Role And Mechanism Of IPO11 High Expression In Bladder Urothelial Carcinoma Progression

【Background】Bladder urothelial carcinoma is still the most common malignant tumor of urinary system in China, which is divided into non-muscle invasive bladder cancer(NMIBC) and muscle invasive bladder cancer(MIBC). Approximately 70-80% of newly diagnosed patients with bladder cancer(BCa) have NMIBC, which is generally managed via the minimally invasive surgical treatment method of transurethral tumor resection. The NMIBC can be cured if it will not progress to the MIBC. However, about 30% of NMIBC will progress to MIBC, eventrually. Radical cystectomy with pelvic lymph node dissection is the standard treatment option for local MIBC, however, there are still some 50% of MIBC patients will develop local recurrence within 2 years, the 3-year survival rate is less than 50% even when they have undergone the radical cystectomy and pelvic lymph node dissection, the prognosis of the patient with MIBC is poor. But, the molecular biomarkers in predicting the risk for clinical progression of NMIBC are still lacking, not to mention the targeted drugs in blocking the progress of NMIBC. Therefore, the study on molecular mechanism of bladder cancer invasion and progression is of the great significance and very necessary.Importin11 encoded by IPO11 gene is one of the cytoplasmic nuclear transport receptor(karyopherins) family members, specifically mediating biological macromolecules through the nuclear pore complex into the nucleus. Recent evidences suggest protein and nucleic acid shuttling between the nucleoplasm and cytoplasm is not only for structural need but also a kind of regulation method of cellular processes and also being closely associated with the tumor progression. Cytoplasmic nuclear transport receptors play an important role in regulation of nuclear transport process and the abnormal expression of karyopherins are closely related to the progression, but there is no published report regarding the relationship between IPO11 and tumorigenesis and tumor progression.We have previously detected the increased DNA copy number of IPO11 in basal tumors compared with their corresponding superficial tumors through whole exome sequencing in 2 of 3 MIBCs. We therefore proposed that the increased copy numbers in basal tumors of MIBCs contribute to the progression of bladder urothelial cancer.【Objective】To investigate the role played by the increased IPO11 copy number and its possible molecular mechanism in promoting the progression of bladder urothelial carcinoma.【Materials and methods】 1. Exome sequencing was performed in superficial tumor, basal tumor and their corresponding normal bladder mucosa of 3 cases of MIBC. 2. Through the real time q PCR, the absolute copy numbers of IPO11 were detected in the sequencing samples and 25 superficial tumors and matched adjacent normal mucosa from 25 patients undergoing radical cystectomy. 3. The IPO11 copy number level in the tumor paraffin embedded sections of the above-mentioned 25 cases of superficial bladder were detected by FISH technique. 4. The IPO11 m RNA expression of above-mentioned 25 cases of superficial bladder tumor and matched adjacent normal mucosa were detected through the real time q PCR technique. The relationship between the IPO11 copy numbers and IPO11 m RNA expression status was analyzed. 5. The expression of importin11 in the paraffin embedded sections was detected through immunohistochemical staining in the 134 patients with bladder cancer who had undergone radical cystectomy(including 3 sequencing patients and 25 patients detecting IPO11 copy number variation). The relationship of importin11 expression and IPO11 copy number was analyzed. The relationships between importin11 expression and clinicopathological parameters and its influence on patients’ survival were also evaluated. 6. IPO11 m RNA expression was detected in 8 strains of human bladder cancer cell line. 7. IPO11 knockdown was conducted by RNA interfere technique in EJ and 5637 bladder cancer cell line, CCK-8 test, cell scratch assay, transwell migration and invasion experiment and flow cytometry detection for apoptosis and cell cycle were performed to evaluate the effect exerted by the downregulation of IPO11 gene expression on the cell proliferation, migration, invasion, apoptosis and cell cycle. 8. In in vivo test, cholesterol-conjugated si RNA(si RNA in vivo) was locally injected into the subcutaneous xenograft tumor mass of EJ cells to analyze the effect on the xenograft tumors’ proliferation by the downregulation of IPO11 through in vivo RNA interference experiment. 9. Transcriptome sequencing was performed in the wild type EJ cells and EJ cells with IPO11 knockdown for the KEGG pathway analysis of the different expressing genes. 10. The IPO11 m RNA relative expression of the significantly different expressing genes found by transcriptome sequencing were detected by real time q PCR in the wild type EJ cells and EJ cells with IPO11 knockdown.【Results】 1. We detected the increased DNA copy numbers of IPO11 in basal tumors compared with their corresponding superficial tumors through whole exome sequencing in 2 of the 3 cases of MIBC, which were confirmed by real time q PCR and FISH detection in the paraffin embedded sections. The IPO11 copy number variation was also discovered by real time q PCR in 17 of 25 superficial bladder tumors. Meanwhile, the copy number of IPO11 was found correlated with IPO11 m RNA and protein expression. 2. In immunohistochemistry experiment, high expression of importin11 was significantly correlated with advanced stage, high grade, lymphatic invasion and lymph node metastasis in 134 cases of bladder carcinoma. High expression of importin11 was also significantly related to the reduced 3-year overall survival rate, tumor-specific survival and tumor-free survival. High expression of importin11 is an independent risk factor of poor prognosis for patients with BCa. 3. The non-invasive bladder cancer cell line BIU-87 and papillary bladder carcinoma cell line RT4 were detected a low level of IPO11 m RNA expression, which were significantly increased in the 6 invasive bladder cancer cell lines T24,5637, UM-UC-3, J82, HT1376 and EJ. 4. Scratch assay and transwell invasion assay showed the significantly declined migration ability in EJ and 5637 cells after IPO11 gene knockdown. Transwell invasion assay detected that knockdown of IPO11 gene significantly inhibited the invasion ability of EJ and 5637. The results of CCK-8 cell proliferation experiment showed that IPO11 RNA interference had no significant effect on 5637 cell proliferation ability compared with the negative control group, only causing a reduction of cell proliferation on the 96 th hour after RNA interference for EJ cells. In xenograft tumor experiment, the average tumor volume and tumor weight were decreased in experiment group, but there was no significant difference. Down-regulation of IPO11 had no significant effect on the distribution of cell cycle and apoptosis of EJ and 5637 cells. 5. KEGG pathway analysis regarding the significantly different expressing gene discovered by transcriptome sequencing in wild type EJ cells and EJ cells with IPO11 knockdown showed the two significantly expressing genes CDKN1 A and THBS1 gene in Bladder Cancer pathway. The m RNA expression of two genes was verified by q PCR experiment in the wild type EJ cells and EJ cells with IPO11 knockdown.【Conclusions】 Importin 11 encoded by IPO11 is highly expressed in human bladder urothelial cancer tissue. High expression of importin 11 is correlated with advanced stage, high grade, lymphatic invasion and lymph node metastasis of BCa. High expression of importin 11 is an independent risk factor of poor prognosis for the patients with BCa. High expression of importin 11 plays an important role in promoting invasion and progression of bladder carcinoma. Increased IPO11 gene copy number is an important cause of high expression of importin 11, which contributes to the BCa progression probably through the upregulation of CNKN1 A and THBS1 gene expression.