| Liver cancer is the world's fifth most lethal malignant tumor,and is known for its high invasiveness and high recurrence.Among them,hepatocellular carcinoma(HCC)accounts for more than 90%.The ongoing research on anoikis resistance(AR),which is closely related to tumor invasion and metastasis,in liver cancer has always been in the marginal field.As an essential coagulation-related fibrinolytic factor in the development of HCC,PAI-1 has been shown to be closely related to the invasion and metastasis of HCC,but its clinical prognostic significance and basic research are often contradiction with each other.Whether PAI-1 factor plays an important regulatory role in HCC infiltration and metastasis,caused by the AR phenotype of tumor cells,there is no related report to date.Objective To explore the biological significance of PAI-1 in liver cancer and whether it regulates the anoikis resistance phenotype and specific regulatory mechanisms of liver cancer cells.Methods 1.Immunohistochemistry(IHC)was used to analyze the expression of PAI-1 in HCC tissues,and the relationship between PAI-1 and clinical prognosis(overall survival rate,OS)was analyzed combined with clinical baseline data;2.Western blot and RT-PCR were used to verify the relative mRNA and protein expression levels of PAI-1 in HCC cells;and the regulation of PAI-1 on the expression level of apoptosis protein in HCC cells was verified;to verify that PAI-1 regulates the expression levels of apoptotic proteins(cleaved caspase-3)and EMT protein expression levels(E-cahdarin,N-cahdarin,Vimentin);and to verify that PAI-1 regulates the expression of signal pathway proteins in HCC cells AR(PTEN,PI3K,p-85?,AKT and p-AKT);3.Stable cell lines of PAI-1 RNAi and their ovexpression in HCC progression were constructed by lentiviral vector tools to further verify that PAI-1 regulates the expression levels of mRNA and protein in HCC cells;4.Annexin V-FITC/PI method was used to detect the anoikis phenotype of HCC cells and PAI-1 to regulate HCC anoikis phenotype;5.Cell scratch healing experiment,trans well experiment and CCK-8 experiment were utilized to verify that PAI-1 regulates proliferation,infiltration and metastasis phenotype of HCC cells;6.Bioinformatics technology was also utilized to analyze the regulation of PTEN/PI3K/AKT signal in HCC by PAI-1 The downstream protein molecules and clinical significance of the pathway;7.In vivo HCC intrahepatic metastasis model was used to verify that PAI-1 regulates phenotypes of HCC infiltration and metastasis in vivo and related regulatory mechanisms.Results 1.Through the analysis and statistics of the clinical data and IHC results of 174liver cancer patients by this research group,it was found that the expression of PAI-1was correlated with the survival prognosis of HCC patients.The lower the PAI-expression,the worse the prognosis of liver cancer patients(Log-rank,p=0.027);Measure the Mean-IOD value of IHC slices of patients with liver cancer to verify the Para cancerous vs cancerous tissue(p<0.000),and with the upgrade of clinical staging,the expression of PAI-1 showed a significant downward trend;using COX single-factor regression analysis PAI-1 expression(HR=1.662,95%CL=1.380-2.546,p=0.000),multivariate COX regression analysis of PAI-1 expression(HR=2.303,95%CL=1.852-2.654,p=0.000),suggesting comparison and other factors,PAI-1 could be used as an independent prognostic factor,low expression was 1.662 times/2.303 times the predicted risk of high expression.2.The expression of PAI-1 gene in HCC cell line was low expression at protein and mRNA expression level(VS L02);the stable transgenic strain of lentivirus was constructed:PAI-1 high expression cell group(Hep G2,Huh 7)had three Knockdown targets,the lowest knockdown efficiency reached 80%or more(Hep G2 sh-PAI-1#53.3%,sh-PAI-2#84%,sh-PAI-3#23%;Huh sh-PAI-1#71%,sh-PAI-2#83.3%,sh-PAI-3#66.1%),the overexpression efficiency of the low expression group(SMMC7721 and MHCC-97H)reached more than 70%(MHCC-97H 84%,SMMC 772176.2%).Furthermore,the expression of PAI-1 in the high-expressing cell line was significantly decreased after being suspended for 48 hours,while the low-expression group sus 48h showed a significant upward trend(p<0.001,VS adherent 48h group).After knocking down the PAI-1 gene in the high-expressing cell line,The expression of PAI-1 in the cells suspended for 48h decreased significantly(p<0.001,VS sh-NC sus 48h).After overexpressing the PAI-1 gene in the low expression group,the expression of PAI-1 in the cell suspension for 48h increased significantly(p<0.001,VS OV-NC sus 48h);to verify the expression of cleaved-caspase 3,the apoptotic rate sus 48h decreased significantly in knockdown process of PAI-1 cell line(VS sus 48h sh-NC,p<0.001),while the overexpression of PAI-1 cell line was in opposition(VS sus 48h OV-NC,p<0.001).To explore EMT phenotype protein(N-cadherin,E-cadherin,Vimentin)phenotype protein changes,it was found that the higher the expression of PAI-1,the worse the infiltration and metastasis ability of suspension cells,and vice versa after 48 hours of cell suspension(VS sh-NC sus 48h/OV-NC sus 48h,p<0.001);PTEN,PI3K,PI3K phosphorylated protein p85?,AKT,and AKT phosphorylated protein p-akt expression levels as were analyzed respectively.After the cells in the sh group were suspended for 48 hours,the PTEN expression had an obvious decrease(VS sh-NC sus 