Role And Mechanism Of Gankyrin In The Invasion And Progression Of Bladder Cancer

?Background?Bladder cancer(BC)is the most common malignancy of the urological system in China.In the light of the pathological stages,BC is classified as non-muscle-invasive BC(NMIBC)and muscle invasive BC(MIBC).70% of newly diagnosed BC are NMIBC,mainly treated with TURBT,with 5 year survival rate beyond 90%.15% of patients with NMIBC could gradually develop into MIBC,where patients have low survival rate after surgery resection of bladder tumor.The remaining 20-40% were MIBC,treated mainly with radical cystectomy,50% of which will develop metastatic disease after surgery,with 5 year survival rate around5%.Obviously,high-invasion BC pose a threat to human health and it is pretty critical to clarify the mechanism of BC invasion and progression for surgical treatment and prognosis of BC patients.Gankyrin plays a vital role in lots of tumorigenesis.Gankyrin was first found in the study of hepatocellular carcinoma and then with the further study,it was discovered that Gankyrin was high-expressed in colon,pancreatic,cervical,lung and esophageal cancers,being associated with malignant phenotypes.At present,the specific molecular mechanism of Gankyrin about the occurrence,development and infiltration of BC has not been clearly defined and clarified.In the early stage,we sequenced the transcriptome of 10 patients with BC and the results showed that there were high expression of Gankyrin in BC tissues compared with normal tissues.Traditional theory of molecular origin of BC and the results from recent High-throughput sequencing suggest a crucial role of p53 related pathway in invasion of BC.Gankyrin was proved to be the upstream regulator of p53.Thus,it is speculated that the high expression of Gankyrin may be related to the invasion and progression of BC.?Objective?This study aimed to research the role and mechanism of Gankyrin in the invasion and progression of BC,which could shed light on targeted therapy and prognosis of BC.?Materials and Methods?1.The transcriptome of 10 patients with BC tissues and normal tissues were sequencedby RNA-seq technology.Gankyrin was screened out as candidate gene to further verify and explore its function according to literature review.2.The expression levels of Gankyrin in tumor tissues and normal tissues were detected by qRT-PCR from 90 BC patients and by western bolt from 20.3.The high expression of Gankyrin in tumor tissues of BC patients was verified by mining data from public database called Oncomine.4.The expression of Gankyrin was observed by immunohistochemistry in 3 MIBC tumors locating at the mucosa,superficial muscular layer and deep muscular layer of a single BC tissue slide.Combining with immunohistochemical results of 130 BC patients and follow-up results,the relationship between Gankyrin and pathological stages,grades and prognosis of BC were explored.5.The expression of Gankyrin in normal urothelium 2 NMIBC cell lines and 2 MIBC cell lines were detected by Western blot and qRT-PCR.6.Constract Gankyrin stably high-expressed BIU87 cell line and Gankyrin stably knock-down UMUC3 cell line.The effects of gankyrin on the proliferation of BC cells were detected by the EdU cell proliferation assay and subcutaneous tumor model in NOD/SCID mice.The effect of Gankyrin on the migration and invasion of BC cells were detected by cell scratch assay and Transwell chamber migration and invasion assay.The effect of Gankyrin on apoptosis of BC cells were detected by flow cytometry.7.The expression of Gankyrin was detected by immunohistochemistry and its significance was discussed by the BBN-induced spontaneous bladder cancer of mice.8.The possible mechanism of Gankyrin involved in BC was detected by correlation analysis of Gankyrin and MDM2 expression by immunohistochemistry assay in 32 paraffin embedded sections of BC patients.The effect of Gankyrin overexpression and knock-down on MDM2 expression was determined by western bolt.Immunofluorescence and co-immunoprecipitation were applied to explore the interactions of Gankyrin and MDM2.?Results?1.The results of RNA-seq showed that the expression of Gankyrin gene in bladder cancer was significantly higher than adjacent normal urothelium(P<0.05),with a