| Several previous reports have demonstrated that microRNAs affect gene expression through interaction between their seed region and the 3'-untranslated region of the target mRNA,resulting in post-transcriptional regulation by classical pathway.However,a dual mechanism of action,microRNA-744would affect both transcription andtranslation,have been proved recently.The aim of this study was to identify that microRNA-744 targets c-FOS,a protumorigenic gene,by directly interacting with itspromoter and thereby regulating malignancy in lung cancer.The potential mechanism from the aspects of the gene-chip analysis,database mining methods,cellular level,protein levels,animal experiments,clinical datawere described.MicroRNA-744 overexpression promotes proliferation,migration and invasion of lung cancer A549,SPC-A-1,H1299 and H520 cells,and the high expression were unfavorable for survival.Compared transfected antagomir-744 group with transfected antagomir-NC group,the IC50 of paclitaxel monotherapy was 4.104 vs 5.191 nmol/Lon lung cancer cells A549,P<0.05,and lung cancer cells H520 shows the same trend.Silence of microRNA-744 enhanced radiosensitivity of A549 andH520 cells as evidenced by reduced ability to form colonies after radiation exposure.The binding sites between microRNA-744 and c-FOS promoter were demonstrated by luciferase reporter geneassays.Furthermore,MAPK signaling pathways was activatedwith overexpressed microRNA-744.This newly identifid miR-744/c-FOS pathway providesfurther insight into the progression and metastasis of lung cancer,while theseobservations suggest that recognition of gene promoters by miRNAs may be a natural and generalmechanism for regulating gene transcription.INTRODUCTIONLung cancer is the leading cause of cancer mortality across the world.Despite progression in surgery,radiotherapy,chemotherapy,survival improvement is still a big challenge in lung cancer[1].The biotherapy of lung cancer developed quickly,especially the molecule targeted therapy,which had achieved graveness progression and the curative effect was striking[2].Biochemotherapy had possessed increasingly considerable position in the therapy of lung cancer as a firenew treatment model[3].Various molecular targets have been reported,but only a few have demonstrated the clinical effect.Some ongoing studies focus on EGFR-TKI alone or combination chemotherapy as first line therapy in benefit population[4].Some of noel molecular targets may be much better than existing ones,so we still need further validation.MicroRNA is abnormal expression in most tumor cells,and it is an ideal target for cancer molecular targeting therapy[5].MicroRNAs(miRNAs)are short(17-25 nucleotides)non-codingRNA molecules.They can silence gene expression by binding to 3'untranslated region(3'UTR)sequences in their target messengerRNAs(mRNAs),resulting in the inhibition of translation or mRNAdegradation[6].Recently,several cases in which miRNAs target 5'UTRs,codingregions,promoters,or sequences downstream ofgene termini to silence gene expression have also beenreported,raising the possibility of multiple modes ofaction for miRNAs[7-9].Furthermore,there is more or less molecular interaction between the non-coding RNAs[10].Intensive research in past two decades has uncovered the presence and importance of noncoding RNAs(ncRNAs),which includes microRNAs(miRs)and long ncRNAs(lncRNAs).These two classes of ncRNAs interact to a certain extent,as some IncRNAs bind to miRs to sequester them.Such lncRNAs are collectively called 'competing endogenous RNAs' or 'miRNA sponges'[11].Consequently,microRNA will be one of the most active research topics again for several reasons,as its potential applications inclinical treatmentand a richer persistence mechanism.In previous miRNA microarray analysis,wedemonstrated for the first time that the expression ofmiR-744 was decreased signifiantly by metastasisassociated gene 1(MTA1)knockdown[12].Meanwhile,we also ver:ifid theoncogenicpotential