| BackgroundLung cancer is one of the most common and lethal cancers in the world,and Non-small cell Lung Cancer(NSCLC)is the dominant histopathology.It is found that GATA2 is crucial for survival and growth of those NSCLC cells with mutations in KRAS and other oncogenes on the RTK/RAS pathway,and the deletion of GATA2 reduces survival of KRAS mutant NSCLC cells significantly inhibit the development of NSCLC.In addition,GATA2 can maintain the survival of NSCLC cells by regulating simultaneously proteasome complexes,IL-1/NF-κB and Rho signaling pathways.GATA2 is known to be a member of GATA family,which is a transcription factor that regulates many developmental processes,and the interaction of GATA transcription factors plays an important role in regulating gene expression in GATA family.GATA2binds at GATA2 gene promoter region and activates its own transcription.However,GATA1 can displace GATA2 from this position,and instigates GATA2 repression by means of disruption of its positive autoregulation.Therefore,GATA1-mediated displacement of GATA2 is tightly coupled to repression of GATA2 transcription.c-Myc is constitutively expressed in many types of cancer cells,which combined with the E-boxes sequence in the promoter region,and then activates the transcription of numerous oncogenes involved in abnormal cell proliferation,leading to the formation of malignant tumors.Constitutive up-regulation of c-Myc gene has been discovered in a lot of cancers,such as gastric,cervical,colon and non-small cell lung cancer.Therefore c-Myc has been identified as a potential target for cancer treatment.During recent years,long non-coding RNA(lncRNA)has been found to play an important regulatory role in a variety of tumorigenesis mechanisms including NSCLC.LncRNA can target transcriptional activators or inhibitors to regulate the transcription and expression of downstream genes.One type of lncRNA accumulates in cis after transcription and regulate adjacent gene expression either by recruiting transcription factors or chromatin modifiers.For example,the lncRNA CCND1,which is transcribed near the chromosome of CCND1 gene,can bind to TLS proteins and transform TLS into the active conformation,which can bind to the CBP/p300 complex on the promoter of CCND1 and ultimately inhibit the transcription of CCND1 gene.ObjectiveTo explore the specific molecular mechanism of lncRNA GATA2-AS1 regulating the growth of NSCLC and its targeted regulation by c-myc,so as to provide experimental basis for exploring new therapeutic targets of NSCLC.Materials and methods1.Cell Culture and transfection:A549 cells(KRAS mutant)and HEK293T cells were cultured in DMEM supplemented with 10%Fetal Bovine Serum,1 mm Sodium pyruvate and 100 mg/ml streptomycin.2.RNA interference:After transfection of HEK293T cells into control group or experimental group,the cells were cultured in Opti-MEM for 12 hours and then in DMEM containing 10%Fetal Bovine Serum for 48 hours.The efficiency of knockdown was assessed by Western blotting and quantitative PCR(qPCR).3.Quantitative real-time PCR(qRT-PCR):Total RNA was extracted using Trizol and then reverse-transcribed into cDNA.qPCR was carried out and analyzed by using SYBR and ROX Reference Dye(ROX).4.Western blot(WB)analysis:Harvested cells,added SDS loading buffer solution,boiled protein at 95°C for 10~15 minutes,and separate protein on SDS-page.5.Chromatin immunoprecipitation(ChIP)assay:A549 cells were crosslinked with 1%formaldehyde at room temperature for 10 minutes.Glycine was added to stop the reaction.The treated cells were overhung with a lysis buffer.The protein coupled with anti-GFP or control IgG was incubated with cell lysates.The eluted product was amplified by PCR.6.RNA immunoprecipitation(RIP):1~3×10~7 cells were lysed and centrifuged with hypotonic buffer.The lysate was pretreated with protein A/G beads and incubated at 4°C for 3 hours with A/G beads coupled with the corresponding antibody.After extensive washing,the bead-bound immunocomplexes were eluted using elution buffer.To isolate protein bound RNA from the eluted immunocomplexes,samples were treated with protease K and RNAs were extracted.The purified RNAs were then subjected to RT-PCR analysis.7.Colony formation assay:A549cells transfected withscramble,GATA2 or GATA2-AS1 siRNAs were plated at a density of 500 cells per well on a six-well plate.After one week,cells were fixed with 10%cold methanol for 5 minutes and stained with crystal violet at room temperature for 30 minutes.After extensive wash,the colonies were photographed.8.Cell cycle analysis:The cells were harvested,washed and fixed with 70%ethanol at4°C overnight.Set and centrifuged for 5 minutes.PBS was rinsed twice and incubated at room temperature for 30 minutes with PBS.Finally,the distribution of cell cycle was detected by Flow cytometry.Results1.GATA2-AS1 inhibits the proliferation of NSCLC cellsLncRNA GATA2-AS1 was a non-coding oncosuppressor gene located on the antisense chain of GATA2 gene on chromosome 3 of the human genome.Firstly,we