Long Non-coding RNA PVT1 Promote Autophagy By Up-regulating ATG5 Expression In Gastric Cancer

Introduction:Gastric cancer(GC)is the second most common cause of cancer-related death worldwide.In 2012,more than 700 000 deaths estimated to have occurred.And it is the most common gastrointestinal malignancy in East Asia,Eastern Europe,and parts of Central and South America and its 5-year overall survival rates are less than 25%.In spite of the progression of treatment for GC in recent years,but the prognosis of gastric cancer is not satisfactory.Therefore,a better understanding of the molecular mechanism and genetic network that controls the initiation and progression of GC may help identify potential diagnostic and prognostic biomarkers and therapeutic targets.Long-noncoding RNA(lncRNAs)is a class of non-coding RNAs with a transcription length greater than 200 nucleotides.It has been found that lncRNAs are involved in a variety of biological processes such as chromatin modification,X chromosome inactivation,transcription,translation,gene imprinting and regulation of protein activity.Recently,a large number of researches have demonstrated that lncRNAs are as the key regulators of tumorigenesis involving a variety of biological processes.lncRNA plasmacytoma variant translo-cation 1(PVT1)is located in the human chromosome 8q24.21 region.The expression of PVT1 was significantly upregulated in a variety of digestive system tumor tissues,such as colorectal cancer,liver cancer and pancreatic cancer and esophageal cancer.These finding suggested that lncRNA PVT1 may play an important role in the regulation of cancer progression and has the prognosis values in detecting cancer disease.In this respect,the potential molecular mechanisms and biological effect of lncRNA PVT1 still need to be researched.Autophagy is a conserved pathway from yeast to human,which is an important intracellar homeostatic process.Autophagy is initiated from the formation of autophagosome to deliver the protein and cell organelle to lysosome for degradation.This catabolic process is involved in a variety of autophagy related genes(ATGs),which could influence the activation of autophagy.Under various pathological conditions such as hypoxia,inflammation and starvation,autophagy can be stimulated to help cell tosurvival from the adverse conditions.Many studies have indicated that autophagy plays a significant and alternative role in the progression of cancer cells.In gastric cancer,several studies have showed that autophagy can affect a wide range of pathological events.More and more researchers have found that lncRNAs can participate in the regulation of autophagy.But the special mechanisms are still not very clear and need to be explored.The aim of this study was to investigate the expression pattern of lncRNA PVT1 in gastric cancer by TCGA database,and to study the effect of lncRNA PVT1 on the proliferation,migration,invasion and autophagy in gastric cancer cells by in vitro experiments.The present study may have contributed to understand the regulation mechniasm between lncRNA PVT1 and autophagy in gastric cancer.2.Materials and methods2.1 Patients and samplesThe gene expression profile of gastric cancer tissues and normal samples were downloaded from TCGA database.The total 373 cases of gastric cancer with gene expression data and corresponding clinical information were analysed in this study.The expression of lncRNA PVT1 was normalized using average(rma)algorithm.2.2 Cell CultureThree GC cell lines(AGS,SGC-7901,BGC-823)and human normal gastric cells(GES-1)were purchased from ATCC.These cells were grown in RPMI 1640 or DMEM(Biowhittaker,Walkersville,MD)with 10% fetal bovine serum(HyClone,Logan,UT)at37°C under 5%CO2.2.3 Cell transfectionTo investigate the role of lncRNA PVT1,lncRNA PVT1 cDNA(GenePharma)were cloned into the mammalian expression vector pcDNA3.1(Invitrogen,Carlsbad,CA),the vectors were transfected into SGC-7901 cells.BGC-823 cells were transfected with either si RNAs targeting lncRNA PVT1 or scrambled negative controls(Gene Pharma,Shanghai,China)next.2.4 Quantitative real-time PCRTRIzol reagent(Invitrogen,Life Technologies,Carlsbad,CA,USA)was used to extract cell total RNA.Using a RevertAid First Strand cDNA Synthesis kit(ThermoScientific,Waltham,MA,USA)First-strand cDNA to synthesize with