| According to the International Agency for Research on Cancer(IARC),lung cancer has far surpassed other types of cancer in the global cancer mortality rate.Pathologically,lung cancer is divided into small cell lung cancer and non-small cell lung cancer(NSCLC),the latter accounting for more than 85% of lung cancers.Non-small cell lung cancer is further divided into lung adenocarcinoma,lung squamous cell carcinoma,large cell lung cancer and adenosquamous carcinoma,among which lung adenocarcinoma is the most common clinical type.Radiotherapy is the main treatment for different stages and different pathological types of lung cancer,which can significantly improve the local control rate and long-term survival rate of lung cancer.However,the phenomenon that lung cancer cells develop radioresistance after radiation and lead to tumor recurrence still exists.Therefore,studying the mechanism of radioresistance in lung cancer cells and finding new radiotherapy targets are crucial for improving the prognosis of patients.It has been reported that the expression of neuropilin-1(NRP1)increases when lung cancer cells develop radioresistance.However,the specific molecular mechanism of NRP1 regulation in the process of radiation tolerance of lung cancer cells is still unclear,especially the regulatory mechanism of its upstream transcription factor needs to be further explored.Research Background:Neuropilin-1(NRP1)is a single-pass transmembrane glycoprotein that is present in all vertebrates and is highly conserved across species and species.NRP1 is expressed in many tumor cells,such as lung cancer,brain glioma,breast cancer and colon cancer.It has been reported in the literature that NRP1 is a novel lung cancer marker and is closely related to the radiation resistance of lung cancer cells.GATA binding protein-3(GATA3)is a member of the GATA family.As a transcriptional regulatory element(TREs),it can affect the occurrence of a variety of tumors,and its specific target genes are important for initiating malignant phenotypes.The transcription program is crucial.For example,the high expression of GATA3 in breast cancer can promote the proliferation and metastasis of epithelial cells in the breast cavity.GATA3 in bladder cancer can be used as a marker for endoluminal typing.High expression of GATA3 in lung adenocarcinoma has also been considered as a poor prognostic factor.However,most of the current research is mainly focused on clinical research,and its internal regulatory mechanism is rarely reported.Recently,more and more scholars have begun to pay attention to the role of epigenetic modification in the occurrence and development of cancer.Epigenetics regulates geneexpression through DNA methylation,histone modifications,chromatin remodeling,and noncoding RNAs,and plays an important role in determining cell proliferation and differentiation.Histone modification can affect the affinity between histones and DNA double-strands,thereby changing the loose or tight helical state of chromatin,which in turn affects the binding of regulatory proteins such as transcription factors to chromatin,and affects gene expression.The activation of chromatin structure is related to H3K4,H3K36,H3K79 methylation,etc.,while the silencing is related to H3K9 and H3K27 methylation.Some literatures have confirmed that H3K4me3 is related to the acquisition of radioresistance in A549 cells,and its methyltransferase MLL5 is also involved in the formation of radioresistance.Preliminary studies of our group have shown that NRP1 is a co-receptor of a variety of growth factors,which can promote the epithelial-mesenchymal transition(EMT)process of lung cancer cells by interacting with TGF-β,thereby enhancing the Invasion and metastasis of cancer cells.And GATA3 has also been reported that its high expression is highly correlated with poor prognosis of lung adenocarcinoma.At present,little is known about the epigenetic regulation mechanism of NRP1 and radioresistance in lung cancer cells,so whether the epigenetic modification H3K4me3 and transcription factor GATA3 are involved in the formation of radioresistance,and whether it affects radioresistance by regulating NRP1 what about? These scientific questions need to be solved urgently.Therefore,elucidating the relationship between the transcription factor GATA3,histone H3K4 trimethylation modification and NRP1,and exploring the complex regulatory role among the three and the mechanism of promoting the formation of radioresistance in lung adenocarcinoma cells will help improve the