of miR-744in nasopharyngeal carcinoma tumorigenesis and metastasis[13].The Fos gene family consists of 4 members:FOS,FOSB,FOSL1,and FOSL2.These genes encode leucine zipper proteins that can dimerize with proteins of the JUN family,thereby forming the transcription factor complex AP-1[14].As such,the FOS proteins have been implicated as regulators of cell proliferation,differentiation,and transformation.In some cases,expression of the FOS gene has also been associated with apoptotic cell death[15].It is defined as an oncogene,which plays a role in the lung cancer progression.However,the potential mechanism of c-FOS in human lung cancer cell invasion is obscure.In this study,microRNA-744 overexpression promotes proliferation,migration and invasion of lung cancer A549,SPC-A-1,H1299 and H520 cells,and the high expression were unfavorable for survival.Furthermore,we identified that miR-744 directly bindsto c-FOSpromoter region,resulting in elevated c-FOS expression aswell as increased migration and invasion of lung cancer cells.Our data first demonstrated the regulatorymechanism ofoncogene c-FOS transcription by miR-744 anduncovered a potential mechanism underlying the upregulation of c-FOS in lung cancer.MATERIALS AND METHODSTissue sampleFifteen pairs of primary lung cancer tissues and their corresponding pericarcinous tissuesamples were included.All samples were immediately placedin liquid nitrogen and stored at-80?.Both pericarcinous tissue and tumor tissueswere verified by histological analysis.Database mining methodsThe Cancer Genome Atlas(TCGA)Data Portal provides a platform for researchers to search,download,and analyze data sets generated by TCGA(http://cancergenome.nih.gov/)[16].Cell lines and cell cultureThe A549 and H520 cells were purchased from American Type Culture Collection(ATCC,America),andthe SPC-A-1,H1299 cells were purchased fromShanghai Cell Bank of Chinese Academy of Science(Shanghai,China).All cultured in RPMI-1640 medium(Corning,NY,USA)supplemented with 10%fetal bovine serum(FBS,GIBCO,SouthAmerica,NY,USA).All these cells were maintained with 5%C02atmosphere at 37?.Stable transfectionThe precursor sequence of miR744(MI0005559)was synthesized,annealed and then inserted into theAgeI/EcoR1 site of GV209 vector(GeneChem,Shanghai,China)to construct a vector expressing miR744,namedLV-miR744.The GFP vector was used for control.PLV-Luci-control was still constructed byGeneChem(Shanghai,China).A549 and H520 cells were performed co-transfection of LV-miR744 with pLV-Luci-control or control vector with pLV-Luci-control.Plasmid and oligonucleotide constructionAntagomiR-744,antagomir negativecontrol(antagoNC),AgomiR-744,agomir negativecontrol(agoNC)weredesigned and synthesized by RiboBio(Guangzhou,China).Si-c-FOS and si-control weredesigned and synthesized by Genepharma(Shanghai,China).Transient transfectionThe A549,SPC-A-1,H1299 and H520 cells were seeded in 6-wellplates at 30-50%density.Transient transfection was performed withLipofectamine 3000 reagents according to the manufacturer's instructions(Invitrogen,USA).For all the experiments,cells were collected at 24-48h after transfection.RNA extraction and quantitative real-time PCR(qRT-PCR)analysesTotal RNA was extracted from cell lines by using TRIzol(?)Reagent(Invitrogen,CA,USA)according to the manufacturer's instruction.Forthe detection of mature miR-744,total RNA was reverse transcribed intocDNA and then used to perform qRT-PCR by using SYBR(?)PrimeScripTM miRNA RT-PCR Kit(TaKaRa,Dalian,China).For the detection ofc-FOS mRNA,total RNA was reverse transcribed into cDNA by usingPrimeScriptTM RT reagent Kit(TaKaRa,Dalian,China).The quantitativereal time PCR for c-FOS mRNA was performed on Mx3005P real-timePCR instrument(Stratagene,USA)by using SYBR(?)Premix Ex TaqTM(TaKaRa,Dalian,China).Small nuclear RNA U6(U6 snRNA)orGlyceraldehyde-3-phosphate dehydrogenase(GAPDH)was used as aninternal control for miRNA or mRNA quantification respectively.Thesequences of the