The Mechanism Of Complement C5a-induced Up-regulated KLF5/GCN5/GDF15 To Promote NSCLC Cell Proliferation

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The plasma C5a concentration and the expression of proliferation related gene in NSCLC patients and the correlation of KLF5,GCN5,GDF15 or C5aR expression with the clinic-pathological parameters of the NSCLC patientsObjective:To detect the C5a concentration in the plasma of NSCLC patients or healthy volunteers,and the gene expression profile change in fresh NSCLC tissues compared with corresponding adjacent tissues.Meanwhile,to examine the expression of transcription factor KLF5,transcription coactivator GCN5,growth differentiation factor GDF15 or C5a receptor(C5aR),and to analyze the correlation between KLF5,GCN5,GDF15 or C5aR expression in 185 NSCLC tissues and clinic-pathological parameters of these NSCLC patients.Methods:The plasma C5a concentration of NSCLC patients and healthy donors was detected by ELISA.The fresh or paraffin embedded NSCLC tissues andadjacent tissue samples with the corresponding clinic-pathological information were collected.Thereafter,high throughput transcriptome sequencing(RNA-seq)was used to screen the differentially-expressed genes,and the expression of selected genes was validated by RT-qPCR in 40 NSCLC samples.Besides,the expression of KLF5,GCN5,GDF15 and C5aR in paired tissues of 185 NSCLC patients was assessed using immunocytochemistry(IHC)staining,and the correlation between these genes mentioned-above and the clinic-pathological features of NSCLC patients was analyzed.Results:(1)The C5a concentration in the plasma samples of 40 NSCLC patients was markedly higher than that of 40 healthy people.(2)The RNA-seq showed that a total of 86 genes were upregulated(?2 fold)in the NSCLC tissues compared with the adjacent tissues,and by GO analysis,17.4%of these genes were related to cell proliferation or survival.(3)The mRNA levels of some proliferation related genes(i.e.KLF5,HMGA1,FOXM1,EHF,HMGB3,SOX4,SOX9,GCN5,GDF15,MDK,TDGF1 and cyclin D1)in the 40 cases of NSCLC tissues were significantly higher than the corresponding adjacent tissues,especially KLF5,GCN5 and GDF15,which were also positively correlated with each other.(4)The KLF5,GCN5 and GDF15 protein expression in 185 NSCLC tissues was remarkably increased in comparison with the paired adjacent tissues,and was closely connected with the tumor size,lymph node metastasis and TNM stage.(5)The C5aR protein expression was higher in 185 NSCLC tissues than in the adjacent tissues,which was also associated with the above-mentioned clinic-pathological features.Conclusion:(1)The upregulated plasma C5a concentration suggests the activation of complement system and the present of C5a in the tumor microenvironment of NSCLC patients.(2)There was a remarkable difference of the gene expression profile between the NSCLC tissues and adjacent normal tissues,and the expression of many proliferation related genes was significantly increased,especially KLF5,GCN5 and GDF15,which were also positively correlated with each other.(3)The upregulated KLF5,GCN5 and GDF15 as well as C5aR in NSCLC tissues were associated with the tumor size,lymph node metastasis and TNM stage,indicating that these genes have a certain link with the occurrence and development of NSCLC.Part ? The role of C5a stimulation in upregulating A549 cell proliferation and KLF5,GCN5 and GDF15 expression as well as the molecular mechanism of KLF5 and GCN5 promoting cell proliferation and GDF15 gene transcription and expressionObjective:To explore the role of C5a in triggering NSCLC cell proliferation and the expression of KLF5,GCN5 and GDF15 genes as well as the effect of C5a-induced KLF5,GCN5 and GDF15 on NSCLC cell proliferation and GDF15 gene transcription and expression with the underlying molecular mechanism.Methods:(1)Firstly,the C5aR expression in six NSCLC cell lines was detected using immunoblotting(IB).Then,the proliferation of A549 cells and the protein expression of KLF5,GCN5 and GDF15 upon C5a stimulation for different time were assessed by CCK8,cell counting and colony formation and IB assays respectively.Besides,the proliferation and fore-mentioned proteins expression in A549 cells treated with C5aR antagonist or C5a neutralizing antibody before C5a stimulation were also examined by using the same methods.