Function And Mechanisms Of LncRNA DMTF1v4（NR₀24549） In Development Of Multidurug Resistance Of Gastric Cancer

【Background】Gastric cancer （GC） is one of the malignances with highest lethality rate;however, the treatment outcome is usually poor because of multidrug resistance（MDR）. MDR refers to the ability of tumors becoming resistant to many kindsof chemotherapeutic drugs with different structure and mechanisms. MDR canbe divided into two types, namely, intrinsic resistance and acquired resistance.Intrinsic resistance means that tumors become resistant to drugs beforechemotherapy is implemented; while acquired resistance means that tumorsbecome resistant to drugs during the process of chemotherapy. Mechanisms ofMDR are very complicated and previous researches from genome transcriptionand translation level failed to illustrate this problem deeply. Herein, moreresearches from a new angle are needed to interrogate mechanisms of MDR ofgastric cancer.long non-coding RNA （lncRNA） is one of the compelling transcpritome members in recent years. It is characterized by its cell and tissue specificexpression patterns and ability of regulating the expression and modification ofprotein-coding genes from multiple perspectives. Moreover, altered expressionof lncRNAs is one of the characteristics in many kinds of cancers, hence, thediscovery of lncRNAs provides an important opportunity to study the biologicalfunction and mechanisms of MDR of malignant tumors. To study the alterationof lncRNA expression profiles between MDR sublines and their parental celllines and, further, to study the effect of differentially expressed lncRNAs onthe expression of MDR related protein-coding genes will provide novelapproaches illustrating the nature of mechanisms of MDR, and exploring newstratigies and targets for reversing MDR.【Aims】To compare the expression profile difference of lncRNA between GCmultidrug-resistant sublines and their parental cell line through high throughputlncRNA microarrays; screen gastric cancer MDR related lncRNAs throughstrategies such as change fold, genome loci blast, neighboring genes analysis,phylop conservation degree and etc; validate some key GC MDR relatedlncRNAs; evaluate the correlation between key lncRNAs expression level anddrug sensitivity examined by histoculture drug response assay （HDRA） in GCclinical samples; investigate the effect of key lncRNAs on the GC MDRphenotype and the mechanisms by which key lncRNAs exert their effet; andprovide a novel approach reversing GC MDR from new perspectives.【Methods】1. High throughput lncRNA microarrays（Human LncRNA Microarray V1.0：Human_HX12_LNC; Genome Build:HG18,Build36; http://www.arraystar. com/Products/product_main.asp?id=239） are used to compare the lncRNAexpression profile difference between GC MDR sublines,SGC7901/ADRand SGC7901/VCR, and their parental cell line SGC7901.2. To screen gastric cancer MDR related lncRNAs through strategies such aschange fold, genome loci blast, neighboring genes analysis, phylopconservation degree and etc3. To evaluate lncRNA DMTF1v4expression level by quantitative PCR in40GC clinical samples in comparison with adjacent non-tumor tissues;examine drug sensitivity of40GC clinical samples by HDRA and analyzethe correlation between DMTF1v4expression level and drug sensitivity.4. To deplete the DMTF1v4expression in SGC7901/ADR and SGC7901/VCRcells by siRNA transfection; validate the effect of depletion of DMTF1v4ondrug sensitivity of GC MDR sublines through MTT assay, plate cloningexperiment and IC50assay.5. To investigate effect of depletion of DMTF1v4on the apoptosis ofSGC7901/ADR cell through cytoflowmetry and western blotting.6. Adriamycin accumulation and retention assays are used to examine theadriamycin accumulation and retention rate of SGC7901/ADR cell atdesignated time after silencing of DMTF1v4.7. Western blotting experiments are implemented to examine the key MDRrelated protein such as P-glycoprotein（P-gp） and multidrug relatedprotein（MRP）expression of SGC7901/ADR and SGC7901/VCR cells atdesignated time after silencing of DMTF1v4.8. Construct DMTF1v4silencing lentivirus vector and stable transfected LV-DMTF1v4SGC7901/ADR cell line; inject LV-DMTF1v4SGC7901/ADRcells subcutaneously in nude mice and examine the effect of depletion of DMTF1v4on the drug sensitivity of GC cells in vivo.9. Examine P-gp expression level of SGC7901/ADR cells at designated timethrough quantitative PCR and western blotting experiments after DMTF1v4is depleted by lentivirus