Experimental Study On The Effect Of TGFBI Regulating The Malignant Phenotype Of Multiple Myeloma And Its Mechanism

Multiple myeloma(MM)is a common hematological malignancy,and its incidence is related to the abnormal proliferation of plasma cells.The main manifestation is due to abnormal overexpression of monoclonal immunoglobulin.In recent years,the incidence rate showing a gradual upward trend,and the median survival was less than 3 years.Although the application of new anti-MM drugs and autologous stem cell transplantation have improved the treatment of remission rate and survival time,the residual cells making the disease recurred nearly in every patients.Studies have shown that the development of MM is closely related to various oncogene overexpression,and tumor suppressor gene inactivation,but the precise molecular mechanism is not well understood.Therefore,the following are urgent problems in clinical that is to further study on multiple myeloma occurrence,development and precise molecular mechanism.Transforming growth factor-beta induced gene(TGFBI)is an extracellular matrix molecule,which involved in intracellular communication and signal transmission;it can regulate the malignant proliferation and the invasive ability of cells.TGFBI has shown frequent deletions and down-regulation in lung cancer,breast cancer,ovarian cancer,prostate cancer,embryonic rhabdomyosarcoma and other malignant tumors.Studies have shown that TGFBI has anti-tumor function,which can inhibit tumor cell proliferation,invasion and metastasis,and can improve thesensitivity and efficacy of chemotherapy drugs to extend the survival of patients.TGFBI,as a tumor suppressor gene,participated in cancer progression and tumorgenesis,is the ideal target for tumor gene diagnosis and treatment.At present,the levels of expression,occurrence,development and mechanism of TGFBI in multiple myeloma are not clearly understood.In this study,TGFBI,as the target,was selected to explore its expression in bone marrow samples of patients with multiple myeloma;and the relationship between the expression level and clinic pathology of TGFBI was analyzed.The expression of TGFBI in myeloma cells was up-regulated by constructing of TGFBI over-expression lentiviral vector,and observed the changes of malignant phenotype such as cell proliferation,apoptosis and invasion and metastasis in vitro and in vivo,also their mechanism of action has been explored.The aim of this study was to determine the potential therapeutic value of TGFBI in multiple myeloma,so as to provide a new target for those patients with multiple myeloma in targeted therapy.Experimental methods and results:Part I: Expression of TGFBI in multiple myeloma and its clinical significanceMethods: 94 patients with multiple myeloma were diagnosed and the clinical data were collected.The expressions of TGFBI were detected by immunohistochemical staining.The relationship between the expressions level of TGFBI and the clinicopathology were analyzed.Results: The expressions of TGFBI were significantly lower in patients with multiple myeloma,and its expression level was negatively correlated with the age,clinical stage and treatment efficacy,but not with the gender.Part 2: Construction and identification of TGFBI overexpression Lentiviral vectorMethods: The full length of CDS of TGFBI gene was obtained by PCR and identified by agarose gel electrophoresis.Using Age I restriction endonucleases digested the GV208 plasmid vectors,the target gene TGFBI was connected into the linear GV208 plasmid vector to construct GV208-TGFBI recombinant plasmid through the T4 DNA ligase,and then transformed into competent DH5? bacteria.The specific primers were used in the fragment which containing GV208,amplified by PCR,and the TGFBI target gene fragment,then choose the positive clones to confirm its sequences.By transfecting the successfully constructed GV208-TGFBI lentiviral vector into 293 T cells,and testing the titer of lentivirus solution by virus packaging,harvesting and concentrating,and then infecting acquired lentivirus with multiple myeloma RPMI8226 cells,the efficiency of cell infection was detected by fluorescence microscopy and flow cytometry.RT-PCR and western-Blot were used to detect the expression of TGFBI mRNA and expression of protein in RPMI8226 cells before and after infection.Results: