| Objective: Neuroblastoma(NB)is the most common malignant extracranial solid tumor in children.It originates from the nerve crest and has the clinical characteristics of rapid progression,strong invasion and early metastasis.Its mortality rate accounts for 15% of the total cancer mortality in children,ranking the first,and is also known as the "king of childhood cancer".It is a serious threat to the growth and survival of children and brings a heavy burden to the society.Therefore,there is an urgent need to find a more effective treatment.The overall survival rate statistics of NB patients showed that the overall survival rates of patients with well-differentiated,poorly differentiated and undifferentiated tumor tissues were 81.4%,61% and 30%,respectively.In addition,the tumor tissues of low-risk and moderate-risk patients are often accompanied by the spontaneous outcome of well-differentiated neuroblastoma or benign ganglioblastoma.Therefore,inducing differentiation therapy is considered to be a potential method for the treatment of NB.Retinoic acid is the most widely used inducer at present.However,because the molecular mechanism of retinoic acid on NB is not clear,and it has side effects such as embryonic teratogenicity,inhibition of bone growth and damage of liver function,it has become an urgent problem to explore the target molecules for the treatment of NB.In our group,All-trans retinoic acid(ATRA)was used to treat NB cell line NGP cells for 48 hours for transcriptomics sequencing.Through bioinformatics analysis and R2 database,the molecule Fibronectin 1(FN1)and Sex determining region Y-box 4(SOX4)were selected as target factors.As an important component of Extracellular matrix(ECM),FN1 plays an important role in embryonic development,wound healing,coagulation,host defense and metastasis.It has been reported that it is related to tumor tissue migration and invasion in gastric cancer,bladder cancer,esophageal cancer,breast cancer and thyroid cancer,but the effect of ATRA on the biological behavior of NB cells has not been reported.SOX4 plays an important role in embryonic development and nervous system.Studies have shown that the high expression of SOX4 is closely related to the prognosis of ovarian cancer and glioblastoma,but the effect of ATRA on the biological behavior of NB cells has not been reported.The purpose of this study is to study the effect of retinoic acid on biological behavior of NB cells through FN1 and SOX4.Methods:1.Cell lines: NGP and SY5 Y NB cell lines were used in this study.2.Drug treatment: NB cells were treated with ATRA,and the cell morphology,cycle,proliferation,migration and invasion were observed.3.Transcriptomics sequencing: NGP cells treated with 5 ?M ATRA for 48 hours were sequenced,combined with R2 database to explore the survival rate and screen the target molecules.4.The expression of Microtubule associated protein-2(MAP-2)and Growth associated protein-43(GAP-43)in NB cells was detected by Western blot method.The expression levels of m RNA and protein of FN1 and SOX4 in NB cells were detected by RT-q PCR and Western Blot respectively.5.The morphological changes of cells were observed by two-photon confocal microscope.6.Flow cytometry was used to detect the changes of cell cycle after PI staining.7.Cell transfection method was used to down-regulate the expression level of FN1 si RNA in NGP and SY5 Y cells,and down-regulate or up-regulate the expression level of SOX4,si RNA or overexpression plasmid in NGP cells for follow-up experiments.8.The cell fusion rate was dynamically observed by Incu Cyte Zoom,and the cell proliferation was detected by CCK-8 and colony formation test.9.Cell migration and invasion were detected by scratch and Transwell chamber,and the migration rate was =(0 hour scratch area-24 hour scratch area)/ 0 hour scratch area ×100%.10.SPSS was used for statistical analysis,P < 0.05.There was statistical significance.Results:Part ?: biological behavior changes,transcriptome sequencing and molecular screening of NB cells treated with ATRA.1.Morphological changes such as axonal dendritic extension were observed in NGP and SY5 Y cells treated with ATRA for 48 hours,and the expression levels of neuronal specific marker molecules MAP2 and GAP-43 were increased.2.Compared with the control group,the proliferation rates of NGP and SY5 Y cells treated with ATRA for 48 hours decreased by 24.15% and 25.88%,respectively(P <0.01).3.Compared with the control group,the percentage of cells in G1 phase increased by 16.44% and 18.32% in NGP and SY5 Y cells treated with ATRA for 48 hours,respectively.4.Compared with the control group,the migration rate of SY5 Y cells decreased by20.48% after ATRA treatment for 24 hours(P < 0.001),but there was no significant difference in NGP cell mobility.The number of invasive cells in SY5 Y cells decreased by14.17%,but there was no significant difference in the number of invasive cells in NGP cells.5.Transcription sequencing of NGP cells treated with 5 ?M ATRA for 48 hours showed that "cell movement" and "cell adhesion" were in the forefront of tumor-related enrichment items,and the protein-protein interaction network between differentially expressed genes in the enrichment items showed that FN1 was one of the core genes,so FN1 was selected as the target molecule.Six of the top 20 enrichment items were related to nerve system.So SOX4,a factor related to neurodevelopment,was selected as the target molecule.Part ?: the role of FN1 in the effect of ATRA on the biological behavior of NB cells.1.Using R2 database to analyze the correlation between the expression level of FN1 and prognosis,it was found that the higher the expression level of FN1,the higher the overall survival rate of