Regulation And Molecular Mechanism Of Histone Acetyltransferase P300 On GINS1 Gene Expression In Lung Cancer Cells

Lung cancer is the most common tumor in the word,which can be divided into small cell lung cancer(SCLC)and non-small cell lung cancer(non-small cell lung cancer)according to the different ways of growth and spread.Current treatment options include symptomatic therapy,chemotherapy,radiotherapy,surgery,as well as newly developed immunotherapy.The above anti-tumor treatment regimens have indeed brought benefits to patients,but the therapeutic effect for patients is still limited.Therefore,to explore the molecular mechanism of lung cancer,and to find specific targets for diagnosis and treatment have received increasing attention.Based on the existing sequencing data of lung cancer,the target gene GINS1 which was abnormally up-regulated in lung cancer tissues was selected,and found that the transcription factor Foxp3 regulates the expression of GINS1.GINS1,also known as PSF1,and GINS2 GINS3 GINS4 together constitute GINS complex.During the initiation of DNA replication in eukaryotes,the GINS complex,along with CDC45(cell division cyclin 45)and MCM2-7(minichromosome maintenance proteins2-7),forms the CMG,which is involved in the initiation and extension of DNA replication.As a key member of DNA replication helicase,GINS protein complex is a necessary factor for rapid cell proliferation,which is closely related to the proliferation of malignant tumor cells.GINS1 in the GINS complex is the most critical subunit,and is involved in the development and progression of lung cancer,colorectal cancer and many other cancers.These data fully confirm the importance of GINS1 in tumor growth,suggesting that GINS1 is a potential target for tumor therapy.The regulation of acetylation in vivo is in a state of dynamic equilibrium under the action of histone acetyltransferases and deacetylases.Acetylated modification can make chromatin structure is looser,forming an open chromatin environment,thus promoting gene transcription.Studies have reported that acetylated modification can not only happen on the histones,can also occur on the transcription factor.Acetyltransferase can enhance the acetylation level of transcription factors,is conducive to the downstream target genes of transcription.In this study,we investigated the effect of common acetyltransferase p300 on GINS1 expression regulation and the specific molecular mechanism of p300 regulating GINS1.Objectives:To explore the mechanism of histone acetyltransferase p300 regulating GINS1 in lung cancer cells.In addition,the regulation effect of p300 on GINS1 gene transcription and the effect on the proliferation of lung cancer cells were investigated.Methods:1.In H1299 and A549 cell lines,the effect of overexpressed Foxp3 on GINS1 expression was detected by Western Blot assay to determine the regulatory effect of transcription factor Foxp3 on GINS1.2.Immunoprecipitation technology was used to detect whether p300 and Foxp3 were combined with each other in lung cancer cell line A549.Truncated Foxp3 and p300 were designed and constructed,and transfected into lung cancer cell line A549 for expression by transient transfection method.The immunoprecipitation technique was used to detect the specific domain of p300 binding in Foxp3.3.In lung cancer cell lines H1299 and A549,we tested whether p300 regulates the acetylation of Foxp3.The changes in the acetylation level of Foxp3 were detected after the co-transfection of p CMVβ-p300-myc and Foxp3 plasmid with Flag tags or only the transfection of Foxp3 plasmid with Flag tags.4.In lung cancer cell lines H1299 and A549,after treatment with deacetylase inhibitor TSA and acetylase inhibitor SGC-CBP30,the effect of GINS1 promoter activity was detected.5.In vitro,si RNA for silencing p300 and p CMVβ-p300-myc for overexpressing p300 were designed to detect the effects of knockdown and overexpression of p300 on GINS1 promoter activity by transient transfection.6.In lung cancer cell lines H1299 and A549,the changes of GINS1 m RNA and protein levels in lung cancer cells with P300 si RNA knockdown and p300 overexpression were detected by PCR and Western Blot.7.CCK8 and plate clone-formation experiments were used to detect the effects of knockdown and overexpression of p300 on the proliferation ability of lung cancer cell lines H1299 and A549.Results:1.In H1299 and A549 cell lines,the expression of GINS1 was increased after overexpression of Foxp3 compared with the control group,indicating that the transcription factor Foxp3 regulates GINS1.2.In lung cancer cell line A549,p300 binds to Foxp3,and Foxp3’s leucine zipper domain and zinc finger domain bind to p300.3.In lung cancer cell lines A549 and H1299,increased levels of acetylation of Foxp3 were found after overexpression of p300.4.In lung cancer cell lines A549 and H1299,GINS1 promoter activity increased after treatment with the deacetylase inhibitor TSA.The activity of GINS1 promoter decreased after treatment