log2-fold change of 1.21,and its RPKM value in tumor samples from 9 patients were higher than in the corresponding adjacent normal urothelium.2.The expression of Gankyrin mRNA in 90 paired samples was further detected by qRT-PCR.The expression of Gankyrin mRNA was confirmed to be up-regulated in 66 samples,and Gankyrin expression in other 24 samples was lower than that in adjacent normal tissues,with a high-expressed rate of 73.3%.Furthermore,twenty paired bladder cancer samples and adjacent normal tissues were validated by western blot.The expression of Gankyrin protein in tumor tissues was significantly higher than that in adjacent normal tissues in 17 paired samples.3.We analyzed the dataset Dyrskjot Bladder 3 from the public database Oncomine and further verified that the expression level of Gankyrin mRNA in bladder cancer tissues was higher than that in normal tissues(P<0.01).4.Gankyrin gradually up-regulated in tumors from mucosa,superficial muscular layer and deep muscular layer in the three slides.Immunohistochemistry analysis in 130 samples showed that the expression level of Gankyrin was significantly higher in MIBC compared to NMIBC(P<0.01)and in high grade BC compared to low grade BC(P<0.01).Furthermore,the overall survival time,disease-specific survival time and progression free survival time in patients with high Gankyrin expression were significantly lower than in patients with low Gankyrin expression(all P<0.01).In multivariable COX regression model,Gankyrin was an independent risk factor for poor prognosis of BC.5.By the analysis of western blot and qRT-PCR,a low level of Gankyrin mRNA and protein expression was detected in normal urothelium cell line SVHUC compared to BC cell lines,and the expression was significantly increased in MIBC cell lines UMUC3 and J82 compared to NMIBC cell lines BIU87 and SW780.6.The BIU87 cell line which was stably overexpressed of Gankyrin and the UMUC3 cell line which was stably knocked down of Gankyrin were successfully constructed.The results of EdU cell proliferation assay and subcutaneous mice tumor model showed that the overexpression of Gankyrin in BIU87 cells or the knockdown of Gankyrin in UMUC3 cells could result in increased or decreased proliferation of bladder cancer cells compared to their controls respectively.The results of cell scratch assay and the Transwell chamber migration assay showed that the overexpression or knockdown of Gankyrin could result in enhanced or inhibited migration of bladder cancer cells compare to their controls respectively.The results of Transwell chamber invasion assay showed that the overexpression or knockdown of Gankyrin could result in enhanced or inhibited invasion of bladder cancer cells compare to their controls respectively.Flow cytometry results showed that the overexpression or knockdown of Gankyrin did not affect the apoptosis of bladder cancer cells.7.We successfully constructed the BBN-induced mice spontaneous bladder cancermodel and found by immunohistochemistry assay that Gankyrin expression was higher in BC tissues than normal urothelium and gradually up-regulated from NMIBC,MIBC to lung metastasis.8.We found by immunohistochemistry assay a positive correlation between Gankyrin and MDM2 expression(R=0.583,P<0.001).Gankyrin overexpression in BIU87 cell and knockdown in UMUC3 cell resulted in up-regulation or down-regulation of MDM2 expression detected by western blot.Co-localization of Gankyrin and MDM2 was found in BIU87 cell and BC tissue by Immunofluorescence.Gankyrin binds to MDM2 in BIU87 cell detected by co-immunoprecipitation assay.?Conclusions?Gankyrin gene and protein were highly expressed in bladder cancer.The expression of Gankyrin was closely related to the pathological stage and grade of bladder cancer.And the expression of Gankyrin was correlated with the prognosis which was an independent risk factor for poor prognosis of bladder cancer.Up-regulation of Gankyrin expression could enhance the proliferation,migration and invasion of bladder cancer cells.Down-regulation of Gankyrin expression significantly inhibited the proliferation,migration and invasion of bladder cancer cells.Gankyrin binds to MDM2 in bladder cancer,which may a potential mechanism for the invasion and progression of bladder cancer.