curative effect of tumor radiotherapy and open up new new basis for the treatment strategy.Research Purposes:This project intends to use A549 and H1299 two different gene backgrounds of NSCLC cell radiation resistance models to study the regulation of GATA3 and H3K4me3 on the transcription and translation levels of NRP1,and to analyze the effects of GATA3 and H3K4me3 in different lung adenocarcinoma cell radiation resistance models.The regulation rules and the effects of the two on NRP1-induced radioresistance are discussed,in order to provide a new target and new idea for the clinical formulation of radiotherapy regimens for different genotypes of lung cancer.Research Methods:Two radioresistance models of lung adenocarcinoma cells(named A549-RR cells and H1299-RR cells,respectively)were established by fractionated high-dose X-ray irradiation.Each irradiation dose was 6 Gy,and the dose rate was 1.02 Gy/min(irradiated A549 cells)and0.75 Gy/min(irradiated H1299 cells),5 times of irradiation,with a total dose of 30 Gy.The success of the model construction was verified by experiments such as clone formation,CCK-8,phalloidin staining,cell morphology,and flow cytometry.q RT-PCR and Western Blot experiments were used to detect the m RNA and protein expressions of GATA3 and NRP1 during the construction of the radiation-resistant cell model.The co-localized expression of GATA3 and NRP1 proteins in radiation-resistant cells and the nuclear entry of GATA3 were detected by immunofluorescence.The upstream transcription factors and binding sites of NRP1 gene were predicted by bioinformatics website.Gene expression profiling chip sequencing was used to analyze differentially expressed genes between parental cells and radiation-resistant cells,and GO analysis and KEGG signaling pathway were used to analyze which functions and important signaling pathways these differential genes were mainly concentrated in.Transient transfection technology was used to knock down and overexpress GATA3 and NRP1 to explore the upstream and downstream regulatory relationship between GATA3 and NRP1.The binding site of GATA3 and NRP gene was predicted by bioinformatics website to construct recombinant plasmid,and the regulation effect of GATA3 on the promoter region of NRP1 gene was detected by dual-luciferase reporter gene experiment.Ch IP-seq and Ch IP-q PCR experiments were used to detect the enrichment of GATA3 and H3K4me3 in the promoter region of NRP1 gene in two radiation-resistant cell models,and to explore the mechanism of GATA3 and H3K4me3 in the formation of radiation resistance.Finally,the effect of GATA3 knockdown on the radiotherapy efficacy of lung adenocarcinoma and its mechanism were evaluated in vivo by nude mouse tumor-bearing experiments.Research Results:1.Construction of radioresistant cell models(A549-RR and H1299-RR)The results of clone formation assay and CCK-8 assay showed that the survival rate and proliferation activity of A549-RR and H1299-RR cells were significantly improved compared with parental cells under different radiation doses.The two radiation-resistant cell models were observed to have longer pseudopodia than the parental cells under light microscope,and the morphology became irregular.The results of immunofluorescence showed that the F-actin protein was thickened in both radiation-resistant models,and changed from punctate filaments to obvious fascicular filaments.Cell cycle results showed that compared with parental cells,A549 radioresistant cells had G1 phase arrest and H1299 radioresistant cells had S phase delay.The above results indicated that the radioresistant cell model was successfully constructed.2.Gene microarray analysis of up-regulated genes in a radiation-resistant cell modelFirst,the bioinformatics website(JASPAR-A database of transcription factor binding profiles(genereg.net))was used to predict the transcription factors that bind to the NRP1 gene,of which 13 factors were positively regulated with a binding probability of more than 95%.They are ETS1,HIC2,GATA3,ID4,SPIB,MZF1,SPI1,TCF4,TFAP2 A,TCF3,NFIC,RBPJ and ZEB1.The binding site of the transcription factor GATA3 to NRP1 is closest to the transcription start position.From the gene expression profile chip results,compared with A549 cells,3888 genes were up-regulated and 4067 genes were down-regulated in A549-RR cells.Heat map analysis showed that GATA3,NRP1 and KMT2 B genes were up-regulated genes.The differential genes found in A549 and A549-RR cells were mainly concentrated in cancer pathway,PI3K-AKT pathway and