Study Of Autophagy Induced By URI Knockdown And Its Mechanism In Gastric Cancer Cells

BackgroundURI（Unconventional prefoldin RPB5 Interactor）is a member of the evolutionarily conservative prefoldin family,and also a RNA polymerase II subtype 5 interacting protein,which is involved in nutrition sensitive and mTOR dependent regulation of the transcription program.A number of studies have shown that URI is an oncogene.Our previous studies have found that URI can promote survival and migration as well as the chemoresistance of gastric cancer cells.Autophagy is a catabolic process that facilitates nutrient recycling via degradation of damaged organelles and proteins through lysosomal mediated degradation.It is complex regarding the biological significance of autophagy with cancer,as quite different conclusions may be reached in studies targeting the same kind of caner.However,it was generally accepted that autophagy play key roles during initiation,progression,chemoresistance,as well as targeted therapy of cancer.Especially,autophay based targeting therapy for cancer has shown promising clinical applications.It has been found that overexpression of PI3K/Akt/mTOR pathway proteins has potential significance for the prognosis of gastric cancer.Due to the fact that URI is associated with mTOR/S6K1 signaling pathway,we therefore hypothesized that URI may be involved in the activation of autophagy in human gastric cancer cells.Here,we investigated the effect and potential mechanisms of URI on autophagy in gastric cancer MGC-803 and HGC-27cells.ObjectiveTo investigate the effect and potential mechanisms of URI on autophagy in gastric cancer cells.Methods（1）Gastric cell culture.The low differentiated MGC-803 and undifferentiated HGC-27 gastric cancer cells were cultured in DMEM medium and RPMI-1640 medium respectively with addition of 10%fetal bovine serum.（2）Knockdown or overexpression of URI.For siRNA gene knockdown experiment,transfection of URI si RNA-A and scramble control sequence was performed using Hiperfect Transfection Reagent and Opti-MEM.To overexpress URI,URI expression plasmid pCMV6-URI,the vector control pCMV6-entry were transfected into gastric cancer cells using the Lipofectamine 2000 and opti-MEM reagent.The levels of mRNA and protein expression of URI in MGC-803 and HGC-27 cells were analyzed by fluorescent real-time quantitative RT-PCR and Western blotting.（3）Autophagy detection GFP-LC3 punctum aggregation was analyzed through GFP-LC3 transient transfection and confocal microscopy.The autophagic vesicles were observed by transmission electron microscopy（TEM）.We further measured the autophagic flux by employing NH₄Cl,a commonly used negative regulator of autophagy,which inhibits autophagosome-lysosome fusion through cidification inside the lysosome.Western blotting was used to measure the expression of LC3-I and LC3-II.（4）Detection of Phosphorylated mTOR/p70S6K by western blotting assay.mTOR,p70S6K,p-mTOR and p-p70S6K proteins were detected in pCMV6-URI transfected cells and URI si RNA-A transfected cells by western blotting assay.Results（1）The expression of URI in gastric cancer cells was markedly increased after transfection with the plasmid pCMV6-URI.The expression of URI in gastric cancer cells was significantly decreased after transfection with the interference fragment si RNA-A.（2）URI siRNA-A transfection induces GFP-LC3-labeled autophagosomes appeared in the cytoplasm and aggregate into puncta.The cells with or without siRNA-A transfection showed different micrographs.In cells transfected with scrambled sequence,GFP-LC3 diffused throughout the cytosol.Numerous puncta were observed following siRNA-A transfection in MGC-803 and HCG-27 cells,but fewer puncta in cells pretreated with 3-MA,a autophagy blocker at an early stage.（3）URI siRNA-A transfection induces autophagic vesicles formation.We observed an increase of autophagic vesicles containing cytosolic contents and disintegrating materials in the URI siRNA-A transfection cells by TEM.In contrast,such vesicles were less frequently seen in the control cells and cells of siRNA-A transfection with3-MA treatment.（4）URI knockdown induces autophagic flux.Compared with the URI silencing only samples and the URI silence plus Rap treatment samples,the ratio of LC3-II/LC3-I was significantly increased with NH₄Cl pretreatment in MGC-803 and HGC-27 cells.These results indicate that URI knockdown activates autophagy and induces autophagic flux.（5）URI enhances expression of phosphorylated mTOR and p70S6K.Overexpression of URI increased the expression of P-m TOR（S2448）and P-p70S6K（Thr389）proteins significantly.On the contrary,URI silence significantly reduced the expression of phosphorylated mTOR and p70S6K in MGC-803 and HGC-27 cells.Conclusions（1）Our results indicate that URI was involved in the activation of autophagy and Knockdown of URI significantly induced autophagy flux in gastric cancer cells.（2）URI knockdown-induced autophagy was associated with its regulation of m-TOR/p70S6K signaling pathway.