Study On The Mechanism Of NKD1 Protein Regulating Hepatocellular Cell Invasion And Proliferation Ability

Research Purpose 1.To explore the expression patterns and clinicopathologic significance of NKD1 in HCC;2.To explore the affection and mechanism of NKD1 on HCC cell migration and invasion ability;3.To study the affection and mechanism of NKD1 on HCC cell proliferation ability;Methods 1.Total protein was extracted from clinical HCC tissue samples,Western blot method was used to detect NKD1 protein express patterns and compared the level in HCC tissues with that in non-tumor tissues;2.Immunohistochemistry was used to detect NKD1 express location and level in HCC tissues.After a evaluation of the immunostaining results,we can get a score for each sample,X2 test was used to study the clinicopathologic significance of NKD1 in HCC;3.“Gain and loss-of-function” study and trans-well assay were used to explore the affection of NKD1 on HCC cell invasion ability.In addition,subcutaneously transplanted tumor metastasis model was used to verify the results get in vivo;4.In the condition of co-transfection of NKD1 and Rac1,we verify the critical function of Rac1 in NKD1 regulating HCC invasion ability through restore experiment;Immunofluorescence analysis was performed to explore the affection of NKD1 on HCC cytoskeleton rearrangement;5.We used Co-immunoprecipitation and Immunofluorescence analysis to explore the combination of NKD1 and Rac1,ubiquitination assay was used to find out the mechanism for the degradation of Rac1;6.Real-time PCR assay has proven that Rac1 can promote the transcription of NKD1.What's more,Luciferase reporter assays and co-transfection has also proven that this function depend on the regulation of EZH2;7.K-M survival analysis find out that abnormal expression of NKD1 and Rac1 is associated with a poor prognosis of HCC patients;8.CCK-8,Ed U and clone formation assays was used to analyze the influence of NKD1 on HCC cell proliferation capacity,and subcutaneously transplanted tumor model was used to confirm in vitro;9.By comparing with the expression level of PCNA protein on the condition of adding p53 inhibitor to the NKD1 over-expression cell,we confirm that NKD1 regulates HCC cell proliferation is partially depend on p53;10.NKD1 m RNA expression pattern in HCC tissues was detected by Real-time PCR assay,and the clinical correlation between NKD1 m RNA expression and tumor size,survival time was analyzed by statistical analysis.Results 1.Western blot assay find out that NKD1 protein expression is down-regulated in HCC tissues compared to adjacent non-cancerous tissues;2.Expression of NKD1 protein was detected in 58 HCC specimens by immunohistochemcal staining,thirty two(55.2%)samples showed a lower level,and the level of NKD1 protein expression in HCC was significantly associated with poor differentiation(p=0.003),intra or extra-hepatic metastasis(p=0.010)and tumor size(p=0.011);3.Migration and invasion experiments showed that ectopic expression of NKD1 significantly inhibited HCC cell migration and invasion ability of SMMC-7721 and Huh7 cells.Conversely,when down-regulation of NKD1 in Hep G2 cells,invasion and migration data showed the opposite results.In addition,the mice injected with SMMC-7721+NKD1 cells displayed less pulmonary and intra-hepatic micro-metastatic nodules and a lower metastasis ratio than that injected with SMMC-7721+Control,all of this reveal that NKD1 negatively regulate HCC cell invasion and migration ability in vivo and in vitro.4.Ectopic expression of NKD1 in HCC cells down-regulated Rac1 protein expression,NKD1 over-expression induced inhibition of invasion and migration was strongly restored by the expression of Rac1.Mechanism study find out that NKD1 had an effect on the cell cytoskeleton arrangement based on the change of Rac1.5.By using immunofluorescence laser scanning confocal microscopy and coimmunoprecipitation,we find out that a significant portion of NKD1 co-localized and combine with Rac1 in the cytoplasm of SMMC-7721 cells.Western blot analysis indicated that NKD1 significantly shortened the half-life of the Rac1 protein and mechanism study uncover that NKD1 interacts with Rac1 and promotes its degradation by inducing polyubiquitination-mediated proteasomal degradation;6.Rac1 can promote NKD1 transcription and up-regulated NKD1 at the m RNA and protein levels in HCC cells,mechanism study uncover that Rac1 over-expression markedly decreased the protein level of EZH2 which contribute to the up-regulation of NKD1 transcriptional activity;7.NKD1 was down-regulated at the m RNA level in HCC tissues compared with non-tumor tissues,whereas Rac1 protein was up-regulated in HCC tissues.Moreover,the patients with relatively lower NKD1 and higher Rac1 expression in tumor tissues compared with paired non-tumor had a shorter overall survival time(P= 0.029);8.By using CCK-8,Ed U and clone formation assay,we find out that downregulation of NKD1 promotes HCC cell proliferation,whereas enhanced NKD1 expression inhibits proliferation ability in vivo and vitro;9.Western blot detection show that the protein expression of p53 and p21 was significantly increased and PCNA expression was decreased in the NKD1 over-expression cells compared with the control group.And this affection was restored by the treatment of p53 inhibitor,pifithrin-a.10.NKD1 m RNA expression in HCC tissues was negatively correlated with tumor size(P=0.002)and poor prognosis(P=0.029).Conclusion 1.NKD1 protein levels were significantly lower in cancerous tissues compared with corresponding normal tissue.In addition,the level of NKD1 protein expression in HCC was significantly associated with tumor size,intra or extra-hepatic metastasis and differentiation;2.NKD1-mediated negative regulation of HCC cell invasion and migration is dependent on Rac1;3.NKD1 interacts with Rac1 and promotes its degradation through the ubiquitin-proteasome pathway;4.Rac1 activates NKD1 transcription in HCC cells by down-regulating EZH2 expression;5.Abnormal expression of NKD1 and Rac1 is associated with a shorter post-operate survival time of HCC patients;6.NKD1 negatively regulate HCC cell proliferation partially via p53;7.NKD1 m RNA expression in HCC tissues correlates with tumor size and a poor prognosis of HCC patients.