| Multiple myeloma(MM)is a plasma cell malignant proliferation disease,accounting for about 10% of the blood tumor.Angiogenesis not only plays an important role in solid tumors,but also in hematological malignancies,especially in myeloma.The process of angiogenesis is very complex,including the proliferation,migration and formation of the endothelium.Recent studies have found that,as an important member of the Rho family,RhoC plays an important role in regulating tumor cytoskeleton,cell motility,cell transformation,tumor cell invasion and metastasis,and is closely related to tumor angiogenesis.The formation of tumor vessels is mediated by micro-environment,tumor cells and various growth factors,and the process is very complex.If myeloma cells was considered as the only target of anti-tumor angiogenesis will not achieve satisfactory results.The study found that RhoC were expressed in multiple myeloma cells and vascular endothelial cells,and the expression of RhoC in tumor tissues is closely related with angiogenesis,suggesting that the expression of RhoC in vascular endothelial cells participates in the regulation of the morphology and the function of vascular endothelial cells,thus promoting angiogenesis in the myeloma tissue.Through the construction of RhoC shRNA plasmid and lentiviral vectors: negative control group(NC group)and experimental group(S group),multiple myeloma vascular endothelial cells(MVECs)and human umbilical vein endothelial cells(HUVECs)were transfected in vitro;Western blot and qRT-PCR were used to detect the expression of RhoC in the cells;Phalloidin labelled by rhodamine was used to conduct the cytoskeleton staining and the cytoskeleton were observed by laser scanning confocal microscopy(LSCM);The endothelial cell morphology and number of pseudopodia were observed by scanning electron microscope;The effect of RhoC on the mobility of vascular endothelial cells was detected by scratch test;The effect of RhoC on the cyclization of vascular endothelial cells was observed on the Matrigel matrix;The endothelial cells were cultured with Cytodex 3 microcarrier,and the number of endothelial buds was observed by inoculating the microcarrier ball on the Matrigel matrix;The expression of ROCK protein and MAPK protein was detected by Western blot technique;The study of the relationship between RhoC and endothelial cell function will further provide theoretical basis for elucidating the angiogenesis mechanism of myeloma and targeting therapy for tumor angiogenesis.Materials and Methods 1 Myeloma vascular endothelial cell isolation and identificationThe myeloma tissue samples were collected,and no necrotic tissue was taken on the ice under aseptic conditions.Anti-CD14,anti-CD45 and anti-CD64 magnetic beads were used for negative screening to remove mixed monocytes,lymphocytes and granulocytes in the samples,Then the cells are cultured.Immunocytochemistry was used to detect CD31,CD34,vWF,CD105 to identify the cells.2 Culture MVECs and HUVECs and detect RhoC expressionVascular endothelial cells were cultured with fetal bovine serum and medium.The expression of RhoC in the isolated and cultured vascular endothelial cells was detected by immunocytochemistry,Western blot and qRT-PCR.The results showed that RhoC was expressed in both cells.3 Transfected MVECs and HUVECs 3.1 Rho C shRNA plasmid construction and transfectionWe designed four interference sequences,that is,S1 RhoC shRNA-1,S2 RhoC shRNA-2,S3: RhoC shRNA-3,S4: RhoC shRNA-4 and one negative control sequence NC RhoC shRNA-NC.RhoC shRNA were transfected into MVECs and HUVECs,and the best interference sequences were screened by fluorescence microscopy and Western blot.Follow-up experiments were performed using the best interference sequence of the S group and the NC group.3.2 The constructed plasmids were transfected into vascular endothelial cells by lipofectionWhen the cells were grown to 60%,the above groups of plasmids were fully mixed with the liposomes,respectively,and then added to each group of cells,and after 12 hours,they were replaced with complete medium.The transfection efficiency was observed at 24 h,48h,and 72 h after transfection,and the interference effects of each group were examined.The efficiency of transfection was calculated as a percentage of the number of fluorescent cells per 200 X field of view.Each group of cells was randomly selected from 5 fields of view.3.3 The constructed plasmids were transfected into vascular endothelial cells by electroporation transfectionThe cultured cells were digested and suspended,and transferred to a 96-well plate.The electroporation solution and the above plasmid were mixed and added to each well,the parameters of the electrorotometer were adjusted,and the cultured cells were electroporated and transfected.Specific observation time and transfection efficiency assessment methods are the same as above.3.4 Transfection of vascular endothelial cells with constructed plasmids using lentiviral vectorsThe lentiviral vector packaging was carried out using the effective plasmid constructed above,and the packaged