primers used for qRT-PCR were listed in SupplementaryTable 1.Western BlottingEqual amounts of proteins were separated by 10%SDS-PAGE gels andblotted onto nitrocellulose membranes.The blots were incubated withprimary antibodies against GRB2,Rafl,ERK1/2,c-FOS(1:2000;Abcam,Cambridge,USA)and GAPDH(1:2000;Santa Cruz Biotechnology,CA,USA)at 4? overnight.The membranes were incubated for 2h with horseradishperoxidase-conjugated goat anti-mouse secondary antibody(1:5000;Santa Cruz Biotechnology,CA,USA)at room temperature.Promoter reporter construction and luciferase assaysThe pGL3 vectors containing wild type or mutant putative miR-744 binding site in human c-FOS promoter were synthesized by HuaAnPingKang Co.,Ltd(Shenzhen,China).The sequences of these wild type or mutant promoterconstructs were listed in Supplementary Table 2-5.All the constructs wereconfirmed by DNA sequencing(HuaAnPingKang Co.,Ltd.,Shenzhen,China).Thecells were plated onto24-well plates at a density of 5×103 cells/well ahead of transfection study.A mixture of 150 ng c-FOS promoter constructs(Luc-C-FOS-A,Luc-C-FOS-B,Luc-C-FOS-C,Luc-C-FOSmut-1,Luc-C-FOSmut-2,Luc-C-FOSmut-3,Luc-C-FOSmut-4)or controlvector,100 nmol AgomiR-744,agomir negativecontrol(agoNC)and 5 ngpRL-TK vector(Promega,WI,USA)were co-transfectedinto A549,SPC-A-1,H1299 and H520 cells per well using Lipofectamine 3000 and incubated for 48 hours.Dual luciferase reporter assay system(Promega,WI,USA)was used to perform the luciferase assay according to themanufacturer's instruction.EdU assayAfter AntagomiR-744,antagomir negativecontrol(antagoNC),AgomiR-744,agomir negativecontrol(agoNC),si-c-FOS and si-control treatmentfor 48 h,cells were used to measure DNA synthesiswith a Cell-LightTM EdU imaging detecting kit(RiboBio,Guangzhou,China)according to the manufacturer' sinstructions.Detect IC50 using CCK8 cell assayAfter transfection,the cells were seeded in 96-well plates according to 5000/well.After the cells were stuck to the cell walls,Paclitaxel was added into each wells.Paclitaxel was added to each wells according to the following concentration gradients(0-16 nmol/L or 0-32 nmol/L)and the cells were cultured for 78 h.After that,10 ?l CCK8 was added into each well and completely shook and put into the incubator.2 h later,96 well plate was put into a pre-heated micro-plate reader.The absorbance values(OD)was measured at a wavelength of 450 nm in each well.Clonogenic assaysTo investigate the radiation effect in vitro,200-10000 cells/well were seeded onto 6-well plate and then radiated with different doses(0,2,4,6 and 8 Gy)with a 6MV X-ray source at 2 Gy/min.After incubated in RPMI-1640 medium with 20%FBS treatment at 37?,5%C02 for 10 days,cells were fixed and stained with 0.5%crystal violet in 20%methanol.Colony numbers were counted,and plating efficiency and survival fraction were calculated afterwards.Survival fraction curves were determined with a linear-quadratic model[S = exp(-?D-?D2)]using GraphPad Prism 5.0(GraphPad Prism,San Diego,CA,USA).Cell migration and invasion assaysCell invasion assay was assessed by using transwell chamber(Corning,NY,USA)in 24-well plate.Each group of cells(5-10x104 cells/100 ?l)wasresuspended in serum-free medium and seeded onto the upper chamberwith 12.5%matrigel-coated membrane(BD Bioscience,San Jose,CA)at 48h post-transfection,while the lower chamber was filled with 500?l fresh complete medium.After incubated for 18-24h at 37? with 5%C02,non-invading cells were removed from the upper surface of the filter byscraping with a cotton swab.Invading cells that adhered to the lowersurface of the chambers were fixed in methyl alcohol and stained withhematoxylin.The invading cells were manually counted at 200xmagnification in three random fields by using inverted microscope.Similar inserts without matrigel were used to perform the migrationassay.Wound healing assayWhen cells were grown to approximately 90%confluency(after 48h oftransfection),an artificial wound was created with