48h,p<0.001),while the expression of p85?and p-akt increased(VS sh-NC sus 48h,p<0.001).Compared with the sh-NC group,the expression of PTEN was significantly reduced and the expressions of p85?and p-akt were increased after the cells of the sh group adhered to the wall for 48 hours.However,compared with the suspension group,the activation of downstream proteins in the signaling pathway was more obvious.Compared with the OV-NC group,the expression of PTEN increased,and the expression of p85?and p-akt decreased(VS OV-NC sus48h,p<0.001)after the cells were suspended for 48h in the OV group.There was also an increase,the expression of p85?and p-akt decreased,the change trend was not obvious(VS OV-NC sus 48h,p>0.05),the unphosphorylated protein PI3K,AKT did not change significantly compared with the NC group.3.Use flow cytometry Annexin V-FITC/PI to detect the apoptosis of highly expressing cells after PAI-1 gene knockout,the apoptosis rate of sus 48h decreased significantly(sus 48h sh-NC-Hep G2 43.32%±4.23%VS sus 48h sh-PAI-1-Hep G2 15.38±1.68%,sus 48h sh-NC-Huh 7 53.7%±7.23%VS sus 48h sh-PAI-1-Huh 7 15.35.7%±3.33%,p<0.001),and for the opposite was true in low-expressing cell lines(sus 48h OV-NC-SMMC 7721 21.41%±3.36%VS sus 48h OV-PAI-1-SMMC 7721 44.41±4.35%,sus48h OV-NC-MHCC-97H 17.21%±3.63%VS sus 48h OV-PAI-1-MHCC-97H 32.16%±3.88%,p<0.001).4.Scratch experiment showed that PAI-1 highly expressing cells,when PAI-1 gene was knocked out,the healing speed of the relative area of Hep G2 scratch was significantly faster than that of the sh-NC group(0.86 cm~2±0.21 cm~2 VS 0.14 cm2±0.06 cm~2,P<0.001),the trend of Huh 7 was the same as before(0.98 cm~2±0.16 cm~2 VS 0.57cm~2±0.21 cm~2,p<0.001).In addition,for the cells with low PAI-1 expression,compared with the OV-NC group,the trend was just in opposition as well.The healing speed of MHCC-97H scratches was automatically reduced(0.12 cm~2±0.02 cm~2 VS0.38 cm~2±0.11 cm~2,p<0.001),and the trend of SMMC 7721 was the same as before(0.29 cm~2±0.17 cm~2 VS 0.74 cm~2±0.18 cm~2,p<0.001)).Trans well experiment showed that when the PAI-1 gene was knocked out,the infiltration and metastasis ability of Hep G2 in cells with high PAI-1 expression was significantly enhanced compared with the sh-NC group(invasion:32±4 VS 8±3,p<0.001;migration:22±2 VS 6±3,p<0.001),the trend of Huh 7 was the same as before(invasion:298±14VS 78±13,p<0.001;migration:324±22 VS 85±16,p<0.001);PAI-1 low-expressing cells,when the PAI-1 gene was overexpressed,compared with the OV-NC group,the trend was just the opposite.The infiltration and metastasis ability of MHCC-97H was significantly reduced(invasion:23±5 VS 107±21,p<0.001;migration:21±4 VS 94±15,p<0.001),SMMC 7721 has the same trend as before(invasion:112±22 VS 198±13,p<0.001;migration:124±18 VS 237±26,p<0.001).Using cck-8 to detect the proliferation rate of HCC,the cell proliferation phenotype was basically the same in the NC group(OV-MHCC 97H VS OV-NC group,p=0.017;OV-SMMC7721 VS OV-NC group,p=0.024).5.According to various online bioinformatics prediction websites,we predicted that AKT1,as the main regulatory protein of PTEN and PAI-1,might participate in the activation/inactivation of PTEN by PAI-1 and regulate the PI3K/AKT signaling pathway.6.In order to construct a model of liver cancer metastasis regulated by PAI-1 in vivo,we performed HE staining on liver specimens of OV-NC and OV-PAI-1 MHCC-97H tumor-bearing NOD/SCID mice,and compared two groups of liver cancer metastases with photographs Correct.Compared with the OV-PAI-1 group,the OV-NC group had significantly more liver metastases and an unquestionably harder texture.Utilizing a small animal imaging technology to visualize the intensity of mouse CDX tumor formation and analyzing the average ROIs of tumors,significant results were obtained.It could be seen that the imaging intensity of the OV-NC group was significantly higher than that of the OV-PAI-1 group.Compared with the OV-NC group,the ROIs of the OV-PAI-1 group were significantly lower(3648±457 VS 9874±688,p<0.001).In order to verify the prognosis of HCC regulated by PAI-1 in vivo,a Kaplan-Meier survival curve was drawn.Compared with the OV-NC group,the survival period and duration of the OV-PAI-1 group was significantly prolonged(VS OV-NC,p=0.0037).Western-blot was used to verify the expression levels of PAI-1,PTEN,PI3K,p-85?,AKT and p-AKT protein in the tumor-bearing tissue proteins of the OV-NC and OV-PAI-1 groups of mice.Compared with the OV-NC group,the expression of PAI-1 in the OV-PAI-1 group significantly increased,and the expression of PTEN protein increased as well,the expression of p-85?and p-AKT protein decreased(p<0.001)marginally.However,while the expression levels of PI3K and AKT portrayed no significant changes.Conclusion This research and investigating team has systematically analyzed and verified the regulatory role of PAI-1 in liver cancer through clinical,in vivo and vitro experiments,and confirmed that:1.PAI-1 is generally low in HCC and is correlates with patient prognosis 2.PAI-1 is involved in the infiltration,metastasis,and proliferation of HCC phenotypes,which is closely related to AR phenotypes of HCC;3.PAI-1 mediates the AR phenotype of liver cancer cells by regulating the PTEN/PI3K/AKT signaling pathway to promote the invasion and metastasis of liver cancer. |
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