Preliminary Study On The Effect Of NRAGE Gene On The Growth And Autophagy Of Liver Cancer Cells

Hepatocellular carcinoma(HCC)is the third most common cause of death and the sixth most common cancer in the world.Since cancer mortality was over 80%in developing countries,liver cancer has been a serious public health problem in these parts of the world.Studies show that autophagy level is decreased in HCC,suggesting that decreased levels of autophagy may act as a promoter in liver tumorigenesis.Autophagy is a significant conserved catabolic process which has critical functions in homeostasis maintenance of cell under normal growth conditions and in preservation of cell viability under various stresses.Autophagy and tumor are closely related.Defects in basal autophagy result in toxic accumulation of protein aggregates,damage organelles,impair genome integrity and stability,limit cell survival,and ultimately led to disease states,such as cancer,aging and neurodegeneration.But in the process of cancer development,autophagy can promote cancer survival,improve metastasis and resistance with helping cancer cells to resist the stress of anoikis,chemotherapy,radiotherapy and so on.As a member of Type II MAGE family members,except MAGE domain MHD,NRAGE contains a unique domain consisting of 25 tandems repeats of the hexapeptide WQXPXX.A large numbers of reports showed that NRAGE,a protein that directly interacted with the p75NTR cytosolic region,induced cell death through a JNK-dependent mitochondrial apoptotic pathway.Consistent with its proapoptotic function,NRAGE may mediate neural progenitor apoptosis via the BMP signaling pathway.And NRAGE could regulate anti-apoptotic factor Che-1 degradation to induce cell death.Previous studies have showed that overexpression of human NRAGE could significantly suppress cell proliferation,migration and invasion.So it was generally recognized as a tumor suppressor because of its contribution to cell apoptosis and metastasis.Nevertheless,recent studies have shown that the expression of NRAGE was higher in esophageal cancer tissues,and it can stabilize PCNA to promote cell proliferation,suggesting that NRAGE is a novel factor of great importance participating in tumorigenesis.In previous studies,our group found that the expression of NRAGE was higher in HCC than normal cells,which suggested that NRAGE may be related to the occurrence and development of HCC.To study the effects of NRAGE on HCC,three oligonucleotides of NRAGE siRNA were designed and synthesized.They were transfected to knockdown expression of NRAGE.The protein level of NRAGE was measured by Western blot.The level of mRNA was detected by QPCR.The cell cycle of HepG2 was examined by FCM.The ability of proliferation and migration of HepG2 cell in vitro were assessed by MTS and wound healing test,respectively.We found that 100nmol/L siRNA could significantly silence the expression of NRAGE.100nmol/L siRNA could decrease the level of mRNA.The ability of cell proliferation decreased after transfection.Cell number of G0-G1 phase increased,cell number of S and G2-M phase decreased after transfection by siRNA2.The migration of HepG2 cell was not changed significantly after transfection with siRNA.Our study demonstrated that knockdown of NRAGE inhibited cell proliferation,blocked cell cycle at G0-G1 phase,but had no significant effect on cell migration.During study,our group also found that autophagy significantly increased in MEF cell from Nrage-/-mouse,suggesting that NRAGE may have some connections with autophagy that the silencing NRAGE will increase autophagy activity.In order to clarify the effect of NRAGE on autophagy,we silenced NRAGE by 100nmol/L siRNA.The LC3B fluorescence spot numbers were analyzed by laser scanning confocalmicroscope.Autophagy marker proteins were detected by Western blot.Co-localization of NRAGE and Beclin1 was detected by immunofluorescence.We found that the number of LC3B fluorescent spots increased,the ratio of LC3B II/I increased,the expression of P62 decreased,which indicated that the level of autophagy increased after NRAGE silencing.NRAGE was mainly distributed in nuclear and Beclinl was mainly distributed in cytoplasm.Treatment HepG2 cell with D-Hanks for 15,30 and 60min,NRAGE went out of nuclear and co-location of NRAGE and Beclinl was increased,which suggested that NRAGE may inhibit autophagy by binding to Beclin1.It was worth noting that Beclin1 expression level was decreased in those tumors with high expression of NRAGE,such as breast cancer,prostate cancer,lung cancer,malignant melanoma tumor and esophageal cancer.Therefore,we speculated that NRAGE may be combined with Beclinl to inhibit autophagy and the decreased autophagy may induce tumorigenesis in liver.