a 10 ?l pipette tip.Thecells were then cultured in fresh medium without FBS.To visualize wound healing,images were taken at Oh and 36h.The relative percentage of woundhealed was calculated as(the width of wound at Oh-the width of woundat 36h)/the width of wound at Oh.In vivo tumorigenicity assayThe experimental protocols were approved by the Animal ResearchEthics Committee of NanfangHospital and we complied with the rules of the Specific pathogen Free(SPF)animal laboratory of the Nanfang Medcal University.Four-to-six-week-old male athymic BALB/c nu/mice were purchased from the Guangdong MedicalLaboratory Animal Center(GDMLAC,Foshan,China).5-8F cells(4 x 106)stably overexpressing miR-744 or control and antagomir-744 or antagomir-744-NC were injectedsubcutaneously into the dorsal flnk of mice(n = 5 pergroup).After 8 days of implantation of tumor cells,tumor size was measured every 3 days and tumorvolumes were calculated with the following formula:V =(L x W2)/2,V,volume(mm3);L,biggest diameter(mm);W,smallest diameter(mm).At the end of experiments,the mice were sacrified and tumors were dissected andweighed.In vivo metastasis assayCancer cells(2×106)stably stably overexpressing miR-744 or negative control were injected into the tail veins of immunodeficient mice(n = 5).After 30 days,the lung colonization capacity of the cancer cells was analyzed based on the normalized photon flux.Thenthe mice were killed and the lung tissues weredissected and fied in 4%formaldehyde overnight.Thefied samples were embedded in paraffi and stainedwith hematoxylin and eosin.Lung metastasis index wascalculated.Statistical analysisData are expressed as the mean ± standard error of triplicates.Eachexperiment was repeated at least three times.Statistical analyses wereperformed using SPSS 13.0(SPSS Inc.,Chicago,USA).The student'st-test was used to assess differences among groups in in vitro studies.Spearman's correlationwas used to analyze the relationship between miR-744and c-FOS mRNA expression.Inall cases,P<0.05 was considered to be statistically significant,and*p<0.05,**p<0.01,***p<0.001.RESULTSmiR-744 is upregulated in lung cancer and associated with overall survivalLevel 3RNA-seq V2 and miRNA-seq isoform quantification data were obtained from the TCGA data of lung cancer.The samples were divided into 2 groups,high-expression group(n=237)and low-expression group(n=219),according to the most optimal cut-off value of miR-744 estimated by the ROC curve.MiR-744were well-associated with OS in lung cancer for high-expression vs low-expression(p=0.0016;Fig.1A).Patientcharacteristics are summarized in Table 1.Further analysis showed thatthe expressing rate of miR-744 was significant different betweensquamous carcinoma and adenocarcinoma(p=0.007;Fig.1B).The stratified analysis by type of cancer showed that high-expression of miR744 was associated with poor survival in squamous carcinoma(HR,2.685;95%CI,1.596-4.517;P ?0.000;Table 2).We analyzed the expression levelof miR-744 in sevenlung cancer cell lines.Similarly,95D,A549,H1299,H520 celllines showed a signifiant increasedexpression of miR-744 with respect to immortalizedcell HBE(Fig.1C).We then examined miR-744 expression level in fifteen pairs of primary lung cancer tissues and their corresponding pericarcinous tissue samples by quantitative real-time PCR(qRT-PCR),andthe sequences of the primersused for the PCR are listed in Supplementary Table 1.Theoverall average expression level of miR-744 in primary lung cancer tissues were increased compared with the levelof expression in their corresponding pericarcinous tissue samples(fold difference = 2.053,P = 0.0329,Fig.1D).These data suggested thatmiR-744 is upregulated in lung cancer and may be involved inthe overall survival.miR-744 promotes lung cancer cells migration,invasionand proliferation in vitro To evaluate the biological functions of miR-744in the development of lung cancer,we conducted loss-andgain-of-function studies in A549,SPC-A-1,H1299 and