random primers.Using a SYBR Premix Ex Taq II(Takara,Dalian,China)on a CFX96 real-time PCR System(Bio-Rad,Hercules,CA,USA)to carry out Quantitative real-time PCR and the results were normalized with 18 s as an internal control.2.5 Cell Proliferation AssayAfter SGC-7901 and BGC-823 cells transfected with plasmid or siRNA lncRNA PVT1 PVT1,the proliferation of GC cells was determined using MTT assay(Sigma-Aldrich,St Louis,MO,USA).Briefly,we seed cells(1×104 per well)in a96-well plate and added 10 mL MTT(Sigma-Aldrich)to each well.Then,we incubated the 96-well plate at 37 1C for 4 hours.Then,dimethyl sulfoxide(150 mL;Sigma-Aldrich)was added to solubilize the MTT formazan.The optical density was determined by absorbance spectrometry at 490 nm.2.6 Colony-forming growth assaysCells(1,000 cells/plate)were seeded in six-well plates and cultured two weeks until visible colonies appeared.The colonies were then stained with crystal violet(Santa Cruz Biotechnology,Inc.)and were counted.2.7 Migration and invasion assaysA 24-well Millicell chamber containing a Matrigel-coated membrane was used for invasion assay.The migration assay was carried out in a similar fashion without the Matrigel coating.Seeding Cells(5×104)in the top chamber and the bottom wells were filled with complete medium.Wipe off cells on the upper membrane surface and the lower membrane surface was fixed with methanol,stained with 0.1% crystal violet,and counted in five random fields.2.8 Western blot analysisWe used RIPA containing protease inhibitingor(Roche,Nutley,NJ,USA)to lyse Cells.Using SDS-PAGE separated protein samples,then transferred protein to polyvinylidene difluoride(PVDF)membranes(Millipore,Billerica,MA,USA).The membranes were incubated with LC3 [LC3B isoform,3868],P62,ATG5 and ?-actin(Abcam,Hong Kong,China),primary antibodies overnight at 4 ?.Using ECL chemiluminescent regents(Pierce,Rockford,IL,USA)assess protein expressio,then using densitometry(Image Lab software,Bio-Rad,Hercules,CA,USA)assess theintensity of the protein bands which was quantified by and normalized to the corresponding ?-actin bands.2.9 Immunofluorescence microscopyPlate cells on 6-well plates,and then use phosphate-buffered saline to wash.Add 4%polyformaldehyde.Then,the primary antibody(1:100,specific for active LC3 and ATG5)was added and incubated at 4 ? overnight.Then wash three times(5mins/time).Add fluorescent secondary antibody(1:100;Santa Cruz Biotechnology,Santa Cruz,CA,USA)to incubate for 2 hours.Wash three times(5mins/time).Counterstain the cells with diaminophenylindole(Santa Cruz Biotechnology)and Use a confocal microscope to visualize.2.10 Transmission electron microscopyUse 3% glutaraldehyde to fix SGC-7901 and BGC-823 cells and store at 4 ?.Then,use 0.1 M sodium cacodylate buffer to fix the cells.Then dehydrate the cells in a gradient of 50–100% ethanol and embedded in araldite.Obtain ultrathin sections(50–60 nm)and stain with uranyl acetate and lead citrate.Use a JEM-1400 transmission electron microscope to view images at 80 kV.2.11 Statistical analysisUse SPSS 13.0(SPSS,Inc.,Chicago,IL,USA)to perform statistical analysis.Use the mean ± standard deviation to present data.Use Student's t-test to compare two independent groups.P value < 0.05 was considered statistically significant.3.Results3.1 The expression of lncRNA PVT1 was upregulation in GC tissues and cell linesTo explore the expression profile of lncRNA PVT1 in GC,we analysed the PVT1 expression in total 373 cases by STAD RNA-seq data downloaded from TCGA database.The result showed that lncRNA PVT1 was significantly upregulation in GC tissues than normal tissues.To study the relationship between lncRNA PVT1 expression and clinical stage,we sorted the cases into two groups according to the different stages and then analyzed the expression profile.We found that lncRNA PVT1 was up-regulated in III+IV stage than I+II stage.This result showed that lncRNA PVT1 expression was associated with high clinical stage.To assessed the level of lncRNA PVT1 expression in GC cell lines.The expression of lncRNA PVT1 in three GC cell lines and one normal gastric cellline was analysed by PCR.We found that the levels of lncRNA PVT1 expression were higher in the BCG-823,SGC-7901 and AGS cells than in the normal gastric cell line GES-1.3.2 Long non-coding RNA PVT1 promotes GC cell