Functional Mechanism Of GLDC Gene Alternative Splicing In Non-small Cell Lung Cancer

Background and objective:Lung cancer is one of the common malignant tumors,with non-small cell lung cancer accounting for 85%.The treatment includes surgical resection,platinum-based chemotherapy,radiotherapy and targeted therapy,however,the occurrence mechanism is not yet fully understood.Therefore,the exploration of new molecules is of great significance to clarify the mechanism and targeted therapy of lung cancer.Glycine decarboxylase(GLDC)is a metabolic oncogene linked to glycine metabolism and tumorigenesis.It is overexpressed in cancer stem cell-rich primary non-small cell lung cancer(NSCLC),driving NSCLC cancer stem cells,and the growth and tumorigenicity of cancer stem cells are also rely on high levels of GLDC expression.Therefore it can be used as stem cell surface markers to identify cancer stem cells.At present,GLDC has been found in a variety of clinical tumor specimens,especially in non-small cell lung cancer,and is positively related to the prognosis of patients.Alternative splicing(AS)is one of the most common mechanisms of human gene regulation,which is crucial in the physiological processes and cell development of tumors.However,the mechanism of AS regulation for GLDC gene has not been reported so far,fully understand the biological role of GLDC and its regulatory mechanism is to develop GLDC as the target antitumor drugs and in-depth study of tumor occurrence mechanism.This study aims to clone GLDC gene AS and analyze biological activity,so as to provide guidance for the molecular mechanism and targeted therapy of non-small cell lung cancer.Methods:1.Construction of cloning vector:The primers were designed according to cDNA of human GLDC,and the expression of GLDC AS(GLDC variant1)mRNA in A549 and MRC5 were detected by RT-PCR combined with sequencing at the mRNA level.GLDC full-length(GLDCfl)and GLDC splice variant1(GLDCV1)were over-expressed in MRC5 cells,and the protein expression were detected by Western blot.2.In vitro:MTT was used to detect the viability of cells,BrdU,soft agar clone and plate cloning assay showed the effection on MRC5 cell proliferation.Using RT-PCR to detect the stem cell marker gene expression and whether AS affects the expression of lactic acid.3.In vivo:Screening stable transfer MRC5 cells then injected into nude mice,the empty plasmid as negative control,to observe whether it has tumorigenic ability.4.Simultaneously,the stable transfected cells were used to verify the effect of overexpression on PI3K/AKT,JAK/STAT,and ERK/MAPK pathway proteins and the effect on cyclin expression was also verified.Results:1.In A549 and MRC5,GLDCfl and GLDCV1 were expressed at RNA level.Western blot showed only A549 expressed at protein level.2.After transfection of 48 h,the MTT revealed the cell viability increased.The BrdU,cell soft agar clone and plate cloning assay demonstrated that the transfection group could promote the proliferation of MRC5 cells,RT-PCR detection of stem cell related gene markers showed the transfection group promoted the expression of CD44 and ALDH1,after transfection of 24 h,the results of lactic acid conveyed that the transfected group could promote the production of cell lactic acid.3.In vivo tumor-bearing experiments demonstrated that stabilizing splice cells had tumorigenicity.4.The mechanism of overexpression promoting cell proliferation was studied.It was found that overexpression GLDCfl and GLDCV1 could increase the expression of P-ERK and P-P38 in ERK/MAPK signaling pathways,indicating that it may promote cell proliferation through ERK/MAPK signaling pathways.Then overexpression can also increase the expression of Cyclin D1.Conclusions:1.This study analyzed the expression patterns of GLDC alternative splicing in tumor cells and revealed the biological roles of GLDC in non-small cell lung cancer.2.It's clear that overexpression GLDCfl and GLDCV1 could enhance MRC5 cell viability and promote cell proliferation?cell soft agar clone formation assay and plate cloning assay?gene marker expression,luciferase acidity,and the tumorigenicity of non-small cell lung cancer in vivo.3.It has also been found that overexpression may play a role in promoting cell proliferation through the ERK/MAPK signaling pathway.4.Overexpression of GLDCfl and GLDCV1 promotes cell proliferation by affecting the expression of CyclinD1.