(2)The expression plasmids(pIRES2-KLF5,pIRES2-GCN5 and pIRES2-GDF15)and short hairpin RNA plasmids(shKLF5,shGCN5 and shGDF15)were constructed and transfected into A549 cells(with C5a stimulation if shRNAs were transfected),and the cell proliferation and KLF5,GCN5 or GDF15 expression were measured subsequently.Furthermore,the upstream and downstream relationship between KLF5,GCN5 and GDF15 expression was determined using IB.(3)The potential response elements of KLF5 binding to GDF15 promoter(RE1-RE5)were predicted by online software JASPAR,and then the full length(FL)as well as truncated GDF15 promoter reporter plasmids were constructed and transfected into A549 cells.By luciferase reporter assay,the GDF15 promoter activity in A549 cells stimulated with C5a or transfected with KLF5 overexpression or shRNA plasmids was examined,and the approximate KLF5 binding site on GDF15 promoter was determined.(4)The exact site of KLF5 and GCN5 binding to GDF15 promoter was identified by ChIP and Re-ChIP assays.Results:(1)The expression of C5aR was found in six NSCLC cell lines,significantly in A549 and PC9 cell lines.C5a stimulation could not only promote the proliferation of A549 and PC9 cells(especially A549 cells),but also enhance the expression of KLF5,GCN5 and GDF15 in A549 cells exposed to C5a for 3h.(2)The C5a-induced cell proliferation and KLF5,GCN5 or GDF15 expression were notably reversed by C5aR blocking or C5a neutralizing.(3)The overexpression of KLF5,GCN5 and GDF15 could boost the proliferation of A549 cells,while silencing KLF5,GCN5 and GDF15 genes could suppress the cell proliferation induced by C5a.(4)The expression of GDF15 increased or decreased when KLF5 or GCN5 was overexpressed or silenced respectively,but the change of GDF15 expression could not affect the protein levels of KLF5 or GCN5 in A549 cells.(5)The activity of GDF15 promoter was significantly augmented or inhibited in A549 cells upon C5a stimulation or KLF5 gene overexpression or knockdown separately.Moreover,the KLF5 response elements were located in the region of-401 to-55 nt on GDF15 promoter,which contained the RE4 and RE5.(6)The KLF5 binding to the-103 to+58 nt region(containing RE5)on GDF15 promoter was revealed by ChIP and Re-ChIP assay.(7)The GCN5 and KLF5 could form a complex and bind to the above-mentioned region on GDF15 promoter in a KLF5 dependent manner.Conclusion:C5a could induce the A549 cell proliferation via C5aR,enhancing the expression of KLF5 and GCN5,and further promoting the transcription and expression of GDF15 gene through the KLF5-GCN5 complex binding to GDF15 promoter.Part ? The effect of the GCN5-KLF5 binding and the acetylated KLF5 on C5a-induced A549 cell proliferation as well as GDF15 gene activation,transcription and expressionObjective:To explore the effect of KLF5 acetylation by GCN5 on the proliferation and GDF15 gene transcription and expression of the C5a-stimulated A549 cells.Meantime,to determine the acetylation site of KLF5 by GCN5,and to clarify the role of GCN5 HAT domain mutation or KLF5 acetylation site mutation in A549 cell proliferation and GDF15 gene expression in response to C5a.Methods:(1)Immunoprecipitation(IP)and IB were used to assess the binding of KLF5 and GCN5 protein as well as KLF5 acetylation level in A549 cells treated with C5a or in the cells pretreated with C5aR antagonist or C5a neutralizing antibody before C5a stimulation.(2)By using mass spectrometric technique,the KLF5 acetylation sites modified by GCN5 were analyzed.(3)Using IP and IB assays,the KLF5 acetylation level was detected when the A549 cells were transfected with HAT domain deleted GCN5 expression plasmid(?GCN5)or KLF5 acetylation site mutation plasmids(KLF5-335R,KLF5-391R and KLF5-335R/391R).(4)ChIP,Re-ChIP and ChID assays were performed to identify the binding level of acetylated KLF5 modified by GCN5 on GDF15 gene promoter.(5)Luciferase reporter assay,qPCR and IB were used to examine GDF15 promoter activity as well as GDF15 mRNA and protein level in A549 cells transfected with above-mentioned mutation plasmids of KLF5 and GCN5.Meanwhile,the proliferation of cells with the same treatments was also measured by CCK8,cell counting and colony formation assays.Results:(1)The binding of KLF5 and GCN5 as well as the acetylation of KLF5 were markedly upregulated in the A549 cells after C5a incubation for 2h,and peaked at 3h.However,this phenomenon was reversed when C5aR was blocked or C5a was neutralized.