Discovery And Preliminary Fuctional Study Of Novel GLDC Variants And The Suppressive Effects On Tumor Stem Cells Of Compounds Inhibiting GLDC Expression

Altenative Splicing(AS)is one of the important mechanisms of eukaryotic gene expression regulation,founding it is closely related to the occurrence of tumor.The mechanism of AS regulation for GLDC gene has not been reported so far.It is the first time founding a novelGLDC gene variant and a missense mutation site have been identified in the ovarian cancer cell line and a related functional study has been carried out.Glycine decarboxylase(GLDC)is an oncogene associated with lactate metabolism,highly expressed in tumor initiation cells(or tumor stem cells),and plays an important role in maintaining tumor cell survival and proliferation and even tumorigenesis effect.GLDC is highly expressed in a variety of clinical tumor tissue specimens,especially in ovarian cancer,and is positively related to the mortality of patients.Therefore,GLDC may be an effective therapeutic target for tumor stem cells and effective control of tumor progression.And fully understand the biological role of GLDC and its regulatory mechanism is to develop GLDC as the target of anti-tumor drugs and in-depth study of tumor occurrence and development mechanism of the important foundation.On the other hand,we used the GLDC promoter-regulated luciferase system to screen the natural compound TI13,which could effectively inhibit the expression of GLDC.In this study,we investigated the effect of TI13 on ovarian cancer stem cell-related anti-Biological effects.The main findings are as follows:1.Discovery of novel variants of GLDC gene and construction of individual variant expression vectors.A novel AS variant of the GLDC gene was found by RT-PCR combined with sequencing at the mRNA level and the missense mutation sites of the 1577 C> G heterozygote were found.Then there was G C doublet at the corresponding position of mRNA 1577 by RT-PCR combined with sequencing.The eukaryotic expression vectors of GLDCV1,GLDCV1 mut,GLDCf1 and GLDCf1 mut were successfully constructed whichis facilitatedto the subsequent study.2.The function of each variant of GLDC gene.First,in vitro experiments,by overexpressing GLDC eukaryotic expression vectors in ovarian cancer cells,founding that GLDC novel AS variant enhances the cell viability of ovarian cancer cells,promotes luciferase acitity,enhances cell anchors non-dependent clonal formation and plate cloning ability,and promote the expression of ovarian cancer stem gene markers.In vivo experiments,all the variants of the GLDC gene could enhance the tumorigenic ability of the ovarian cancer cell SKOV3 by demonstrating the tumor-bearing tumor of the GLDC gene,and demonstrated that the 1577 C>G heterozygous mutations had no significant effect on GLDC function.3.Effects of compound TI13 on GLDC expression and cell viability in ovarian cancer cells.By RT-qPCR and Western Blot founding that TI13 could significantly inhibit the expression of GLDC in ovarian cancer cell lines A2780 and SKOV3 in a concentration-dependent manner.MTT assay was used to detect the viability of cells founding that TI13 could inhibit the cell viability in a concentration-dependent manner,and the inhibitory effect on GLDC-expressing A2780 cells was stronger than that of SKOV3,which was lower in GLDC.4.Effects of compound TI13 on ovarian cancer stem cells.First,in vitro experiments,compound TI13 significantly inhibited A2780 and SKOV3 luciferase acitity,cloning ability and ovarian cancer stem cell marker gene expression;Then in vivo nude mice tumor test,TI13 treated SKOV3 cell tumor formation ability was significantly reduced,suggesting inhibition of GLDC expression by TI13 can specifically inhibit ovarian cancer stem cells.5.Confirm the role of GLDC in the tumor suppressor effect of compound TI13.GLDC overexpression can partially reverse the inhibitory effect of TI13 on luciferase acitity and plate cloning ability of ovarian cancer cells,suggesting that TI13 inhibits ovarian cancer stem cells at least in part by inhibiting GLDC expression.In conclusion,GLDCV1.GLDCV1 mut.GLDCf1 and GLDCf1 mut had similar effects on the cell viability,luciferase acitity,clonal formation and surface markers of ovarian cancer stem cells in ovarian cancer cells.The results provide new ideas and methodsto pathogenesis of GLDC-related diseases as well as disease diagnosis and treatment.In addition,the compound TI13,in vivo and in vitro experiments can be targeted to inhibit ovarian cancer stem cells,the future development of TI13 into ovarian cancer stem cells for the effective treatment of drugs laid the theoretical basis.