Studies Of The Influence Of T24Bladder Cancer Cell’s Biological Behavior By Silencing The Vitamine-D Binding Protein Gene

Background:Bladder cancer is the most common highly malignant tumor of the male genitourinary system, according to the United States in2013new estimate of the top10cancers of the largest number of cases, male incidence of bladder cancer ranks the fourth, eighth in the mortality, in which the pathological type of bladder transitional cell carcinoma accounts for more than90%. According to the report in some cities in tumor incidence in recent years, the incidence of bladder cancer increased., Cystoscopy and biopsy remains the gold standard for the diagnosis and follow-up of bladder cancer, but this check is invasive, patients will bring a certain amount of pain and fear, and clinical applications will be subject to certain restrictions, and urinary cytology non-invasive, high specificity in the diagnosis of bladder cancer, but the sensitivity is low. Due to the lack of specificity of tumor markers, bladder cancer is difficult to achieve early diagnosis, explore new method for early and effective diagnosis of bladder cancer has become the urgent need to address the practical problems in the clinical and medical research has been hot and difficult. Modern malignant tumors showed that tumor occurrence and development of multi-factor, multi-stage formation, its mechanism lies in the interior of the cell gene structure and function of an unexpected change occurs, resulting in the biological characteristics of malignant tumor cells without limit proliferation, infiltrative growth and multi-directional transfer. In this multi-stage process of carcinogenesis, the activation of oncogenes, tumor suppressor gene inactivation that the two processes are considered to be the most important changes in molecular. Which the two mechanisms in the process of tumor formation has played a non-negligible effect:First, changes in the DNA nucleotide sequence of a gene mutation genetics; another epigenetic changes in gene expression in, this change mainly through genetic meiosis, does not change the sequence of the gene DNA, but by DNA itself chemically modified manner, from the level of transcription of gene expression, especially protein expression, thus regulating the function of the DNA, more and more attention in the process of tumor formation. Although the improvement in medicine and monitoring of bladder cancer early detection is still a huge challenge. While we have recently found that the vitamin D binding protein (Vitamin-D Binding Protein, also known as DBP or GC protein, hereinafter referred to as GC protein) can be used as a new urine-derived markers161, in the early stage of the urinary bladder skin cancer detection and monitoring plays an important role, and is likely to become an important method of early diagnosis of bladder cancer. Vitamin D binding protein in bladder cancer cells have not yet been elucidated, it is necessary to further study and discussion of the role of the protein in bladder cancer cells.Purpose:This study was designed to silence vitamin D binding protein gene for the vitamin D binding protein expression by RNAi interference contrast differences invasive bladder cancer cell lines vitamin D binding protein with normal bladder cells andfurther observed changes in the biological characteristics of invasive bladder cancer cell lines. Materials and Methods:Part1:cell culture:Select T24bladder cancer cell lines and normal bladder epithelial cells for cell culture as a control group, divided into a group of bladder cancer cells and normal bladder epithelial cells group.2, respectively, two groups of cells extracted total protein immunoblotting, protein electrophoresis, transferred to a membrane, closed hybridization and exposure detection GC protein expression in T24bladder cancer cell lines with the protein in normal bladder epithelial cell lines differences in the expression.Part2:1cell culture:Select bladder cancer cell line T24is divided into the GC-siRNA group as the experimental group, negative control group and blank control group of three groups, GC-siRNA group application of RNAi method, the liposome transfection siRNA interference clips, silence bladder cancer T24cells vitamin D binding protein gene (referred to as GC gene) expression; negative control group liposomal transfection of the negative control fragment does not interfere with the GC gene expression; blank control group neither turn dye.2, respectively, electron microscope with fluorescence interference fragment GC-siRNA group and negative control group, the transfection efficiency.3were extracted from the three groups of cells, total protein, Western blot after electrophoresis, transferred to a membrane, sealed hybridization, exposure, three groups of cells in the GC protein expression differences were detected contrast, to detect silence GC GC protein gene in the experimental group of down the situation.Part3:cell culture:Select bladder cancer cell line T24divided into GC-siRNA group, negative control group and blank control group of three groups, GC-siRNA group application of RNAi, with liposomal transfection of siRNA interference segment, silence GC gene expression; negative control group with liposome transfection negative control fragment does not interfere with the GC gene expression; blank control group, neither the transfection process.2cells in each group using the MTT cell proliferation, apoptosis of using Annexin-V-PI double staining flow cytometry using Transwell assay cell migration changes, observe the silence GC gene the biological properties of T24cells.Results:Part1:GC protein in bladder cancer T24cells showed high expression state than normal bladder epithelial cells was significantly increased and there are significant differences.Part2:application of liposomal transfection of siRNA interference segment silence GC genes significantly down-regulated in human bladder transitional cell carcinoma T24cells GC protein expression, the GC protein expression by GC-siRNA group significantly higher than the negative control group and blank control group decreased significantly different; no significant difference between the negative control group and blank control group.Part3:1.MTT experimental results show that the proliferation of cells of each group:GC-siRNA group were compared with the negative control group and blank control group, the level of T24bladder cancer cell proliferation was inhibited, there are significant differences in the negative control group and cell proliferation was no significant difference in the control group;2.Annexin-V-PI double staining detected by flow cytometry cell apoptosis:GC-siRNA group of bladder cancer T24cells apoptosis compared with the negative control group and blank control group was significantly increased and there are significant differences between the negative control group and blank control group showed no significant difference;3.Transwell experiments:GC-siRNA group T24bladder cancer cell migration compared to the negative control group and blank control group decreased significantly and there is a significant difference between the negative control group and blank control group showed no significant difference.Conclusion:GC protein was highly expressed in human bladder cancer T24cells by targeting silence GC gene significantly lowered T24cells GC protein, can significantly inhibit the proliferation and migration of T24cells, and to promote bladder cancer T24apoptosis GC protein highly expressed in human bladder cancer may be a series of malignant characteristics of cancer cells in the bladder.