Using PCR to Obtain the Full Length of TGFBI CDS.By using binding reaction,it was successfully linked with GV208-TGFBI overexpressed lentiviral vectors,and by PCR amplification,electrophoresis and DNA sequencing,we successfully constructed the GV208 lentiviral vector.After transfected the 239 T cells,the titer of lentivirus was 2.0 ×108TU / ml by virus packaging and concentration.When infection the GV208-TGFBI lentiviral vector with the RPMI8226 cells of multiple myeloma,the flow cytometry transfection rate was more than 90%.RT-PCR and Western-blot were used to detect the expression of TGFBI mRNA before and after infection,and we find that in 48 hours theexpression of TGFBI mRNA in RPMI8226 cells of multiple myeloma,and the scramble lentiviral vectors were significantly higher than that in the scramble lenti-viral vectors of GV208 group,the expression level was 4.8and 4.6 times higher,and compared with the uninfected group and scramble lentiviral vectors group GV208,the difference was statistically significant(P <0.01).Part III: Study of TGFBI gene regulation of malignant phenotype of multiple myeloma cells and its mechanism in vitroMethods: The experiment was divided into two groups: GV208 group as the control group,GV208-TGFBI group as the experimental group.Multiple myeloma RPMI8226 cells were transfected with the recombinant plasmid of lentivirus.The cell proliferation was detected by CCK-8 method.Cell cycle and apoptosis were detected by flow cytometry.Transwell assay was used to detect the invasive ability of the cells.Western blot was used to test the protein expression of CyclinD1,MMP2 and VEGF in the cells.Results: The results showed that the proliferative ability of multiple myeloma in the group of RPMI8226 cells was significantly lower than that in GV208 group,and the difference was more obvious with the prolongation of time.The proportion of cells at G0 / G1 phase was increased,while,the number decreased significantly in S phase and the apoptosis rate was significantly increased to 35.02%.Compared with GV208 group,the difference was statistically significant(P <0.05).The invasion ability of cells exposed that: the number of multiple myeloma RPMI8226 cells in the GV208-TGFBI group that passes through the membrane pores was 98 ± 7 per visual field,and the number of transmembrane cells in the GV208 group was 255 ± 12 / per visual field,the difference was also statistically significant(P <0.05).Meanwhile,the protein expression of CyclinD1,MMP2 and VEGF in the myelomaRPMI8226 cells was significantly down-regulated in the GV208-TGFBI group,and compared with the GV208 group,the expression levels were respectively 59.0%,67.4% and 80.0%,the difference was statistically significant(P <0.05).Part IV: Study of TGFBI gene regulation of malignant phenotype of multiple myeloma xenografts in nude mice and its mechanism in vivo.Methods: Inoculated the GV208 and GV208-TGFBI multiple myeloma RPMI8226 cells into the skin to construct nude mice xenograft model.To observe the growth of tumor cells,and measure the long and short diameter of the tumor,calculate the tumor volume and tumor inhibition rate.The transmission electron microscope was used to observe the ultrastructure tissue of tumor cells.The protein expression of TGFBI,PCNA,Cyclin D1,MMP2 and VEGF were detected by Western blot.Results: The multiple myeloma RPMI8226 cells' xenograft models were successful constructed the in nude mice.The tumor formation time of GV208 group and GV208-TGFBI group were respectively 6 days and 10 days.The growth rate of GV208-TGFBI group with multiple myeloma in nude mice was slow.And with the prolongation of time,the tumor volumes were smaller and the weights were lighter,and the tumor inhibition rate reached to 62.03%.The results of transmission electron microscopy showed that the morphological changes,such as cell necrosis and apoptosis,were increased in the multiple myeloma cells of GV208-TGFBI group,and the proliferation of tumor cells were decreased.The expression of TGFBI protein in the multiple myeloma tissue of GV208-TGFBI group was significantly increased,the protein expression increased by 3.74 times,while,the expressions of PCNA,CyclinD1,MMP2,VEGF protein were significantly decreased,the protein expressions were inhibited by 52.1%,55.1%,71.4% and 77.8%,the difference was statistically significant(P<0.05).