Identification Of Gins1 As A Marker Of Non-small Cell Lung Cancer And Study On Its Transcriptional Regulation Mechanism

BACKGROUND:Lung cancer is a malignant tumor with high morbidity and mortality rates,which seriously endangers human health.Non-small cell lung cancer(NSCLC)is the most common form of lung cancer,accounting for 80 to 85%of all cases of lung cancer,the number of its incidence is increasing year by year,and most of them have already developed to the middle and advanced stage when diagnosed,which brings great challenges to the treatment of NSCLC.Therefore,searching for new biomarkers of accurate diagnosis of NSCLC is crucial for its effective treatment.In recent years,gene expression microarray has provided convenience for gene targets screening in NSCLC.However,basically more accurate analysis results can be obtained by cross-platform integration analysis of multiple microarray datasets and then discover more reliable target genes for the prevention and treatment of NSCLC.Finally,we selected GINS Complex Subunit 1(GINS1)as a target gene for following study.GINS1 is a member of the GINS complex,is part of Cdc45-Mcm-GINS(CMG)helicase at DNA replication forks,and plays a pivotal role in DNA replication initiation and elongation.GINS1 is critical for proper DNA replication and maintenance.Recently,there has been an increasing number of studies on GINS1 in malignant tumors.OBJECTIVE:The aim of this study is to screen and verify potential biomarker with diagnostic value for NSCLC,and to research its influence on cell proliferation,and further to explore the regulatory mechanism of its transcription,then to provide theoretical evidence for the diagnosis and treatment of NSCLC.METHODS:(1)Screen differential expressed genes in gene expression profile of NSCLC and verify them.In this study,the inclusion and exclusion criteria of gene expression profile were first developed.The quality of each microarray dataset was evaluated,and the candidate genes that might be therapeutic targets for NSCLC were screened by standard normalization and differential analysis,and then GINS1 was obtained.Meanwhile,the TCGA database was used to verify GINS1 expression and prognosis survival.Then,qPCR and Western blot were used to detect the expression of GINS1 in 73 NSCLC tissues.Finally,We examined the association between GINS1 expression and clinicopathological parameters as well as overall survival in 73 NSCLC patients.(2)The effect of GINS1 on cell proliferation in NSCLC.CCK-8 assay and colony formation assay were carried out to detect the regulatory effect of GINS1 on NSCLC cell proliferation.Then we evaluated the effects of GINS1 on growth of NSCLC in vivo.(3)To investigate the transcriptional regulation mechanisms of GINS 1.The potential the transcriptional regulation mechanisms of GINS1 were comprehensively analyzed based on literature and sequencing data of silenced MALAT1.Firstly,qRT-PCR was used to detect the expression of MALAT1 in 73 NSCLC tissues,and to explor the correlation between MALAT1 and GIN S1 expression in NSCLC,and to determine the relationship of MALAT1 and GINS1 protein expression with NSCLC clinicopathologic parameters.Then,experimental techniques including RNA interference,and plasmid DNA transfection were performed,and the regulation of GINS1 by MALAT1 was detected through qPCR,Western blot and luciferase reporter assay,and the cell proliferation was also detected in vitro and in vivo.Thirdly,the transcription factors through which MALAT1 regulates GINS1 were predicted by bioinformatics analysis,and verified by qPCR,Western blot and luciferase reporter assay.Finally,RIP,RNA pull down,ChIP,EMSA,CO-IP and Western blot experiments were performed to explore the specific molecular mechanism of MALAT1-regulated GINS1 transcription.RESULTS:(1)Screen differential expressed genes in gene expression profile of NSCLC and verify them.We screened 46 potential up-regulated genes that may be therapeutic targets for NSCLC through comprehensive analysis of 6 gene expression profiles,and the candidate gene GINS1 was obtained.Then verified the expression and survival of GINS1 candidate genes using TCGA database,and the results shown that GINS1 overexpression in tumor tissues and predicted poor survival.In addition,GINS1 expression was investigated by qPCR and Western blot in 73 NSCLC,and the results shown that GINS1 overexpression was detected in tumor tissues,and closely related to tumor size and clinical stage,and predicted poor survival.(2)The effect of GINS1 on cell proliferation in NSCLC.To explor the effects of GINS1 on cell proliferation in NSCLC,we downregulated and upregulated GINS1 expression in NSCLC cell lines,and CCK-8 results showed that the cell proliferation ability was significantly decreased in the shGINS1 group and increased in GINS1 overexpression group compared with the control group and the similar results were observed in colony formation assay.In addition,silencing GINS1 significantly inhibited tumor cell proliferation compared with the control group in vivo.(3)To investigate the transcriptional regulation mechanisms of GINS1.Based on the literature and sequencing data analysis,we hypothesized that the mechanism of GINS1 is regulated by upstream MALAT1.First,the relationship between MALAT1 and GINS1 was investigated in NSCLC tissues,and the results showed that MALAT1 and GINS1 were highly expressed and positively correlated in NSCLC tissues,and both MALAT1 and GINS1 overexpression was correlated with clinical stage and tumor size and predicted poor survival.To validate whether MALAT1 regulated GINS1 expression in NSCLC cells,we through qPCR,Western blotting,luciferase assay,CCK-8 assay,colony formation assay and xenograft assay.These results not only showed that knockdown of MALAT1 can reduce GINS1 expression and thus inhibit NSCLC proliferation,but also revealed that upregulation of GINS1 attenuated the loss of GINS1 expression in MALAT1 knockdown NSCLC cells and thus rescued the decreased proliferation abilities induced by MALAT1 knockdown.Next,based on bioinformatics analysis,we speculated that MALAT1 mediates GINS1 transcription by regulating FOXP3,then affecting GINS1 expression.To validate whether FOXP3 regulated GINS1 transcription in NSCLC cells,we used ChIP,EMSA and luciferase assay.These results showed that FOXP3 could bind to the promoter of GINS1 and FOXP3 overexpression could increase the GINS1 promoter activity and final expression of GINS1.To further confirme whether MALAT1 regulates FOXP3,we used qPCR and Western blotting.These results showed that silencing MALAT1 inhibites the protein levels of FOXP3,but couldn’t change the mRNA levels.And silencing MALAT1 cells were treated with CHX and MG132,the Western blot results showed that knockdown of MALAT1 could shorten the half-life of FOXP3 protein and CO-IP assay revealed that silencing MALAT1 could enhance the ubiquitination level of FOXP3.Finally,we through CO-IP,RIP and RNA pull down assay to further explore the specific molecular mechanism of MALAT1 regulating FOXP3 ubiquitination.The results showed that 1)E3 ubiquitin ligase STUB 1-mediated ubiquitination of FOXP3 also occur in NSCLC cells;2)although silencing MALAT1 did not have an effect on STUB1 expression,they did enhance the interaction of STUB1 with FOXP3;3)both MALAT1 and STUB1 interacted with the same ZF and LZ domains of FOXP3,suggesting that MALAT1 functioned as a mediator,which disturbed STUB1-FOXP3 interaction,thereby decreasing STUB 1-mediated ubiquitination and degradation of FOXP3.CONCLUSIONS:(1)MALAT1 and GINS1 were highly expressed and positively correlated in NSCLC tissues,and both MALAT1 and GINS1 overexpression was correlated with clinical stage and tumor size and predicted poor survival.(2)Silencing MALAT1 promotes ubiquitination and degradation of FOXP3 by enhancing the binding between FOXP3 and STUB1,and then suppresses the GINS1 transcription and expression levels,and finally inhibits the proliferation of NSCLC cells.