Study On The Function And Mechanism Of FOXD1 In Promoting Progression Of Nasopharyngeal Carcinoma

Background and objective: Nasopharyngeal carcinoma(NPC)is a highly invasive squamous cell carcinoma of head and neck,distant metastasis or recurrence is the leading cause for treatment failure or death in NPC patients.Several studies have shown that forkhead box transcription factors(FTFs)play an important role in the malignant progression of many human cancers through transcriptional activation or inhibition regulation of human genes.Forkhead Box D1(FOXD1)is upregulated in various tumors and plays important role in promoting cancer.However,the function of FOXD1 and its regulation mechanism in NPC remain unclear.Therefore,it is significant to investigate the function of FOXD1 and its molecular mechanism in the progression of NPC.Materials and methods:(1)q RT-PCR and western blotting(WB)were used to detect the expression level of FOXD1 in NPC cells;The expression of FOXD1 in NPC and adjacent normal tissues was detected by q RT-PCR,Western blot and IHC assays;(2)The effects of FOXD1 on the proliferation,apoptosis,invasion and migration of NPC cells were detected by using CCK8,cloning,flow cytometry,scratch test and Transwell assays;(3)WB,CHIP,and q RT-PCR assays were used to verify that FOXD1 could transcriptionally activate LDHA,PKM and ENO1 expression;The effects of FOXD1 on ATP levels,glucose consumption and lactate production in NPC cells were detected by ELISA;(4)RNA FISH,q RTPCR,WB,RNA stability test,CHX protein stability test,RNA pulldown and RIP assays were used to detect the effect of FOXD1-AS1 on FOXD1expression;(5)The nude mouse tumorigenesis experiment further confirm that FOXD1-AS1 enhances glycolysis levels by stabilizing FOXD1 expression to promote tumorigenesis of NPC in vivo;(6)JC-1 assay was used to detect the effect of FOXD1 on the mitochondrial membrane potential of NPC cells;LC3B and TOM20 double fluorescence localization experiment,as well as Mtphagy Dye and Lyso Dye double staining methods were used to detect the effect of FOXD1 on the level of mitophagy in NPC cells;The effect of FOXD1 on mitophagy-related gene expression was detected by q RT-PCR and WB;CHIP-q PCR was used to detect whether FOXD1 is a transcription factor of BNIP3 and verify the specific transcription binding sites;(7)CCK8,cloning,flow cytometry,scratch test,Transwell,LC3 B and TOM20 dual fluorescence localization experiment,as well as Mtphagy Dye and Lyso Dye double staining method were used to determine whether FOXD1 induced mitochondrial autophagy to reduce the drug sensitivity of NPC cells to gemcitabine through BNIP3;(8)TM,Endo H,and PNGase F were used to treat NPC cells or tissues to detect the presence of N-linked glycosylation modifications in FOXD1;Immunofluorescence and CHIP assays were used to detect the effects of N-linked glycosylation modifications on localization and transcriptional activity of FOXD1;After point mutation plasmid transfection,WB was performed to detect the specific N-linked glycosylation modification site of FOXD1;(9)Cellular immune-fluorescence assay was used to detect the effect of Nlinked glycosylation at Asn-176 site of FOXD1 on mitophagy in NPC cells;WB assay was used to detect the effect of ALG3 on N-linked glycosylation modification of FOXD1 protein;Co-IP assay to detect whether ALG3 could directly bind to FOXD1;Immunofluorescence assay was used to detect the effect of ALG3 on FOXD1 distribution;(10)Nude mouse tumorigenesis further verify that ALG3 regulates the FOXD1/BNIP3 axis to increase the mitophagy level of NPC cells,thereby promoting the proliferation,invasion,migration,as well as reduce drug sensitivity to gemcitabine of NPC cells.Results:(1)FOXD1 is highly expressed in NPC tissue and negatively correlated with patient prognosis;FOXD1 could promote the proliferation,invasion,migration of NPC cells,and inhibited its apoptosis.(2)FOXD1-AS1 could stabilize the m RNA and protein expression of FOXD1;(3)FOXD1-AS1 could stabilize FOXD1 expression to transcriptionally activate the expression of key glycolytic genes LDHA,PKM,and LDHA,which promoting the level of glycolysis and malignant progression of NPC cells;(4)In-vivo assay confirmed that FOXD1-AS1 promotes tumorigenesis of NPC through FOXD1/glycolysis pathway.(5)Asn176 is an N-linked glycosylation modification site of FOXD1 protein,and Nlinked glycosylation modification at this site could increase the stability and nuclear localization of FOXD1 protein.(6)ALG3 mediated N-linked glycosylation of FOXD1 could increase the stability and nuclear localization of FOXD1 protein,thereby transcriptionally activating BNIP3 expression to promote mitochondrial autophagy in nasopharyngeal carcinoma cells,which promoting proliferation,invasion,migration,and reduce the drug sensitivity of NPC cells to gemcitabine.(7)Subcutaneous tumorigenesis experiment confirmed that ALG3 regulates FOXD1/BNIP3 axis to promote tumorigenesis in NPC and reduce its drug sensitivity to GEM.Conclusions:(1)FOXD1-AS1 can improve the level of tumor glycolysis by stabilizing the expression of FOXD1,promoting the proliferation,invasion,migration of NPC cell and tumorigenesis of NPC in vivo;(2)ALG3 regulates the FOXD1/BNIP3 axis to increase mitophagy levels to promote proliferation,invasion and migration of nasopharyngeal carcinoma cells,as well as reduce its drug sensitivity to GEM.76 figures,1 table,109 references...