PinX1 Serves As A Prognostic Indicator For Renal Cell Carcinoma And Inhibits Its Metastasis Via NF-?B/MMP-2 Signaling Pathway

Objective This project intends to study PinX1 expression status and its correlation with the clinicopathological features in human renal cell carcinoma?RCC?via two independent human RCC tissue microarray?TMA?and study the molecular mechanism that PinX1 regulates metastasis of RCC in vitro and in vivo.Methods 1.The clinical relevance of PinX1 in RCC was evaluated using tissue microarray and immunohistochemical staining in two independent human RCC cohorts which contained 353 cases RCC tissues altogether.Immunohistochemistry staining was utilized in TMA slides to evaluate the PinX1 expression in normal renal tissues,renal cell carcinoma tissues and paired adjacent non-tumor tissues.Kaplan-Meier survival curves were constructed using 5-year overall or disease-specific cumulative survival to compare the patients with high PinX1 staining to those with low PinX1staining.Cox regression analysis was used to evaluate if PinX1 could be a prognostic indicator for renal cell carcinoma patients.Univariate and multivariate Cox regression analysis indicated that PinX1 may serve as a molecular prognostic marker for this aggressive disease.2.We transiently transfected 786-O and ACHN cells with pEGFP-C3-control and pEGFP-C3-PinX1 plasmids or control siRNA and PinX1 siRNA,respectively.Forty-eight hours after transfection,PinX1 protein expression was detected by Western blot.The proliferation ability of RCC cells with PinX1 overexpression or suppression was detected by CCK-8 assay.The migration and invasion assay were used to detect the migration and invasion abilities of 786-O and ACHN cells after PinX1 knockdown or overexpression.Gelatin zymography or Western blot was used to detect MMPs'activities or protein expressions.Migration and invasion assays were used to investigate the migration and invasion abilities when MMP-2 pathway was interrupted by MMP-2 inhibitor in renal cancer cells with PinX1 knockdown.Western blot and RT-PCR were used to examine the protein and mRNA expression of NF-?B p65 subunit in PinX1 overexpressed and suppressed RCC cells.Western blot was used to examine p65nuclear translocation in RCC cells with PinX1 knockdown.Migration and invasion assays were used to investigate the migration and invasion abilities of PinX1 and p65co-suppressed RCC cells.3.The PinX1 overexpressed 786-O cell line(PinX1^OE-786-O),PinX1 suppressed786-O cell line(PinX1^KD-786-O)and control 786-O cell line?Ctrl-786-O?were established by infecting with lentivirus packing PinX1 expression vector,PinX1shRNA expression vector and control vector respectively.Target cells were infected with lentivirus for 48 hours then selected with puromycin for 3 weeks.PinX1 protein levels of these cell lines were confirmed by western blot.Three groups of nude mice were injected through tail vein with PinX1^OE-786-O,PinX1^KD-786-O and Ctrl-786-O cells respectively.After 2 months,three groups of mice were sacrificed and their lungs were resected and fixed in 10%buffered formalin for metastatic nodules counting and further histopathological analysis.Immunohistochemical staining was used to detect PinX1,MMP-2 and p65 expression in metastatic nodules on lungs resected from nude mice.Results 1.PinX1 expression is decreased in RCC tissues compared with paired adjacent non-tumor tissues and normal renal tissues.Fisher's exact analysis revealed that PinX1 expression in the carcinoma tissues of the training cohort conspicuously correlated with some clinicopathological features,such as depth of invasion-pT status?P=0.018?,lymph node metastasis-pN status?P=0.043?,and TNM stage?P=0.013?.These findings were confirmed in the validation cohort of RCC patients.Fisher's exact analysis revealed that PinX1 expression in the carcinoma tissues of the validation cohort conspicuously correlated with some clinicopathological features,such as depth of invasion-pT status?P=0.001?,lymph node metastasis-pN status?P=0.006?,and TNM stage?P=0.002?.Kaplan-Meier survival curves were constructed using 5-year overall or disease-specific cumulative survival to compare the patients with high PinX1staining to those with low PinX1 staining.Our data revealed that low PinX1 staining correlated with both worse overall and disease-specific survival in RCC?P=0.002 and P=0.002,respectively,log-rank test?.2.We transiently transfected 786-O and ACHN cells with pEGFP-C3-control and pEGFP-C3-PinX1 plasmids or control siRNA and PinX1 siRNA,respectively.Forty-eight hours after transfection,PinX1 protein was significantly overexpressed or knockdown in RCC cancer cells,respectively.Overexpression or silence of PinX1 had no effect on the proliferation of RCC cells?P>0.05?.In the migration assay,we found that PinX1 knockdown in 786-O and ACHN cells significantly enhanced the ability to migrating through transwell filter inserts respectively?***,P<0.001?.Inversely,PinX1 overexpression in 786-O and ACHN cells dramatically suppressed the migration ability of RCC cells?***,P<0.001?.In cell invasion assay,we got the similar conclusion:PinX1 knockdown can enhance the invasion ability of 786-O and ACHN cells?***,P<0.001?;PinX1 overexpression in 786-O and ACHN cells can suppress their invasion ability?