The Role And Regulating Mechanism Of PIGU In Hepatocellular Carcinoma

Background:Hepatocellular carcinoma(HCC)is the sixth most common malignancy and has high mortality rate.Many risk factors including infection with hepatitis B virus and hepatitis C virus,diabetes,excessive alcohol consumption,obesity,and smoking are associated with HCC.Although great progress has been made in the diagnosis and treatment of HCC,morbidity and mortality rates continue to increase in many areas.Most patients were diagnosed at an advanced stage usually.Therefore,it is of great significance to explore new therapeutic targets and strategies for patients with HCC.Phosphatidylinositol glycan anchor biosynthesis class U(PIGU)is an important subunit of glycosylphosphatidylinositol transaminase(GPI-T)complex.There is increasing evidence that the GPI-T complex plays a vital role in the development of cancer.It has been reported that PIGU was overexpressed in HCC,and overexpression of PIGU indicated poor survival in HCC patients.while the function of PIGU in HCC remains unknown.Previous studies have shown that the NF-?B signaling pathway is a critical participant in the initiation and progression of cancer.NF-?B signaling pathway was abnormally expressed and activated in HCC cells specifically.Therefore,in present study,we explored the function of PIGU in HCC progression and its mechanism.We hypothesized that PIGU may play a role in regulating the biological function of HCC cells,possibly by activating the NF-?B signaling pathway.Object:To observe the expression of PIGU in HCC cells,explore PIGU's influence on the proliferation,migration,invasion and other biological functions of HCC,and clarify the function of PIGU in the occurrence and development of HCC and its mechanism.Methods:Part 1: Bioinformatics analysis of PIGU in HCC and immunohistochemical detection of clinical HCC specimensFrom the Gene Expression Profiling Interactive Analysis(GEPIA)website,PIGU expression and related clinical data from TCGA liver cancer(LIHC)dataset were extracted,and bioinformatics analysis was performed.10 Clinical specimens were collected randomly to detect PIGU protein expression levels using immunohistochemistry,from patients with primary HCC who underwent liver tumor resection in the Affiliated Hospital of Xuzhou Medical University from September 2019 to October 2019.Part 2: Vitro experiment1.To detect the expression levels of PIGU in HCC cells and the effect of downregulation of PIGU on the biological processes of HCC cells(1)The expression levels of PIGU in 1 normal liver cell line and 6 HCC cell lines were detected by Western blot,and two cell lines(Hep-3B and Huh-7 cell lines)with relative high-expressing of PIGU were selected for subsequent experiments,meanwhile immunofluorescence was performed to determine PIGU localization in the two cell lines.(2)2 pairs of PIGU small interfering RNAs(si1-PIGU and si2-PIGU)were designed and synthesized,si-PIGUs or its negative control(si-NC)were transfected into Hep-3B or Huh-7 cell lines for 48 h,the knockdown efficiency was verified by RT-PCR and Western blot respectively.0,24,48 72,96 h after transfection,cell proliferation was detected by CCK-8 experiment.48 hours after transfection,cell cycle distribution and apoptosis were detected by flow cytometry,the expression levels of c-myc,PCNA,Cyclin D1,c-caspase 3 and c-PARP were detected by Western blot.?48 hours after transfection of 2 pairs of si-PIGUs into Hep-3B or Huh-7 cell lines,the cell migration ability was detected by scratch experiment,the cell invasion ability was detected by transwell experiment.Subsequently,the expression levels of MMP2 and MMP9 were measured by Western blot,and the activity of MMP2 and MMP9 in cell supernatant was measured by gelatin zymography method.2.To verify that PIGU regulates the biological function of HCC cells through NF-?B signaling pathway? 48 hours after transfection of two PIGU si-RNAs into Hep-3B or Huh-7 cell lines,the protein expression levels of total p65,p-p65(Ser536),p-p65(Ser 276),I?B? and p-I?B?