vector transfection stably or siRNAs transfectiontransiently.10. Construct nude mice xenograft models by LV-DMTF1v4SGC7901/ADRcells; tumors are obtained and made into paraffin slides forimmunohistochemistry, immunofluorescent and western blotting to examineP-gp expression level difference between DMTF1v4silencing and controlgroups.11. pGL4.24[luc2P/minP] vector encodes the luciferase reporter gene luc2Pand contains a multiple cloning region for insertion of a response element ofinterest upstream of a minimal promoter and the luc2P gene. pGL4.11[luc2P]vector lacks minP compared with pGL4.24[luc2P/minP].Inserts wereamplified and recombined from genomic DNA and cDNA, and they werecloned into the BamHI and SalI sites5’ to the luciferase gene. Assays wereperformed in96-well white plates using Dual-Glo （Promega） according tothe manufacturer’s protocol. ncRNA inserts were cloned and recombinedfrom genomic DNA and cDNA by three steps. First,1500bp upstream andthe first exon was cloned from genomic DNA using corresponding primersas the segment-1; second, other exons were cloned from cDNA usingcorresponding primers as the segment-2; third, segment-1and segment-2were linked together and cloned into the luciferase reporter vector accordingto the principle of fast PCR clone for the control vector（pGL4.74-TK-control）.The primers （GGG GAT CCA GCA GGC TTAGGG GAA AGA G and GGG TCG ACC CCT TGC TCA GTC AAG AAGC） were used to amplify a4kb fragment from genomic DNA not showingany evidence of transcriptional activity. 【Results】1.1499and1420differentially expressed lncRNAs betweenSGC7901/ADR, SGC7901/VCR and SGC7901respectively wereobtained through high throughput lncRNA microarrays. Among them,627differentially expressed lncRNAs were in common and NR₀24549（DMTF1v4） may play the key role in the development of GC MDR.High throughput lncRNA microarrays were used to compare the expressonprofile difference between GC MDR sublines, SGC7901/ADR andSGC7901/VCR, and their parental cell line, SGC7901. Change fold>=2.0wasseen as significantly different.1499and1420differentially expressed lncRNAsbetween SGC7901/ADR, SGC7901/VCR and SGC7901respectively wereobtained through high throughput lncRNA microarrays. Among them,627differentially expressed lncRNAs were in common and were referred tolncRNA-Cs. Furthermore, there were32lncRNAs with change fold>=4.0which included lncRNA-Cs upregulated in GC MDR sublines compared withSGC7901such as NR₀24549,NR₀24074,uc002hfi,uc001vtj,uc003wbn,NR₀26673,NR₀15379,uc002odt,uc010heq,NR₀27241,uc002wcb,uc010hhg,uc001aqd,uc002jmb,ucoo3wig and uc003whs; and lncRNA-Cs downregulatedin GC MDR sublines compared with SGC7901such as NR₀26867，NR₀27339， NR₀27282,uc002uov,uc003gnd,uc001ras, uc002rvu,uc003dhh,uc010jya, uc001ekz,uc003tbi,uc001jtu,uc010itd,uc002ued,uc010dga anduc010cny。Of them, NR₀24549（DMTF1v4）was found to be the highestupregulated lncRNA in both MDR GC sublines compared with SGC7901.（SGC7901/ADR-SGC7901change fold=25.89SGC7901/VCR-SGC7901change fold=21.42）。 2. It showed that DMTF1v4may be a key GC MDR related lncRNAthrough strategies such as change fold, genome loci blast, neighboringgenes analysis, phylop conservation degree and etc.1) Results of lncRNA microarray indicated that NR₀24549（DMTF1v4）is the highest upregulated lncRNA in both MDR GCsublines compared with SGC7901.（SGC7901/ADR-SGC7901change fold=25.89SGC7901/VCR-SGC7901change fold=21.42）2) It was found that approximately5%（31/627）,21%（132/627） and12.3%（77/627） of the lncRNA-Cs were located in regions nearMDR-related genes, oncogenes and tumor suppressors, respectively.Furthermore, P-gp was located307Kb downstream of DMTF1v4.3) phylop conservation analysis indicated that lncRNA DMTF1v4represented a relative high conserved gene （Mean±Sd：0.245989±1.14945）.4) DMTF1v4expression level was markedly upregulated inSGC7901/ADR and SGC7901/VCR cells compared with SGC7901and was the highest upregulated one among other15lncRNAs（change fold>=4.0）upregulated in GC MDR sublines.3. The lncRNA DMTF1v4expression of primary GC specimens and drugsensitivity examined by HDRA were negatively correlated.The expression levels of DMTF1v4in40GC tissues and non-tumormucosa by qRT–PCR were examined. The relative change-fold was used todescribe the alteration of DMTF1v4expression level, and forty GC tissues weredivided into DMTF1v4high expressors group and low expressors groupaccording to the change-fold median. Then the inhibition rates of two groups of GC specimens were calculated using Histoculture Drug