knocked down GATA2-AS1 by siRNA in A549 cells and found mRNA level of GATA2 was up-regulated after GATA2-AS1 silencing,and the level of GATA2 protein was up-regulated in GATA2-AS1 deficient cells.Then the expression of GATA2 targets gene in GATA2-AS1 knock-down cells was examined through proteasome complex,IL-1/NF-κB and Rho signaling pathways.Subsequently,we used siRNA to knock down GATA2-AS1 in A549 cells,and found that the absence of GATA2-AS1 accelerated the proliferation of A549 cells,while the absence of GATA2-AS1 and GATA2 did not.These results demonstrate that GATA2-AS1 represses GATA2 expression and NSCLC cells growth.2.GATA2-AS1 binds to GATA1The subcellular localization of GATA2-AS1 in A549 cells was analyzed by means of nuclear-cytoplasmic separation.Then RIP assay demonstrated that ectopically expressed GATA1 was able to interact with GATA2-AS1,whereas endogenous GATA2-AS1 was capable of co-precipitate with GATA1,indicating that GATA2-AS1 and GATA1 protein physically interact with each other in the nucleus.Then two-step RIP assay was used to analyze whether GATA family coregulator FOG1 is included in the GATA2-AS1 and GATA1 complex.In the first phase IP we used anti-Flag IgG which captured high levels of FOG1 and GATA2-AS1 with Flag-GATA1,and in the second IP we used anti-FOG1IgG co-precipitated Flag-GATA1 and GATA2-AS1 in addition to FOG1.The results show that GATA1,FOG1 and GATA2-AS1 form a trimer at the GATA2 promoter,and then regulate its transcription.3.GATA2-AS1 enhances repressive function of GATA1Further ChIP assay using GATA1 antibody showed that there was a GATA binding sequence located at about 2.8 kb upstream of GATA2 gene,and a lower GATA1 occupancy on GATA2 promoter region was detected in GATA2-AS1 silencing cells.This suggests that GATA2-AS1 enhances GATA1 binding to GATA2 promoter and therefore enhances the repressive function of GATA1 on GATA2 expression.It was also found that suppressive effect of GATA2-AS1 on GATA2 expression was rescued by GATA1silencing,which further confirmed that GATA2-AS1 regulated GATA2 through GATA1.4.GATA2-AS1 inhibits the proliferation of NSCLC cells through GATA2Firstly,GATA2 targets expression in A549 cells with or without in GATA2-AS1 siRNA transfection were examined,and it was found those alterations in GATA2-AS1 silencing cells could be reversed by GATA2 siRNA treatment,suggesting GATA2 indeed mediated the effect of GATA2-AS1 on NSCLC cells growth.Then it was demonstrated that the phenotype of GATA2-AS1 silencing A549 cells could be rescued by GATA2 siRNA,which was further confirmed by cell cycle analysis between GATA2 knock-down,GATA2-AS1 and GATA2 double knock-down cells,illustrated that GATA2-AS1regulates the growth of lung cancer through GATA2.5.c-Myc transcriptionally regulates GATA2-AS1Through bioinformatic analyses,c-Myc response element sequence(CACGTG)was determined at the-1964 site of GATA2-AS1 promoter.c-Myc silencing could induce up-regulation of GATA2-AS1 level in NSCLC cells,and in contrast the level of GATA2-AS1reduced gradually when exogenous c-Myc was transfected into A549 cells.Then anti-c-Myc was used to carry out the ChIP assay,and confirmed c-Myc indeed bound to the region including the c-Myc response element at-1964 of the GATA2-AS1 promoter.Furthermore,MIZ-1was knocked down by siRNA silencing and it was found MIZ-1deficiency was able to rescue the GATA2-AS1 down-regulation induced by c-Myc over-expression,indicating c-Myc regulated transcriptional repression of GATA2-AS1 through interacting with MIZ-1.6.GATA2-AS1 expresses at a lower level in NSCLC tissuesTo further understand the function of GATA2-AS1 in the development of NSCLC,the expression of GATA2-AS1 was examined in clinical NSCLC samples.Analysis of surgically removed NSCLC samples showed that the expression level of GATA2-AS1 in most cancer tissues was lower than that in adjacent tissues.At the same time,these cancer tissures also showed relatively higher expression levels of c-Myc and GATA2.However,there was no obvious change in GATA1 level between cancer tissues and adjacent tissues.These results suggest that GATA2-AS1 could inhibit the proliferation of NSCLC cells.Conclusions1.LncRNA GATA2-AS1 was a non-coding cancer suppressor gene,which was located in the antisense chain of GATA2 gene on chromosome 3 of human genome.Its transcription direction was opposite to GATA2,which could inhibit the occurrence and development of NSCLC cells.2.LncRNA GATA2-AS1 binded to GATA1 protein in the promoter region of GATA2,thereby inhibiting the transcription of GATA2 and ultimately preventing the proliferation of NSCLC cells.In addition,GATA2-AS1 had been shown to inhibit the growth of NSCLC cells by regulating the proteasome complex,IL-1/NF-κB and Rho signaling pathways.3.LncRNA GATA2-AS1 could be regulated by transcription inhibition of c-Myc in NSCLC cells.4.This Study proved the role of c-Myc→GATA2-AS1→GATA1→GATA2 signaling pathway in the development of NSCLC and provide potential targets for the treatment of NSCLC. |