H520 cells bytransient transfection with miR-744 agomir or antagomir.The transfection effiiency was verifid by qRT-PCR(Fig.2A).Overexpression of miR-744 in lung cancer celllines remarkably increased the number of migratory andinvasive cells,as determined by Transwell and Boydenassays,demonstrating the promoting effect of miR-744on cell migration which was further confimed bywound-healing assays.The down-regulated miR-744 in lung cancer celllines remarkably decreased the number of migratory andinvasive cells,except H520(Fig.2B and Fig.2C).The EdU assay furthershowed that miR-744 agomir enhanced,while miR-744 antagomir reduced DNA replication in A549,SPC-A-1,H1299 cells,except H520(Fig.2D).Chemosensitizing effect and radiosensitizing effect are mediated via miR-744 To investigate how the miR-744modulates chemosensitivity of lung cancer cell lines,both A549and H520cells were transfected withagomir-744 or antagomir-744(Figure 3A and B).By performing the CCK-8 assay,we observed that knockdown of miR-744 significantly decreased IC50 of Paclitaxel.Compared transfected miR-200c NC group with transfected antagomir-744 group,the IC50 of paclitaxel monotherapy was 5.191 vs 4.104 nmol/L on lung cancer cell A549,P<0.05;similarly,11.130 vs 9.000 nmol/L on lung cancer cell H520,P<0.05.To investigate the effects of miR-744 on radiation sensitivity,we performed colony formation assay in lung cancer cell lines A549 and H520 incubatedwithagomir-744 or antagomir-744.As shown in Fig.3C and D,silence of mir-744 significantly reduced the survival fractions in both cell lines,suggesting antagomir-744increased the sensitivity of these cells towards radiation.The biological parameters of A549 and H520 cells in different treatment groups with "Multitarget-single hitting" model show in Table 3.MiR-744 functions as a tumor promoter in lung cancer metastasis and growth in vivoTo further characterize the oncogenic potential of miR744 in vivo,we established miR744 inhibition cell lines and control cell lines by transfecting A549 cells with either antagomir744 or an antagomir negative control,and a continuing decrease in miR744 and c-FOS expression was verifid by qRT-PCR after transfection(Figure 4 A and B).A549 and H520 cells were transfected with LV-miR-744 or control vector to establish miR744 overexpression cell lines,and aincrease in miR744 and c-FOS expression was verifid by qRT-PCR(Figure 4 C and D).As a result of the downregulation of miR744,the growth ability of A549 were signifiantly decreased compared with the control group(Figure 4 E and F).As a result of the overexpression of miR744,the growth ability of H520 were signifiantly increased compared with the control group(Figure 4 G and H).As a result of the overexpression of miR744,the metastasis ability of A549 and H520 were signifiantly increased compared with the control group(Figure 41,J and K).c-FOS was a direct transcriptional targetof miR-744 in lung cancer cellsC-FOS was proved to promote cellsproliferation,invasion and migration.As shownin Fig.5A,Based on bioinformaticsanalysis fromTargetScan,RNAhybrid,complementarysequence of miR-744 was found in the promoter regionbut not in the 3'UTR of c-FOS gene.Four putative miR-744 binding sites with minimalminimum free energy(MFE)(Site 1,-1227 to-1195;Site 2,-358 to-332;Site 3,-323 to-298;Site 4,-221 to-192;)in c-FOS promoter werepredicted using RNAhybrid database.As shownin Fig.5B,ectopic expression of miR-744 increased c-FOS expression at both mRNA and proteinlevels in A549,SPC-A-1,H1299 and H520 cells,whereas miR-744downregulation reduced mRNA and protein expressionof c-FOS,indicating that c-FOS is a potentialtarget gene of miR-744.The mRNA-seq and miRNA-seq data for lung adenocarcinoma andsquamous carcinoma were downloaded from TCGA websites.BothmiR-744 and c-FOS expression were retrieved from the datasets.ThemRNA and miRNA expression data were processed level 3 data(RNA-seq V2).The samples were sorted from high to low according tothe