ResponseAssay（HDRA）. The lncRNA DMTF1v4expression of primary GC specimensand drug sensitivity examined by HDRA were negatively correlated.Furthermore, the inhibition rate and relative change-fold had a negativecorrelation in two groups.4. Silencing of DMTF1v4increased sensitivity to P-gp related drugs ofSGC7901/ADR and SGC7901/VCR cells.To study the roles of DMTF1v4in development of GC MDR phenotype,siRNAs transient transfection was performed to deplete DMTF1v4in GC MDRsublines and examine the function of silencing of DMTF1v4through severalexperiments.1)Results of MTT assay showed that the growth rate of SGC7901/ADRand SGC7901/VCR cells were significantly lower than controlgroups in the presence of P-gp related drugs such as adriamycin andvincristin.2) Results of plate colony formation experiments showed that the colonyformation rate of SGC7901/ADR cells was significantly inhibitedcompared with control groups in the presence of adriamycin.3) Results of IC50assay showed that sensitivity of SGC7901/ADR cellsto P-gp related drugs such as adriamycin, vincristin and paclitaxelwas significantly increased compared with control groups.5. Silencing of DMTF1v4resulted in remarkable increase of apoptosis ofSGC7901/ADR cells.The results of cytoflowmetry indicated that silencing of DMTF1v4resultedin remarkable increase of apoptosis of SGC7901/ADR cells72h later compared with control groups. The results of western blotting also indicated that Bcl-2expression of DMTF1v4-silencing SGC7901/ADR cells was decreasedremarkably while Bax expression was significantly upregulated and ratio ofBcl-2/Bax was markedly reduced compared with control groups.6. Depletion of DMTF1v4led to more accumulation and less relax ofadriamycin in SGC7901/ADR cellsIntracellular adriamycin accumulation and retention assays indicated thatdepletion of DMTF1v4led to more accumulation and less efflux of adriamycinin SGC7901/ADR cells compared with control groups.7. Depletion of DMTF1v4resulted in decrease of P-gp expression ofSGC7901/ADR and SGC7901/VCR cells.The results of western blotting showed that Depletion of DMTF1v4resulted in decrease of P-gp expression of SGC7901/ADR and SGC7901/VCRcells, however, MRP expression was not changed significantly.8. Silencing of DMTF1v4inhibited tumor growth in vivo.Construction of nude mice xenograft models by LV-DMTF1v4SGC7901/ADR cells and caudal vein injection of adriamyicn by0.5mg/Kg wasperformed daily. Tumor volumn and weight were calculated at designated time.Silencing of DMTF1v4inhibited tumor growth in vivo compared with controlgroup.9. Silencing of DMTF1v4caused decline of P-gp expression ofSGC7901/ADR cellsSGC7901/ADR cells were transfected stably by LV-DMTF1v4cells ortransiently by siRNAs-DMTF1v4, and DMTF1v4expression were reducedsignificantly48h later respectively. P-gp expression examined by quantitative PCR and western blotting were decreased72h later with the depletion ofDMTF1v4.10. Depletion of DMTF1v4resulted in reduction of P-gp expression ofSGC7901/ADR cells in vivoConstruction of nude mice xenograft models by LV-DMTF1v4SGC7901/ADR cells was obtained and the tumors were made into paraffinslides for immunohistochemistry, immunofluorescent and western blottingexperiments to examine P-gp expression level difference between DMTF1v4silencing and control groups. The results showed that Depletion of DMTF1v4resulted in reduction of P-gp expression of SGC7901/ADR cells in vivo.11. Reporter assay indicated that DMTF1v4may promote P-gp expressionthrough an enhancer-like roleDualluciferase reporter assay was performed to investigate whetherDMTF1v4could exert an enhancer-like role and the results indicated thatDMTF1v4, especially the fragment from540bp-1400bp of exon, may promoteP-gp expression through an enhancer-like role.【Conclusion】Based upon high-throughput screening of lncRNAs microarrays, wereported a group of gastric cancer multidrug resistance related lncRNAs for thefirst time. Further, DMTF1v4may be the key lncRNA playing important role inthe development of gastric cancer multidrug resistance. DMTF1v4couldpromote multidrug resistance phenotype of gastric cancer through increasingefflux and reducing accumulalation of P-gp related chemotherapeutic drugsintracellularly, providing a novel approach illustrating the mechanisms by whichP-gp is upregualted in gastric cancer multidrug resistance sublines on the transcriptional level. Thus, DMTF1v4may be regarded as a new target ofreversing multidrug resistance phenotype of gastric cancer.