| Background and Objective:Hepatocellular carcinoma?HCC?presents a high incidence and mortality:HCC is the sixth most common tumor and the second most frequent cause of cancer death worldwide[1,2].However,most importantly,half of these cases and deaths were estimated to occur in China[3].Although treatment platforms have improved over the past decade and diagnostic standardization has been better,managing patients through treatment challenges is difficult[2].It is important to understand the mechanism of HCC progression for the development of HCC therapies.Glucose-regulated protein 78?GRP78?is a class of the HSP70 protein,which confirmed functions as an endoplasmic reticulum?ER?chaperone protein and a regulator of the ER stress signaling pathway[4].GRP78 as a multifunctional proteins that play a key role in physiological and pathological stress[5].A lot of studies have shown that GRP78 may play a crucial part in the tumorigenesis and progression of HCC[6,7].Studies have demonstrated that FAT10 is involved in a number of important cellular development processes,including apoptosis,immune-mediated inflammation,and the regulation of cell cycle and proliferation[8-11].Glucose-regulated protein 78?GRP78?and the ubiquitin-like protein FAT10each promote proliferation in hepatocellular carcinoma?HCC?.In this study,we found that GRP78 and FAT10 were significantly overexpressed in HCC tissues compare with adjacent non-cancerous tissues,and a positive correlation was found between their expression and associated proliferation characteristics.GRP78knockdown reduced FAT10 expression and suppressed HCC proliferation in vitro and in vivo.The effects of GRP78 knockdown were rescued by FAT10 up-regulation,whereas FAT10 knockdown reduced HCC proliferation enhanced by GRP78up-regulation.Furthermore,GRP78 modulated FAT10 expression by regulating the NF-?B pathway,direct activation of the NF-?B pathway increased the expression of FAT10.Taken together,these results suggest that this newly identified GRP78–NF-?B–FAT10 axis will provide novel insight into the understanding of the regulatory mechanisms of proliferation in human HCC.Method:1,A total of 112 paired patients specimens and corresponding adjacent non-tumor tissues were collected from tumor surgical resection at the Second Affiliated Hospital of Nanchang University between January 2006 and June2012.We displayed the GRP78 and FAT10 protein levels in 112 HCCs samples and the corresponding adjacent livers tissues by IHC,and analyzing the results statistically,follow-up patients with phone,and retrospectively analyzing the clinical and pathological features in all patients,summarizing the relationship between GRP78 and FAT10 expression and clinicopathological features and survival rate in patients of hepatocellular carcinoma.2,RT-qPCR,Western blot method were performed to detect the expression levels ofGRP78 and FAT10 mRNA and protein in 112 cases of HCCs samples and the corresponding adjacent livers tissues,and the relationship between GRP78 and FAT10 mRNA and protein were analyzed by scatter plots.3,The levels of GRP78 and FAT10 in Huh-7,MHCC97H,HepG2,HCCLM3,SMCC7721 and HL-7702 were examined by qRT-PCR and Western blotting.Then,we manipulated GRP78 expression levels by stably transfecting cells with GRP78shRNA into Huh-7,MHCC97H,HCCLM3,and SMCC7721 cells.,RT-qPCR,Western blot method were performed to detect the expression levels of GRP78 and FAT10 mRNA and protein,the colony formation assays and Edu proliferation assays were performed to detect the cell proliferation's change.In addition,we increased the expression of GRP78 in MHCC97H,HCCLM3,SMCC7721,and HepG2 cells,T-qPCR,Western blot method were performed to detect the expression levels of GRP78 and FAT10 mRNA and protein.We stably transfected a GRP78-specific LV-shGRP78 lentivirus into MHCC97H and HCCLM3 cells,a nude mouse tumorigenicity assay was performed.,and observe the growth change of mice subcutaneous tumors.4,We upregulated FAT10 in GRP78-knockdown HCC cells,and measured GRP78 and FAT10 proteins expression levels and cell-proliferation abilities by Western blotting and Edu proliferation assays.Next,we knocked down FAT10expression in GRP78-overexpressing MHCC97H cells and analyzed GRP78 and FAT10 protein levels and cell-proliferation ability.6,Using co-immunoprecipitation,Western blotting and Immunofluorescence method to confirm whether the regulation of FAT10 by GRP78 was achieved through NF-?B pathway.Result:1,The IHC results indicated that the GRP78 protein was overexpressed in66.07%?74 of 112?of the HCC tissue samples and FAT10 protein was overexpressed in 63.39%?71 of 112?of the HCC tissue samples.The results demonstrated that both GRP78 and FAT10 overexpression were involved in tumor size,TNM stage,vascular invasion,and tumor encapsulation?P<0.05?.The clinical follow-up data for the HCC patient cohort demonstrated that overexpression of GRP78 and FAT10 positively correlates with poor overall survival?OS??P<0.05?.Furthermore,multivariate Cox regression analyses further displayed that upregulated GRP78 and vascular invasion was the independent predictive factors for poor OS in HCC?P<0.05?.2,Western blotting analysis and qRT-PCR experiments indicated that the expression levels of GRP78 and FAT10 were markedly increased in 112 HCCs samples?P<0.05?.Furthermore,scatter plots suggested that the expression levels of GRP78 and FAT10 mRNA and protein were positively correlated in HCCs?P<0.001?.3,the levels of GRP78 and FAT10 in in Huh-7,MHCC97H,Hep G2,HCCLM3,SMCC7721 and HL-7702 were examined by qRT-PCR and Western blotting,and GRP78 and FAT10 expression in HCC cells were higher than that in HL-7702 cells,and the GRP78 levels were positively correlated with FAT10 levels.Western blotting and qRT-PCR experiments indicated that GRP78 knockdown could reduce FAT10 expression levels in Huh-7,MHCC97H,HCCLM3,and SMCC7721cells?P<0.01?.The colony formation assays and Edu proliferation assays indicated that that GRP78 depletion inhibited proliferation in four HCC cells compared to the NC group?P<0.01?.In addition,qRT-PCR and Western blotting experiments indicated that the upregulation of GRP78 could increase FAT10 expression levels in the HCCLM3,MHCC97H,SMCC7721,and HepG2 cells?P<0.01?.Moreover,TumorsformedafterinjectionofMHCC97H-LV-shGRP78and HCCLM3-LV-shGRP78 cells demonstrated less volume and lower weight compared to tumors formed after the injection of control cells,demonstrating that the down-regulation of GRP78 significantly inhibited tumor growth?P<0.01?.4,The Western blotting results indicated that the down-regulation of GRP78reduced FAT10 expression,whereas overexpressed FAT10 decreased the loss of FAT10 expression in GRP78-knockdown HCCLM3 cells,and overexpressed GRP78markedly up-regulated FAT10 expression in MHCC97H cells,whereas this effect was significantly attenuated by FAT10-knockdown.5,Western blotting and Immunofluorescence method to confirm that the overexpression of GRP78 in MHCC97H and HCCLM3 cells accelerated nuclear NF-?Bp65 protein expression and increased FAT10 expression.Furthermore,GRP78activate NF-?B signaling by modulating the phosphorylation of IKK??14??and positively regulates FAT10 expression.Conclusion:GRP78 facilitates HCC proliferation by modulating FAT10 expression.GRP78modulated FAT10 expression via the NF-?B signaling pathway.Moreover,we found that GRP78 activated IKK??14??and I?B?and promoted the degradation of I?B?,releasing bound NF-?B p65 to translocate to the nucleus and thereby increasing FAT10 expression.The newly identified GRP78–NF-?B–FAT10 axis provides new insight into the proliferation of HCC and represents a valuable target for HCC therapy. |
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