MAPK pathway.Gene function analysis revealed that the differential genes were mainly enriched in biological processes related to the plasma membrane.This suggests that GATA3 and NRP1 are important genes for the development of radiation resistance.Validation of q RT-PCR and WB results showed that the m RNA and protein expressions of NRP1,GATA3 and KMT2 B were increased in A549-RR cells.In H1299-RR cells,the m RNA and protein expressions of NRP1 and GATA3 were increased,but the m RNA expression of KMT2 B was decreased,and the protein expression did not change significantly.3.The expression changes and nuclear entry of GATA3 and NRP1 during the construction of the radiation-resistant cell modelq RT-PCR and WB experiments were used to detect the m RNA and protein expressions of NRP1 and GATA3 during the construction of the radiation-resistant cell model.The results showed that the m RNA and protein expressions of NRP1 and GATA3 during the formation of A549-RR and H1299-RR cells were significantly different at each time.Both increased and decreased in different degrees after irradiation,but the m RNA and protein expressions of NRP1 and GATA3 in A549-RR and H1299-RR cells were significantly higher than those in parental cells after the last irradiation.KMT2 B m RNA and protein expression increased in A549-RR cells,while KMT2 B m RNA expression decreased in H1299-RR cells,and protein expression was unchanged.The results of immunofluorescence showed that GATA3 was nuclearized in both radiation-resistant cells,but NRP1 was only observed in H1299-RR cells,but not in A549-RR.The above results showed that the expression of GATA3,NRP1 and KMT2 B increased and the nuclear entry of GATA3 increased when A549 cells developed radioresistance.When H1299 cells developed radiation resistance,the expressions of GATA3 and NRP1 increased,while the expression of KMT2 B did not change or even decreased,and both GATA3 and NRP1 were nuclearized.4.The regulatory effect of transcription factor GATA3 on NRP1 when two kinds oflung cancer cells develop radioresistanceq RT-PCR and Western Blot experiments were used to detect the m RNA and protein expression of GATA3 and NRP1 in two radiation-resistant cells after knockdown and overexpression.The m RNA and protein expressions decreased;after overexpression of GATA3,the m RNA and protein expressions of NRP1 increased.Conversely,after knockdown and overexpression of NRP1,the m RNA expression of GATA3 fluctuated,but the protein expression did not change significantly.However,in the H1299 cell model,the opposite results were observed with the A549 cell model.After knockdown of GATA3,the expression of NRP1 protein increased;after overexpression of GATA3,the expression of NRP1 protein decreased.On the other hand,after knockdown and overexpression of NRP1,neither the m RNA nor protein expression of GATA3 was significantly changed.Taken together,the results showed that when A549 cells developed radioresistance,GATA3 was located in the upstream to positively regulate the expression of NRP1;when H1299 cells developed radioresistance,GATA3 was located in the upstream to negatively regulate the expression of NRP1.5.Explore the regulatory role of transcription factor GATA3 in the promoter region of NRP1 geneFirst,use the JASPAR website to predict the binding site(AGATTG)of the transcription factor GATA3 and the NRP1 gene promoter region,select a fragment of the NRP1 promoter region according to this site,and connect it into the p GL3-Basic plasmid for firefly-sea cucumber luciferase.Reporter gene experiments showed that the activation of GATA3 on NRP1 gene was enhanced in A549-RR cells,while the activation effect of GATA3 on NRP1 in H1299-RR cells was weakened.Ch IP-seq and Ch IP-q PCR experiments showed that the transcription factor GATA3 was enriched in the NRP1 gene promoter region in the A549 radiation-resistant cell model,but decreased in H1299-RR cells.The above results indicate that one of the downstream target genes of GATA3 is NRP1.Moreover,the enrichment was increased in A549-RR cells and decreased in H1299-RR cells.Combined with result 4,it can be analyzed that the enrichment of GATA3,which plays a role in promotion in A549-RR cells,increases in NRP1 gene,which leads to the high expression of NRP1.It also resulted in increased NRP1 expression.6.Explore the regulatory role of H3K4me3 in the NRP1 gene promoter regionThe expression levels of KMT2 B protein and H3K4me3 protein in the two radiation-resistant cell models were detected by WB experiment,and the results showed