NB patients.The expression levels of m RNA and protein of FN1 in NGP and SY5 Y cells treated with ATRA were increased.2.Down-regulation of the expression of FN1 had no significant effect on cell proliferation and cell cycle: the expression of FN1 was down-regulated by transfecting FN1 si RNA(# 1,# 2)into NGP and SY5 Y cells,and the cells transfected with control si RNA were used as the control group for follow-up experiment.Compared with the control group,the proliferation rate,cell fusion rate,colony formation number and cell cycle of the cells with lower expression of FN1 had no significant difference(P > 0.05),and there was no significant change in cell morphology under microscope.3.Down-regulating the expression of FN1 promoted the migration and invasion of NB cells: compared with the control group,the cells with lower expression of FN1 accelerated scratch healing.The 24-hour migration rate after scratching was calculated:in NGP cells,the migration rate increased by 15.27%(FN1 si RNA#1,P < 0.001)and27.04%(FN1 si RNA#2,P < 0.001),respectively.In SY5 Y cells,the mobility of cells with decreased expression of FN1 increased by 44.44%(FN1 si RNA#1)and 48.69%(FN1 si RNA#2)(P < 0.001).In NGP cells,the number of invasive cells with decreased expression of FN1 was 1.75 times(FN1 si RNA#1)and 1.69 times(FN1 si RNA#2)(P <0.01respectively)than that of the control group.In SY5 Y cells,the number of invasive cells with decreased expression of FN1 was 1.15 times(FN1 si RNA#1)and 1.23 times(FN1 si RNA#2)(P < 0.05)than that of the control group.4.Down-regulation of FN1 expression level had no significant effect on the inhibitory effect of ATRA on NB cell proliferation and cell cycle arrest in G1 phase:Compared with ATRA treatment group,there was no significant difference in proliferation,cell fusion rate,colony formation number and cell cycle of ATRA-treated transfected FN1 si RNA cells.5.Down-regulation of the expression level of FN1 attenuated the inhibitory effect of ATRA on the migration and invasion of SY5 Y cells;in SY5 Y cells: compared with the ATRA-treated group,the migration rate of ATRA-treated transfected FN1 si RNA cells increased by 19.80%(ATRA + FN1 si RNA#1)and 25.96%(ATRA + FN1 si RNA#2)(P both < 0.001).Compared with ATRA treatment group,the number of invasive cells increased by 8.58%(ATRA + FN1 si RNA#1)and 14.49%(ATRA + FN1 si RNA#2)(P <0.05).Part ?: the role of SOX4 in the effect of ATRA on the biological behavior of NB cells.1.Using R2 database to analyze the correlation between the expression level of SOX4 and prognosis,it was found that the higher the expression level of SOX4,the higher the overall survival rate of patients with NB.The expression levels of m RNA and protein of SOX4 were increased in NGP and SY5 Y cells treated with ATRA.2.Down-regulating the expression of SOX4 to promote the proliferation of NB cells:the expression of SOX4 was down-regulated by transfecting SOX4 si RNA(# 1,# 2,# 3)in NGP cells,and the cells transfected with control si RNA were used as the control group for follow-up experiment.Compared with the control group,the proliferation rate of NGP cells with decreased SOX4 expression increased by 24.41%(SOX4 si RNA#1),29.12%(SOX4 si RNA#2)and 32.35%(SOX4 si RNA#3)(P < 0.001).The rate of cell fusion and the number of cells increased under microscope.3.Down-regulation of SOX4 expression level had no significant effect on cell cycle:compared with the control group,the cell cycle of cells with decreased SOX4 expression level had no significant difference(P > 0.05).4.Down-regulation of SOX4 expression level attenuated the morphological changes of NB cells induced by ATRA to neuron-like cells,inhibited cell proliferation and blocked cells in G1 phase: compared with ATRA treatment group,the proliferation rate of ATRA-treated transfectied SOX4 si RNA cells increased by 19.37%(ATRA+SOX4si RNA#1),24.44%(ATRA+SOX4 si RNA#2)and 29.05%(ATRA+SOX4 si RNA#3)(P <0.001).The rate of cell fusion and the number of cells under microscope increased.In ATRA-treated transfectied SOX4 si RNA cells,the percentage of cells in G1 phase decreased,and there was significant difference compared with the group treated with ATRA-treated cells(P < 0 01).5.Up-regulating the expression level of SOX4 promoted the neuron-like changes of NB cells,inhibited cell proliferation and blocked NB cells in G1 phase;compared with the control group,the proliferation rate of NGP cells with increased SOX4 expression decreased by 17.89%,the cell fusion rate and the number of cells decreased,and the percentage of cells in G1 phase increased.Conclusion:1.ATRA treatment of NB cells causes morphological changes,inhibits cell proliferation,blocks cells in G1 phase,inhibits cell migration and invasion,and the inhibition effect is cell-dependent.FN1 and SOX4 were screened by biological information analysis of NB cells treated with ATRA.Their high expressions are closely related to the good prognosis of NB.2.Down-regulating the expression of FN1 promotes the migration and invasion of NB cells and weakens the inhibitory effect of ATRA on the migration and invasion of SY5 Y cells,but has no significant effect on the proliferation and cycle of NB cells,the inhibitory effect of ATRA on the proliferation of NB cells and the arrest of cells in G1 phase.3.Down-regulating the expression of SOX4 could promote the proliferation of NB cells and attenuate the morphological changes,inhibition of proliferation and G1 arrest of NB cells induced by ATRA,but had no significant effect oncell cycle of NB cells.4.Up-regulating the expression of SOX4 can promote the morphological changes of NB cells to neuron-like cells,inhibit cell proliferation and block cells in G1 phase. |
PDF Full Text Request