(Ser 32/Ser36)in Hep-3B or Huh-7 cells was detected by Western blot.Nuclear and cytoplasmic separation Western blot was used to detect the expression levels of p65 in the nucleus and cytoplasm,respectively.Furthermore,immunofluorescence was performed to detect the expression levels and location of p65.? Hep-3B and Huh-7 cells were transfected with PIGU overexpression vector(p CMV3-PIGU),followed by PDTC(NF-?B pathway inhibitor)treatment in NF-?B pathway blocking group 24 h after transfection.48 h after transfection,Western blot was performed to verify the PIGU overexpression efficiency.The cell proliferation was detected by CCK-8 experiment,the cell migration ability was detected by scratch experiment,and the cell invasion ability was detected by transwell experiment.Meanwhile,the protein expression levels of c-myc,c-caspase 3,MMP2,p65 in nucleus and p65 in cytoplasm were detected by Western blot.Part 3: Vivo Experiment and cellular immunity experiment1.To verify the effect of PIGU down-regulation on tumorigenicity of HCC cells in vivo2 pairs of PIGU sh-RNAs(sh1-PIGU and sh2-PIGU)were designed andsynthesized,shRNA-PIGUs or their negative control(shRNA-NC)was transfected into Hep-3B or Huh-7 cell lines for 48 h to construct stable PIGU down-regulated HCC cell lines.RT-PCR was performed to verify the knockdown efficiency of PIGU.Selected the cells in logarithmic growth phase,and injected them into the subcutaneous tissue of nude mice(one cell line in three groups).Measured the diameter of the tumor tissue and calculated the volume every 3 days after injection.All the mice were killed 21 days after injection.Tumor tissues and tumor pictures were taken,tumor growth curves and tumor weight were obtained.The protein expression levels of PIGU,c-myc,MMP2,and c-caspase3 in tumor tissues were detected using Western blot and immunohistochemistry.2.Cellular immunity experiment2 pairs of PIGU si-RNAs were transiently transfected into Hep-3B or Huh-7 cells,and the protein expression levels of PIGU were measured 24 h after transfection by Western blot.24 h after transfection,Natural killer(NK)-92 cells were co-incubated with Hep-3B or Huh-7 cells transfected with si-PIGU at a ratio of 1:2,1:1,and 2:1,respectively.After 48 hours of co-incubation,CCK-8 experiment was performed to detect the toxicity of NK cells,Enzyme-linked immunosorbent assay was used to detect the levels of Granzyme B,MIP-1?,IFN-? and TNF in cell supernatant.Results:Part 1: PIGU highly expressed in HCC cells and was associated with prognosis and the protein expression level of p65Bioinformatics analysis showed that PIGU was overexpressed in HCC tissues compared with normal liver tissues,and the expression levels of PIGU and P65 in HCC tissues were positively correlated.Moreover,the overall survival and disease-free survival were significantly shorter in HCC patients with high PIGU expression than in patients with low PIGU expression.The expression level of PIGU was related to HCC staging.PIGU protein expression levels were higher in tumors than those in paired para-tumor tissues of patients with HCC in TMN stage ? ~ ?,as detected by immunohistochemistry.Part 2: Cell experiment in vitro1.The expression of PIGU was elevated in HCC cell lines.Down-regulating of PIGU inhibited proliferation,migration,invasion and promoted apoptosis of HCC cells.?Compared with the normal liver cell line LO2,the protein expression levels of PIGU in other 6 HCC cell lines were significantly higher.Moreover,Hep-3B and Huh-7 cell lines with relative high PIGU protein levels were selected for subsequent experiments.?Si1-PIGU,si2-PIGU or their negative control(si-NC)were transfected into Hep-3B and Huh-7 cells.The m RNA and protein expression levels of PIGU decreased significantly 48 h after transfection,which indicating the transfection was successful.CCK-8 assay was performed after transfection.Results showed that PIGU knockdown significantly decreased the viability of HCC cells at a time-dependent manner.The cell cycle distribution and apoptosis were measured by flow cytometry,and the results showed that the downregulation of PIGU resulted in the accumulation of HCC cells in the G1 phase and promoted cell apoptosis.it was found by Western blot that the protein expression levels of c-myc,PCNA and cyclin D1 decreased,and those of c-caspase 3 and c-PARP increased dramatically after si-PIGU transfection.? 