level of miR-744 expression,and top 50%,40%,30%,20%,10%ofcases were taken respectively.Only from 94 samples(top 11%of cases),a positive correlation between the expression levels of miR-744 and c-FOSwas revealed(r=0.2177;P=0.035;Fig.5C).To further validateand quantify the transcriptional regulation of miR-744on c-FOS promoter,we cloned the wild-typepromoter of c-FOS into a luciferase reporter vector pGL3-basicand performed reporter assays inA549,SPC-A-1,H1299 and H520cells.Three different c-FOSwild-typepromoter constructswere generated:c-FOS A promoter(-500 to-1),c-FOS B promoter(-1300 to-1),c-FOS Cpromoter(-1900 to-1),andthe sequences of the primers andsequences of wild-type promoters are listed in Supplementary Table 2 and Supplementary Table 3.Theempty pGL3-basic vector was used as negative control.The c-FOS region of-1300 to-1 bp upstreamto the transcription start site showed greater potentialfor promoter like sequence.Forced expression of miR-744 only and signifiantly increased luciferase activitycompared with those transfected withmiR controls,and this induction wasn't observed inARHGAP5 C promoter(Fig.5D).Since the-1300 to-1 bp region was validated to be the key promoterregion of c-FOS and be responsive to miR-744,furthermore,four different c-FOSmut-typepromoter constructswere generated:Luc-C-FOSmut-1 promoter(-1227 to-1195),Luc-C-FOSmut-2 promoter(-358 to-332),Luc-C-FOSmut-3(-323 to-298),promoter(-221 to-192),Luc-C-FOSmut-4 andthe sequences of the primers andsequences of mut-type promoters are listed in Supplementary Table 4 and Supplementary Table 5.Forced expression of miR-744 only and signifiantly decreased luciferase activitycompared with those transfected withc-FOS B promoter(-1300 to-1)(Fig.5E).Schematic model of thepotential mechanism between miR-744 and C-FOS in the regulation of lung cancer cell(Fig.5F).c-FOS mediates the promotion of cancer cells migration and invasion by miR-744It has been shown that c-FOS promotes an aggressive phenotype incancer cells,including increased migration and invasion.Also,wepreviously reported that overexpression of miR-744 enhances themigratory and invasive ability of cancer cells.To characterizewhether c-FOS induced cell biological behaviors could be mediated bymiR-744,we performed co-transfection of si-c-FOS plasmid and miR-744 agomir in A549,SPC-A-1,H1299 and H520cells.The c-FOS expression levels were detected by qRT-PCR and westernblot in A549,SPC-A-1,H1299 and H520 cells transfected with both miR-744 agomir or agomir-NC and si-c-FOS plasmid or si-control for 48 h(Fig.6A).We found that suppressionof c-FOS signifiantly reduced lung cancer cells migrationand invasion,and clearly abrogated the miR744inducedenhancement of migration and invasion in A549,SPC-A-1,H1299 and H520 cells,which was further confimed bywound-healing assays(Fig.6 B and Fig.6C).The EdU assay furthershowed that suppressionof c-FOS signifiantly reduced,while miR744reduced DNA replication in A549,SPC-A-1,H1299 cells,except H520(Fig.6D).MiR744can mediate the Mitogen-activated protein kinase(MAPK)signal transduction pathwayTo validate the potential MAPK pathway activated by miR-744,we examined GRB2,Rafl,total ERK1/2andERK1/2phosphorylation,c-FOSlevels in agomir miR-744 transfectedA549,SPC-A-1,H1299,H520cells.A prominent increase in the GRB2,Rafl,ERK1/2phosphorylation,c-FOSlevels was observed,whereas levels of total ERK1/2 remained unchanged upon miR-744 expression(Fig.7).Conclusions1.miR-744 is overexpressed in lung cancer tissues as well as cells,and is associated with OS.2.miR-744 is involved in the progression and metastasis of lung cancer,and its ectopic expressioncan affect proliferation as well as metastasis of lung cancer cells in vivo and in vitro,whichfunctions as a proto-oncogene.3.C-FOS was a direct transcriptional target of miR-744 in lung cancer cells,and miR-744contributes to lung cancer cells migration and invasion via mediation of C-FOS. |
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