| Part one:the expression of USP22and SIRTl and its clinicopathologic significance in clear cell renal cell carcinomaBackground and objective:Clear cell renal cell carcinoma is related to the abnormal of multiple genes, while USP22was a novel tumor stem marker protein which was found in recent years and predicted to be interacted with SIRT1. Therefore, we detected the expression of USP22and SIRT1in the ccRCC and study its clinicopathologic significance.Methods:The expression of USP22and SIRT1were predicted on gene profile Web site and their expressive difference between renal tumor cell line and renal normal cell line were confirmed through western blot. And then, we took full advantage of surgical ccRCC samples to make tissue chip so as to analyze the cellular localization and expression level of USP22and SIRT1using IHC. Finally, we studied the expression of USP22and SIRT1and its clinical significance conbining with clinicopathologic parameter and survival information from telephone follow-up.Results:1. The expression of USP22and SIRT1in ccRCC tissue and renal tumor cell line are all higher than normal renal tissue and normal renal cell line.2. The paried clinical samples display the development of ccRCC often accompany with the high expression of USP22and SIRT13. USP22and SIRT1co-localize the nuclear and their expressions positively correlate with age, depth of tumoral invasion, metastasis, TNM and histopathological grade.4. The follow-up rate is per cent64.36in all available376patients. There are185healthy survivals,16metastasis and41deaths. TNM I, II, III and Ⅳ respectively take up68.2%(165/242),7.4%(18/242),19.8%(48/242) and4.5%(11/242) among follow-up patients. Radical nephrectomy (open266, laparoscopy42) were performed for308patients,58patients were treated with partial nephrectomy (open57, laparoscopy1).5patients underwent radical nephrectomy and venous thrombus drawing. Radical nephrectomy conbining with adrenalectomy were performed for3patients. Radical nephrectomy with splenectomy or right hemicolectomy was respectively performed for1patient. Patients’diseases-specific and diseases-free survival rate to date was83.1%(201/242) and76.4%(185/242) respectively. While patients’5-year diseases-specific and diseases-free overall survival rate was respectively83.5%(202/242) and76.9%(186/242).5. Log-rank univariate analysis indicated the survival closely relate with age less than60years, diameters of tumor small than8centimeters, tumoral depth of invasion, metastasis, TNM, grade, the expression of USP22and SIRT1, but not relate with gender.6. By Cox multivariate regressive analysis we found histopathological grade, USP22and tumoral depth of invasion were independent risk factor of survival, whereas other survival associated factors, such as age, tumor size, depth of tumoral invasion, TNM, SIRT1were non-independent risk factor.7. There is moderate correlations between USP22and SIRT1among tumor or normal tissue in the46paired samples(r=0.586/0.711, p<0.01). At mean time, the close relationship of USP22and SIRT1is found among all376sampeles (r=0.379, p<0.01).Conclusions: USP22and SIRT1co-localize in cell nucleus and their expressions not only closely correlate with each other, but also positively relate with malignancy of clear cell renal cell carcinoma, which implys that independent risk factor USP22may cooperate with the survival associated factor SIRTl to take part in the development of clear cell renal cell carcinoma. USP22and SIRT1should have potiential value for predicting disease’s progress and molecular target therapy. Part two:the molecular mechanism underlying USP22coporating with SIRT1to take part in progress of clear-cell renal cell carcinomaBackground and objective:The first part indicated USP22and SIRT1co-localize in the nucleus and their expressions not only closely correlate with each other, but also positively relate with malignancy of ccRCC, USP22cooperate with SIRT1to take part in the development of ccRCC. Here, we continutely study the molecular mechanism underlying the close relationship between the expression of USP22and SIRT1and its clinicopathological on the ccRCC.Methods:The plasmids of USP22, SIRT1and their mutants (USP22C185S, SIRT1H363Y) and truncations were constructed and transfected into cell to locate on the protein expressive site. The interaction of USP22and SIRT1, modified relationship, modified results were investigated through co-immunoprecipitation assay and reporter gene experience. The expression of USP22and SIRT1was also knockdowned by shUSP and shSIRTl respectively, and confirmed through western blot in order to assay their roles in the proliferation and apoptosis of tumor using CCK-8and FCM.Results:USP22, SIRT1, USP22C185S, SIRT1H363Y and the truncations of USP22and SIRT1are all co-localized in the nucleus. USP22interacts with SIRT1as well as inhibits the ubiquitinated-degradation of SIRT1, which results in functional enhancement of SIRT1. Knockdown of USP22and SIRT1could upregulate the expression of P53and P21and also inhibit cell proliferation and induce cell apoptosis.Conclusions:Nuclear USP22and SIRT1interact with each other, USP22enhances the function of SIRT1through inhibitting ubiquitinated-degradation of SIRT1by dequbiqutination. USP22and SIRT1could cooperate with each other to inhibit P53and P21to play a key role in the development of clear cell renal cell carcinoma. |