48 h after transfection,the cell scratch assay showed inhibited cell migration ability in the PIGU knockdown groups,and the transwell assay showed inhibited cell invasion ability as well.Meanwhile,the results of Western blot and Gelatin zymography assay showed that PIGU silencing significantly decreased the protein expression levels and activity of MMP2 and MMP9 in Hep-3B and Huh-7 cells as compared with si-NC group.2.PIGU affected the biological functions of HCC cells via NF-?B signaling pathway? 48 h after transfection with 2 pairs of PIGU si-RNAs,Western blot was used to detect the protein expression levels.The results showed that protein expression levels of p-p65(Ser536),p-p65(Ser276),and p-I?B?(Ser32/Ser36)in the PIGU knockdown groups were decreased,and that of I?B? was elevated significantly as compared with si-NC group.Meanwhile,Western blot and immunofluorescence staining results showed that compared with si-NC group,the protein expression level of p65 in the cytoplasm was increased,and that in the nucleus was decreased obviously in the PIGU knockdown groups.? 48 h after the transfection with PIGU overexpression vector,the protein expression levels of PIGU in Hep-3B and Huh-7 cells were significantly increased as verified by Western blot.Then CCK-8 experiment was performed to detect cell proliferation.The results showed that compared with the control group,the cell proliferation rate of the PIGU overexpression group increased.The cell scratch experiment and transwell experiment showed that PIGU overexpression increased the cell migration ability and cell invasion ability significantly.In addition,compared with the control group,the protein expression levels of c-myc,MMP2 and p65 in the nucleus were increased,whereas that of p65 in the cytoplasm and c-caspase 3 were decreased as measured by Western blot.But PDTC(NF-?B pathway inhibitor)reversed these changes.Part 3: Animal experiment and cellular immunity experiment1.Downregulation of PIGU negatively regulated the tumorigenicity of HCC cells in vivosh1-PIGU,sh2-PIGU or their negative control(sh RNA-NC)were transfected into Hep-3B and Huh-7 cells.The m RNA expression levels of PIGU were decreased significantly 48 h after transfection.On the 13 th day after Hep-3B and Huh-7 cells injection into the subcutaneous tissue of nude mice,the tumor volume of the mice in sh RNA-PIGU groups were decreased significantly compared with the control group.Additional,the tumor weight on the 21 st day after injection showed the same trend.Results of Western blot assay showed that the protein expression levels of PIGU,cmyc and MMP2 of sh RNA-PIGU groups were significantly decreased but that of the c-caspase 3 were increased in tumor tissues as compared with the control group.The same changes were also confirmed by immunohistochemistry.2.PIGU knockdown prevented HCC cells from escaping from immune surveillance24 h after Hep-3B and Huh-7 cells were transfected with PIGU si RNAs or si-NC,Western blot was used to verify the knockdown efficiency of PIGU.24 h after transfection,NK cells(NK-92)and HCC cells were co-incubated at a ratio of 1:2,1:1,and 2:1,respectively.After 48 h of co-incubation,CCK-8 experiment was used to detect the toxicity of NK cells.It was found that down-regulating of PIGU increased the sensitivity of Hep-3B and Huh-7 cells to NK-92 cells.Results of ELISA assay showed that before co-incubation with NK-92 cells,the protein expression levels of Granzyme B,MIP-1?,IFN-?,and TNF-? remained stable in the knockdown groups.However,after co-incubation with NK-92 cells,Granzyme B,MIP-1?,IFN-?,and TNF-? protein expression levels in Hep-3B and Huh-7 cells were increased by PIGU silencing.Conclusions:In hepatocellular carcinoma,PIGU protein expression level is significantly increased.By affecting the NF-?B signaling pathway,PIGU promotes proliferation,migration,invasion of hepatocellular carcinoma cells and inhibits apoptosis.Moreover,it promotes immune escape of hepatocellular carcinoma cells,thereby promoting the progression of liver cancer.