Research Of Gene Expression And Silence Of Integrin-linked Kinase In Bladder Urothelial Cell Carcinoma

Bladder cancer (BC) is a kind of tumor which is one of the most common cancers in the world, and it is also the first common carcinoma of urinary system in China. About90%of bladder cancers are urothelial cell carcinomas (UCC). About50%-70%of the patients will recur, and about20-30%of the patients will die of it. To date, the main way to detect and observe bladder cancer is cystoscopy test and urine cytology; although the cystoscopy test is easy to find the tumor, since it is an invasive procedure, it’s uncomfortable for patients to follow; the sensitivity of urine cytology is low, and it is a time-consuming procedure. In the field of treatment, no matter surgical operation or chemotherapy can effect a radical cure. Therefore, it is a great challenge for us to identify molecular, genetic and epigenetic mechanisms which is associated with the development, recurrence and progression in BUCC, with the aim of exploring both non-invasive and more sensitive methods for cancer detection and prognosis evaluation, and potential programs for gene-targeting therapy.The integrin-linked kinase (ILK) is a major signaling integrator in mammalian cells. ILK plays critical roles in development, cell motility, adhesion-dependent signaling, cytoskeleton reorganization and tumor invasion. Upon activation by different stimuli, ILK phosphorylates the serine/threonine protein kinase (PKB/Akt) and glycogen synthase kinase3β (GSK-3β) and participates in the related signaling cascades. PKB phosphorylation by ILK contributes to cell survival in cancer cells. With phosphorylation of GSK-3β, ILK induces the activation of activator protein transcription factor. The phosphorylated GSK-3β can inhibit the expression of E-cadherin by inducing the expression of Snail and ZEB1, which enhanced the invasion and migration abillity of tumor. Furthermore, ILK also can stimulate the expression of MMP-2and MMP-9, which can enhanced the invasion and migration abillity of tumor.Recently, researches from foreign scholars revealed that the levels of ILK protiens and mRNAs were significantly higher in BC tissue samples and cell lines while comparing with normal tissue samples and cell lines. And they found the expression of E-cadherin and MMP-9was abnormal while the expression of MMP-2did not change much. In China, Wang etc. found the level of ILK expression was related with BUCC pathological grading and unrelated with clinical stages. The research result of Zhu etc. was not the same with foreign scholars. They found the expression of MMP-2was abnormal as well as E-cadherin and MMP-9. It needs much more researches to find out the true mechanism of ILK in bladder cancer progression.In this study, we aim to explore the correlation between ILK expression and BUCC. The current study is divided into3sections. Section1, Western-blot, RT-PCR are separately performed to detect the expression of ILK, ILK mRNA in BUCC tissue and normal urothelial samples respectively, so as to evaluate the association between the level of ILK expression and the clinical features of BUCC. Section2, Immunofluorescence,Western-blot and RT-PCR are performed respectively to detect the expression of ILK, ILK mRNA and in urothelial cell line T24, urothelial cell line T24with negative ILK-siRNA and urothelial cell line T24with effective ILK-siRNA. The purpose of this section is to investigate the effect of ILK-siRNA in urothelial cell line T24. Section3, flow cytometry, MTT, tumor invasive assay, migrative assay and conglutinative assay will be used to detect apoptosis, proliferation, invasion and migration activity in all the three groups of urothelial cell line T24, which is to identify the correlation between the expression level of ILK knockdown by siRNA and cell biological function in BUCC. Objectives:To clarify the implication of gene expression of ILK in human BUCC tissue.Methods:In this study,22fresh BUCC tissue samples as well as20cases of normal urothelial tissue (as control) were collected. Western-blot and RT-PCR are separately performed to detect the expression of ILK and ILK mRNA in BUCC tissue and normal urothelial samples respectively.Results:Western blotting confirmed that the protein expression of ILK in BUCC samples was apparently higher than normal tissue, RT-PCR shows that the level of ILK mRNA in BUCC samples is also apparently higher than normal tissue, in accordance with ILK protein expression. No significant correlation is found between the high exression of ILK and symptoms, the tumor stages, or tumor occurrence is indicated. However, a correlation between the expression of ILK and tumor pathological grades is observed.Conclusions:According to the results, we conclude that there is an important relationship between the expression of ILK gene and the occurence of BUCC, implicating that BUCC patients with high level of ILK will probably get a worse prognosis. Objectives:To investigate the expression of ILK and ILK mRNA in human BUCC cell line, and to verification the model of ILK-siRNA transfection in human BUCC cell line is useful.Methods:Immunofluorescence, Western-blot and RT-PCR are performed respectively to detect the expression of ILK, ILK mRNA and in urothelial cell line T24, urothelial cell line T24with negative ILK-siRNA and urothelial cell line T24with effective ILK-siRNA.Results:Our results demonstrate extremely high expression of ILK protein and mRNA in control group, and obviously low level of the expression of protein and mRNA in observed group.Conclusions:The model of ILK-siRNA transfection in human BUCC cell line is established successfully. Objectives:To analyse the relationship between the expression level of ILK knockdown by siRNA and biological function in BUCC.Methods:In this section, flow cytometry, MTT, tumor invasive assay, migrative assay and conglutinative assay will be used to detect apoptosis, proliferation, invasion and migration activity in all the three groups of urothelial cell line T24, which is to identify the correlation between the expression level of ILK knockdown by siRNA and cell biological function in BUCC.Results:Flow cytometry indicates that cells in observed group has an apoptosis trend when comparing with the other two groups, and flow cytometry and MTT both show the proliferation activity in observed group is lower than the other two groups. The invasion ablility, migration ability and conglutination ability are lower than the other two groups according to the invasive assay, migrative assay and conglutinative assay obviously.Conclusions:In conclusion, knockdown ILK by siRNA can both promote the apoptosis and inhibit the invasion ablility, migration ability and conglutination ability in BUCC cells.