***,P<0.001?.To investigate the mechanisms of PinX1regulating migration and invasion in RCC cells,we performed western blot to detect the MMPs protein levels in RCC cells and gelatin zymography to detect the MMPs activity.Our data showed that the MMP-2 expression and activity were negatively regulated by PinX1 in 786-O and ACHN cells,but not MMP-9.We also detected the expression of TIMP protein by Western blot.Data showed that MMP-2 expression was up-regulated or down-regulated corresponded with PinX1 knockdown or overexpression,however,the expression of TIMP-2 had not changed correspondingly.TIMP-1 expression was also not changed,it is the same as the expression of MMP-9.We added MMP-2 selective inhibitor?at the same time of PinX1 siRNA transfecting into 786-O and ACHN cells.As expected,the up-regulation of MMP-2expression and activity was blocked by MMP-2 selective inhibitor I.We also validated this phenomenon by migration and invasion analysis.The migration and invasion ability can be enhanced by inhibiting of PinX1 in 786-O and ACHN cells,however,these regulations were blocked by MMP-2 selective inhibitor I?***,P<0.001?.Above all,it was confirmed that PinX1 inhibited RCC cells'migration and invasion by suppressing MMP-2 expression and activity.Our data demonstrated that PinX1inhibited RCC cells'migration and invasion abilities by down-regulating MMP-2expression and activity in vitro,and this regulation seems not depend on TIMP-2.Then we determined the protein and mRNA levels of NF-?B-p65 in 786-O and ACHN cells after PinX1 overexpression or knockdown.Western blot and RT-PCR results showed that the levels of NF-?B-p65 protein and mRNA were increased sharply in 786-O and ACHN cells after PinX1 knockdown and dramatically down-regulated in786-O and ACHN cells after PinX1 overexpression.Moreover,we determined the cellular distribution of p65 in RCC cells by immunoblot after PinX1 knockdown.Inhibition of PinX1 significantly increased the nuclear distribution of p65.Together,these data indicate that inhibition of PinX1 promotes p65 expression and nuclear localization,activates NF-?B-induced transcription.To further confirm whether NF-?B activation induced by PinX1 inhibition caused the up-regulation of MMP-2 in human RCC cells,Western blot analysis showed that NF-?B-p65 siRNA can inhibit the expression of NF-?B-p65 and MMP-2 in 786-O and ACHN cells.The level of MMP-2 protein was up-regulated by PinX1 suppression in RCC cells,but these effects were further blocked by knockdown of NF-?B-p65 expression with p65 specific siRNA.These data provided further evidence that PinX1 may regulate MMP-2 expression via NF-?B transcription factor.We also validated the mechanism by migration and invasion analysis.The migration and invasion abilities can be enhanced by PinX1inhibition in 786-O and ACHN cells,however,this effect was abolished by co-suppressing PinX1 and NF-?B-p65 in RCC cells.3.PinX1^OE-786-O cell line,PinX1^KD-786-O cell line and Ctrl-786-O cell line were established as described previously.After 3 weeks selection following with lentivirus infection,the PinX1 protein levels of these cells were confirmed by western blot.The result showed that PinX1 protein was significantly overexpressed or knockdown in PinX1^OE-786-O cell lines or PinX1^KD-786-O cell lines,respectively.Three groups of nude mice were injected through tail vein with PinX1^OE-786-O,PinX1^KD-786-O and Ctrl-786-O cells respectively.After 2 months,three groups of mice were sacrificed and their lungs were resected and fixed in 10%buffered formalin for metastatic nodules counting and further histopathological analysis.Randomly selected metastatic nodules had been validated by H&E staining.Extensive tumor formation was found in PinX1^KDD group.In contrast,the lungs in PinX1^OE group had fewer and smaller detectable tumor nodules.A statistically dramatic increase in the number of the lung metastases was seen in PinX1^KD group,compared with the PinX1^OE group??² test,***,P<0.001?and these two groups also had significant diversity compared with Ctrl group respectively??² test,***,P<0.001?.Immunohistochemical staining of metastatic nodules in lungs resected from nude mice showed that MMP-2 and NF-?B-p65 expression in PinX1^OEE group were much lower compared with PinX1^KD group and Ctrl group but TIMP-2expression was not change in every group.These had further validated our conclusion which had been demonstrated previously in vitro.These findings indicate that PinX1 suppresses RCC metastasis via NF-?B/MMP-2signaling pathway and may serve as a RCC candidate clinical prognostic marker and a potential therapeutic target.Conclusion Our data demonstrated that PinX1 expression was dramatically decreased in RCC tissues compared with normal renal tissues and paired adjacent non-tumor tissues.Low PinX1 expression was significantly correlated with depth of invasion,lymph node metastasis and TNM stage in patients,as well as with worse overall and disease-specific survival.Cox regression analysis revealed that PinX1expression was an independent prognostic factor for RCC patients.Moreover,PinX1inhibited the metastasis of RCC cells by suppressing MMP-2 expression and activity via NF-?B-dependent transcription in vitro and in vivo.These findings indicate that PinX1 suppresses RCC metastasis and may serve as a RCC candidate clinical prognostic marker and a potential therapeutic target.