lentiviral vector working solution was diluted into three different concentration gradients 1:5,1:10,and 1:50 to transfect each group of cells.The specific observation time The method of assessing transfection efficiency is the same as above.3.5 Observed in each group of MVECs and HUVECs cytoskeleton,pseudopodia formationThe confocal microscopy and scanning electron microscopy observations of the transfected cells revealed that the RhoC S group had more uniform cell outlines,fewer filopodia,and more regular cell borders than the NC group.3.6 Observation of each group MVECs and HUVEC cell migration,two-dimensional tube formation and three-dimensional budding abilityThe migration of cells in each group was detected by cell scratch assay.The cells in each group were inoculated into Matrigel to observe the formation of each group.The cells were inoculated into Cytodex3 on microcarriers to observe the sprouting of endothelial cells.3.7 Detection of each group of MVECs and HUVECs ROCK,MAPK protein and mRNA expressionAfter transfection for 24 h,each group of cells was lysed on ice.Western blot was used to detect the protein expression of ROCK and MAPK in each group.qRT-PCR was used to detect the mRNA expression of ROCK and MAPK in each group.4 Statistical analysisSPSS17.0 statistical software was used to analyze the data.All measurement data were expressed with Mean±SD.The independent-samples t test was used to compare the means between two groups.One-way ANOVA was used to compare the means of three or more groups.The test standard ? = 0.05.Results 1 MVECs in vitro isolation and identificationThe cells were sorted by immunomagnetic beads and identified by immunocytochemistry.The isolated cells were positive for CD31,CD34,vWF and CD105,so they could be considered as vascular endothelial cells.2 RhoC expression in MVECs and HUVECsImmunocytochemistry,Western blot and qRT-PCR were used to detect the expression of RhoC in both cells.The results show that RhoC is expressed in both cells.3 Transfection efficiency and interference effects of RhoC shRNA plasmid and lentiviral vector in myeloma vascular endothelial cells and human umbilical vein endothelial cells 3.1 Transfection efficiency evaluationThe transfection of lipofectamine of MVECs and HUVECs were observed 72 h after transfection with plasmids of different groups under a fluorescence inverted microscope.The liposomal,electroporation,lentivirus vector transfection efficiencies were as follows:(10.3±3.5)%,(5.3±1.5)%,(90.3±3.5)%.lentiviral transfection efficiency is best.3.2 Cells in each group RhoC interference evaluationThe expression of RhoC protein in both groups was detected by Western blot.The results showed that RhoC protein expression in the S group was significantly lower than that in the NC group after 48 hours culture(P < 0.05).Among them,the best interference sequence is S3 group.4 RhoC expression in MVECs and HUVECs on cytoskeletal,pseudopodia formationThe confocal microscopy and scanning electron microscopy observations of the transfected cells revealed that the RhoC of the S group had more uniform cell outlines,fewer filopodia,and more regular cell borders than the NC group.5 Effect of RhoC on migration,two-dimensional tube formation and three-dimensional budding of MVECs and HUVECs in each groupForty-eight hours after transfection of MVECs and HUVECs,the effects of RhoC on the function of the two kinds of cells were detected.The results showed that the ability of cell migration in RhoC of the S group was significantly lower than that in NC group.The number of two-dimensional tube and the number and ability of budding decreased.6 The expression of ROCK and MAPK in MVECs and HUVECs in each groupWestern blot was used to detect the expression of ROCK and MAPK protein.Gray scale analysis by Image J software showed that the expression of ROCK and MAPK protein in S group was significantly lower than that in NC group,and the difference was statistically significant(P < 0.05).qRT-PCR was used to detect the expression of ROCK and MAPK mRNA.The results showed that the contents of ROCK and MAPK mRNA in the S group were significantly lower than those in the NC group,and the difference was statistically significant(P < 0.05).Conclusion1 Down-regulate RhoC expression in MVECs can affect the skeletal structure and pseudopodia formation of MVECs,and inhibit the formation of two-dimensional vascular network structure and three-dimensional blood vessel sprouting ability of vascular endothelial cells.2 Down-regulate RhoC expression in MVECs affects the skeletal structure and pseudopodia formation of MVECs and inhibit the formation of two-dimensional vascular network structure and three-dimensional blood vessel sprouting ability of vascular endothelial cells,and may be related to RhoC/ROCK and RhoC/MAPK signaling pathways.3 Transfection techniques such as liposome transfection,electroporation transfection and lentiviral vector transfection,the lentiviral vector with the highest transfection efficiency and minimal cell damage. |
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