingThe SPSS 21.0 statistical software package was used for data analysis.Each experiment is presented as mean ± standard error(at least n=3).With p<0.05 considered to represent a statistically significant difference.2 Results2.1 Successful construction of MM transplantion tumor models in nude miceTen nude mice were successfully seeded by subcutaneous implantation,and the tumor-bearing success rate was 100%.Fifteen days after the inoculation,tiny bulges were visible at the inoculation site,and no spontaneous regression occurred.With time,tumor nodules gradually formed under the skin.At 21 days after inoculation,all nude mice were successfully tumor bearing.2.2 The growth of myeloma transplantation tumors in each groupAfter 4 weeks of tumorigenesis,the nude mice were killed and the tumor was stripped.The volume of transplanted tumor in S group was smaller than that in NC group,but the difference was not statistically significant(p>0.05).Within 4 weeks of tumorigenesis,the tumor growth curve was drawn,and the volume of tumor in S group was slightly smaller than that in NC group.2.3 RhoC mRNA and protein expression in myeloma transplantion tumorsThe expression of RhoC mRNA was detected by qRT-PCR.The expression of RhoC mRNA in S group was lower than NC group,and the difference was statistically significant(p<0.05).The expression of RhoC protein was detected by Western blot.The expression of RhoC protein in S group was lower than NC group.(p<0.05).2.4 Angiogenesis of myeloma transplantion tumors in each groupNew blood vessels in the Matrigel were counted under the microscope and their length was measured.The number of angiogenesis in S group was lower than NC group,and the length of angiogenesis was shorter than NC group,the difference has statistically significant(p<0.05);MVD value in matrigel of S group was lower than NC group,the difference has statistically significant(p<0.05).2.5 The cell proliferation of myeloma transplantion tumors in each groupCompared with the NC group,the Ki-67 positive rate decreased in S group,and the difference has statistically significant(p<0.05).Conclusions1 MVECs can be obtained by inducing HUVECs with RPMI8226 cells supernatant.2 Down-regulating the expression of RhoC in MVECs can affect the expression of RhoC mRNA and protein in RPMI8226 cells,and reduce the proliferative and invasive abilities of RPMI8226 cells.Its mechanism may be related to PI3K/Akt signaling pathway,cell cycle-dependent kinase CDK/cyclin CyclinDl,MMP2/9.3 Down-regulating the expression of RhoC in RPMI8226 cells can reduce the proliferation of myeloma transplantion tumor cells in vivo and inhibit angiogenesis in MM transplanted tumors. |
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