proliferation,migration and invasion.We examined the affection of PVT1 in proliferation of GC cells next.According to the expression of lncRNA PVT1 in different GC cells,we overexpressed lncRNA PVT1 expression in SGC-7901 cells by transfected pcDNA3.1-PVT1 vector.The expression level of lncRNA PVT1 is significantly increased by 16.4 fold compared with control group.While,we knockdown PVT1 expression in BGC-823 cells by transfecting siRNA.Among three siRNA,siRNA1 knocked down lncRNA PVT1 expression by 54%transfection efficiency and siRNA1(si-PVT1)was utilized for follow-up experiment.MTT assays were performed to detect effect of proliferation.The result showed that overexpressing lncRNA PVT1 promoted the proliferation in SGC-7901,while knocking down lncRNA PVT1 decreased the proliferation in BGC-823.To further explore the ability of clonogenic survival in GC cells,Colony-forming growth assays was preformed and the result indicted that clonogenic survival was obviously increased following overexpressed lncRNA PVT1 in SGC-7901 while decreased following inhibiting lncRNA PVT1 in BGC-823.In addition,Transwell assays with or without matrigel was performed to detected the GC cells ability of migration and invasion.The results showed that overexpression of lncRNA PVT1 could increase the ability both migration and invasion in 7901 cells,however,inhibiting lncRNA PVT1 decreased the ability both migration and invasion in 823 cells,which indicted lncRNA PVT1 can promote GC cells migration and invasion.Given the above,lncRNA PVT1 can promote GC cells proliferation,migration and invasion.3.3 Long non-coding RNA PVT1 promotes GC cell autophagy.As autophagy is a protective factor in GC,we then detected whether lncRNA PVT1 can regulate GC cells autophagy.We first detected the autophagy marker protein LC3I/II and P62 by western blot.The result showed that LC3 II was significantly increased expressed and P62 was decreased expressed in overexpression lncRNA PVT1 SGC7901 cells,which indicted autophagy flux is activated.On the contrary,LC3 II was decreased expressed and P62 was increased expressed in knockdown PVT1 BGC-823 cells,whichindicted autophagy flux is blocked.To further comfier the effect of PVT1 on GC cells autophagy,immunofluorescence assay was performed to detected LC3 II expression and results in keep with western blot,moreover,it was observed that LC3 II express in cytoplasm.Then Transmission electron microscopy was performed to showed autophagy occurrence in high or low expression PVT1 cells,the red arrow point the autophagosomes.In overexpressing PVT1 SGC-7901 cells,autophagosomes was easier observed compared with control group,meanwhile,autophagosmes was harder observed in inhibiting PVT1 BGC-823 cells compared with control group.These findings indicated that PVT1 can promote GC cells autophagy flux activating.3.4 Long non-coding RNA PVT1 promotes GC cell autophagy by inducing ATG5 expression through binding ATG5 directly.The role of ATGs in the regulation of cell autophagy is crucial.Accordingly,we detected several autophagy relative protein mRNA expression by real-time PCR to screen potential gene involving in lncRNA PVT1 induced GC cells autophagy.ATG3,ATG5,ATG7,ATG10,Beclin and LC3 B,which involved different autophagy process was detected in overexpressing PVT1 SGC-7901 cells by real-time PCR.The result showed that ATG5 increased by nearly 2.4 fold compared with control group.Then we detected the ATG5 protein expression in both overexpressing lncRNA PVT1 and inhibiting lncRNA PVT1 cells.The result showed that ATG5 significantly increased in overexpressing lncRNA PVT1 SGC-7901 cells compared with control group,while ATG5 decreased in inhibiting lncRNA PVT1 BGC-823 cells compared with control group.Furthermore,immunofluorescence assay was performed to detected ATG5 expression and results in keep with western blot,moreover,it was observed that ATG5 express in cytoplasm.One of important functions of long non-coding RNA is binding with protein to regulate its expression.So we use RIP to detect the interaction between lncRNA PVT1 and ATG5.The result showed that lncRNA PVT1 can bind with ATG5 directly in both SGC-7901 cells and BGC-823 cells.Taken together,these findings suggest that lncRNA PVT1 promote GC cells autophagy by inducing ATG5 through binding with ATG5 directly.