that the expression levels of KMT2 B and H3K4me3 protein were increased in A549-RR cells.In H1299-RR cells,the KMT2 B protein expression did not change significantly,while the H3K4me3 protein expression decreased.The results of Ch IP-seq and Ch IP-q PCR experimentsshowed that H3K4me3 was also enriched in the NRP1 gene promoter region in two radiation-resistant cell models,H3K4me3 enriched more in A549-RR cells,and H3K4me3 in H1299-RR cells Enrichment decreased.It indicates that H3K4me3 is also involved in the formation of radioresistance in two types of lung adenocarcinoma cells,but the regulatory mechanisms are completely different.7.Explore the effect of GATA3 knockdown on the efficacy of radiotherapy in lung adenocarcinoma and its mechanism in vivoA cell model of A549 and H1299 cells with stable GATA3 knockdown was constructed in vitro.The results of clone formation experiments showed that the radiosensitivity of A549 cells was enhanced after GATA3 knockdown,while the radiosensitivity of H1299 cells changed little after GATA3 knockdown.A549-sh NC cells,A549-sh GATA3 cells,H1299-sh NC cells,and H1299-sh GATA3 cells were transplanted into nude mice subcutaneously for tumor-bearing experiments,and 20 Gy high-dose irradiation was given at the same time.Strikingly,the tumor volume in the A549-sh NC+20 Gy group was smaller than that in the A549-sh NC group,indicating that 20 Gy X-ray irradiation could significantly inhibit tumor growth.The tumor volume in the A549-sh GATA3+20 Gy group was smaller than that in the A549-sh NC+20 Gy group,indicating that knockdown of GATA3 enhanced the effect of X-rays on tumor growth and played a role in radiosensitization.However,20 Gy irradiation had little effect on H1299 tumors,and the tumors in the GATA3 knockdown group and GATA3 knockdown combined irradiation group still showed a downward trend.The results of q RT-PCR,WB and IHC experiments showed that the expressions of GATA3 and NRP1 in the A549 tumor tissues of the four groups gradually decreased.In H1299 cell xenografts,the expression of GATA3 decreased significantly after 20 Gy irradiation,while the expression of NRP1 did not decrease significantly.The expression of NRP1 increased after knockdown of GATA3,and the expression of NRP1 decreased slightly after knockdown combined with irradiation,indicating that knockdown GATA3 combined irradiation had little effect on H1299 tumor growth.The above results indicated that knockdown of GATA3 can increase the radiosensitivity of A549 cells and improve their radiotherapy efficacy,but had less effect on the radiosensitivity of H1299 cells.Conclusions:1.Different cell cycle arrest occurs when two types of non-small cell lung cancer cells develop radioresistance,A549-RR cells have G1 phase arrest,and H1299-RR cells have S phase delay.2.GATA3 and NRP1 were both elevated during the development of radioresistance in two types of lung cancer cells,while H3K4me3 was elevated in A549-RR cells and decreasedin H1299-RR cells.3.The mechanisms by which GATA3 regulates NRP1 are different in the two radioresistance cell models.In A549 and resistant cells,GATA3 is a promoting transcription factor;in H1299 and resistant cells,GATA3 is a suppressor.4.The transcription factor GATA3 binds to the promoter region of NRP1 gene to regulate the expression of NRP1 gene.In A549-RR cells,GATA3 was enriched in the promoter region of the NRP1 gene,which promoted transcription,but decreased in H1299-RR cells,resulting in a weakened inhibitory effect.5.In A549-RR cells,KMT2 B expression increased,which increased the modification of H3K4me3 in the NRP1 gene promoter region and promoted the transcription of NRP1.In H1299-RR cells,KMT2 B and H3K4me3 inhibit NRP1 transcription,and H3K4me3 modification decreases,leading to weakened inhibition,and finally promoting NRP1 transcription.6.Knockdown of GATA3 gene can significantly improve the radiotherapy effect of A549 cell transplanted tumor,but does not affect the radiosensitivity of H1299 cell transplanted tumor.7.The mechanism of radioresistance of two lung adenocarcinoma cells is different.In irradiated A549 cells,transcription factors GATA3 and H3K4me3 both play a positive role in regulating NRP1 and promoting its expression,thereby promoting the formation of radiation resistance.In irradiated H1299 cells,GATA3 and H3K4me3 both negatively regulate NRP1 gene,and the weakening of this negative regulation leads to the increase of NRP1 transcription,thus forming radiation resistance. |
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