with acetylase inhibitor SGC-CBP30.5.GINS1 promoter activity decreased after p300 knockdown in lung cancer cell lines A549 and H1299.After the overexpression of p300,the activity of GINS1 promoter was increased.6.In lung cancer cell lines A549 and H1299,GINS1 m RNA and protein levels decreased after p300 knockdown.Overexpression of p300,GINS1 m RNA levels and protein increased.7.In the lung cancer cell lines A549 and H1299,CCK8 results showed that the proliferation ability of knockdown p300 lung cancer cells was decreased,while proliferation ability of overexpression p300 lung cancer cells was enhanced.The results of plate clone-formation experiments showed that knockdown of p300 lung cancer cells decreased the clone formation ability,and the results of plate clone-formation experiments showed that overexpression of p300 lung cancer cells increased the clone formation ability.After knockdown of p300 and overexpression of GINS1,the proliferation ability and plate clone formation ability of A549 cells and H1299 cells increased,suggesting that the effect of p300 on cell proliferation was realized through GINS1.Overexpression of p300 and knockdown of GINS1 showed that the proliferation ability and plate clone formation ability of A549 cells and H1299 cells were decreased to the same level as that of the untreated control group,which also proved that the effect of p300 on cell proliferation was realized through GINS1.Conclusions:Previous studies in our lab have confirmed the correlation between GINS1 and lung cancer,and found that GINS1 was highly expressed in lung cancer tissues compared with paracancerous tissues.The previous studies in our lab also found that transcription factor Foxp3 had regulatory effect on GINS1.Firstly,in order to determine the effect of Foxp3 on the expression level of GINS1,the protein expression level of GINS1 was found to increase after the overexpression of Foxp3 in H1299 and A549 cells,indicating that Foxp3 promoted the expression of GINS1.Previous studies have confirmed that Foxp3 and p300 bind to each other in Treg cells.Our study found that Foxp3 and p300 also bind to each other in lung cancer cells,and confirmed that the leucine zipper structure and zinc finger structure of Foxp3 bind to p300.Then,we investigated the effect of knockdown of p300 on the acetylation level of Foxp3.The experimental results showed that the acetylation level of Foxp3 increased after the overexpression of p300.The above results indicated that Foxp3 could regulate the transcription of GINS1,and p300 could bind to Foxp3 and promote the acetylation of Foxp3。Next,we investigated whether p300 could regulate the expression of GINS1 by regulating the acetylation of Foxp3.First,to determine whether acetylation regulates GINS1 transcription,we treated lung cancer cells with deacetylase inhibitor TSA and acetylase inhibitor SGC-CBP30.The results showed that GINS1 promoter activity increased after TSA treatment.GINS1 promoter activity decreased after SGC-CBP30 treatment,indicating that GINS1 promoter activity was regulated by acetylation.In order to further determine whether acetyltransferase p300 regulates GINS1,we constructed p300 small interfering RNA and p300 overexpressing plasmid.The results showed that the activity of GINS1 promoter decreased after p300 knockdown.The activity of GINS1 promoter increased after overexpression of p300,indicating that p300 could regulate the activity of GINS1 promoter.In order to further detect the influence of p300 on the expression levels of GINS1 m RNA and protein,we conducted q RCR and Western Blot experiments,and the results showed that:knockdown p300,m RNA and protein levels of GINS1 were decreased.Overexpression of p300 increased GINS1 m RNA and protein levels,indicating that p300 could enhance the expression of GINS1 m RNA and protein.Since GINS1 plays an important role in the initiation and extension of cell replication and is closely related to cell proliferation,we speculated that p300 could affect the proliferation ability of lung cancer cells by affecting the expression of GINS1.CCK-8 and plate cloning results showed that the proliferation ability of lung cancer cells decreased after p300 knockdown,while the proliferation ability of lung cancer cells increased after p300 knockdown and GINS1 overexpression.The proliferation ability of lung cancer cells increased after the overexpression of p300,and the proliferation level after the overexpression of p300 and the knockdown of GINS1 was consistent with that of the untreated control group,indicating that p300 could affect the proliferation ability of lung cancer cells by affecting the expression of GINS1.In summary,we found that p300 combined with the leucine zipper structure and zinc finger structure of transcription factor Foxp3 to regulate the acetylation level of Foxp3,and then regulate the expression of GINS1.We also found that p300 can further enhance the proliferation ability of lung cancer cells by regulating the expression of GINS1.