(2)The binding of KLF5 and GCN5 as well as the acetylation level of KLF5 in A549 cells were remarkably increased or decreased when GCN5 was overexpressed or silenced.(3)Compared with wide type GCN5(GCN5-WT),the KLF5 acetylation was significantly reduced in A549 cells transfected with ?GCN5,and the acetylation sites of KLF5 by GCN5 were K335 and K391.(4)The C5a-induced acetylation level of KLF5 in the A549 cells transfected with mutation plasmids i.e.KLF5-K335R,KLF5-K391R and KLF5-K335R/K391R could be obviously downregulated.Similarly,in the A549 cells co-transfected with ?GCN5 and KLF5-WT or GCN5-WT and KLF5-K335R/K391R or ?GCN5 and KLF5-K335R/K391R,marked reduction of KLF5 acetylation level were found in comparison with the cells transfected with GCN5-WT+KLF5-WT plasmids.(5)The acetylated KLF5 could increase the binding of itself to GDF15 promoter as well as the transcription and expression of GDF15.(6)The deletion of HAT domain in GCN5 protein and the mutation of KLF5 acetylation sites could remarkably suppress A549 cell proliferation as well as GDF15 gene transcription and expression.Conclusion:In the process of C5a-induced A549 cell proliferation,the transcription co-activator GCN5,which has HAT activity,can bind to and acetylate the transcription factor KLF5,and the acetylated KLF5 could greatly activate GDF15 promoter,elevating the transcription and expression of GDF15 gene and prompting the proliferation of A549 cells.Part ? The effect of silencing KLF5,GCN5 or GDF15 in A549 cells on the growth of and the proliferation related protein expression in the A549-formed nude mice xenograft tumorsObjective:To in vivo investigate the impact of KLF5,GCN5 and GDF15 knockdown in A549 cells on the growth of nude mice subcutaneous xenograft tumors,and to explore the changes of KLF5,GCN5 and GDF15 expression and the KLF5 acetylation by GCN5 as well as the expression of proliferation related proteins(i.e.cyclin D1,PCNA and Ki67)in above-mentioned xenograft tumors.Methods:(1)The shRNA vectors(LV-shKLF5,LV-shGCN5,LV-shGDF15 and LV-shCTR)were packaged into lentivirus.A549 cells were stably transfected with the above-mentioned vectors,and then the corresponding protein expression was detected using IB.(2)The A549 cells with the stable transfection of LV-shKLF5,LV-shGCN5,LV-shGDF15 or LV-shCTR were subcutaneously inoculated into nude mice to establish the subcutaneous lung adenocarcinoma models(N=8),and the formation and growth of the graft tumors were monitored at indicated time.(3)On the 28th day after inoculation,nude mice were sacrificed,and the expression of KLF5,GCN5 and GDF15 in the graft tumors was examined using IB,and meanwhile,the acetylation level of KLF5 in the graft tumors beween LV-shGCN5 and LV-shCTR groups was identified by IP and IB.(4)The morphologic changes and expression of proliferation related proteins(cyclin D1,PCNA and Ki67)of the graft tumors in four group mice were observed by HE staining or IHC staining,respectively.Results:(1)The expression of corresponding protein expression was markedly decreased in the A549 cells stably transfected with LV-shKLF5,LV-shGCN5,LV-shGDF15 or LV-shCTR vectors.(2)The volume,size,and weight of xenograft tumors in the LV-shKLF5,LV-shGCN5 and LV-shGDF15 groups were notably reduced compared with the LV-shCTR group.(3)The expression levels of KLF5,GCN5 and GDF15 proteins were significantly lessened of the graft tumors in LV-shKLF5,LV-shGCN5 and LV-shGDF15 groups,and the GDF15 expression was remarkably decreased in LV-shKLF5 and LV-shGCN5 groups.Moreover,the KLF5 acetylation level of the graft tumors in LV-shGCN5 group was also lower than that in LV-shCTR group.(4)No morphologic change was found among the graft tumors of the fore-mentioned four groups,but the expression level of cyclin D1,PCNA and Ki67 was notably downregulated in LV-shKLF5,LV-shGCN5 and LV-shGDF15 groups in comparison with LV-shCTR group.Conclusion:The silence of KLF5,GCN5 and GDF15 genes in A549 cells could impede the growth and proliferation of xenograft tumors in nude mice,suggesting that the expression of KLF5,GCN5 and GDF15 has a promoting role in NSCLC cell proliferation in vivo.In this process,the GDF15 expression,as a downstream gene,is indeed regulated by KLF5 and GCN5,and GCN5 could acetylate KLF5,finally enhancing the transcription and expression of GDF15 gene.