Preliminary Study On The Effect And Mechanism Of DENND2D On The Proliferation And Migration Of Human Bladder Cancer Cells

Objectives:Bladder cancer is commonly known to be one of the tumors with the highest incidence of urinary systems.Transitional epithelial cell carcinoma of urinary tract is considered as the main pathological type in clinic,the incidence and mortality rate of which stays high.Non-muscular invasive bladder cancer(NMIBC)accounts for Approximately three-quarters of bladder cancer,and the remaining quarter is muscular invasive bladder cancer(MIBC).NMIBC is likely to recur despite its relatively optimistic prognosis.Nevertheless,the poor prognosis of MIBC is attributed to the easy occurrence of metastasis.Metastasis of the bladder cancer explains the leading cause of death in spite of its mechanism of regulating metastasis don’t make clear yet.The elucidation of metastasis mechanism of bladder cancer is deemed to play an important role in exploring new therapeutic targets to improve the survival of patients with bladder cancer.Human DENND2D gene is located on the No.1 chromosomes and is highly conservative in a variety of species.It can encode a protein containing 468 amino acids.As a conversion factor for gorbine nucleotide,DENND2D stimulates GDP to release from Rab9A and Rab9B,which is responsible for the protein transport between Golgi and late intracellular bodies,and participates in the process of cross-membrane transfers.DENND2D is proved to be a cancer suppression gene in malignant tumors such as lung cancer,liver cell carcinoma,gastric cancer,and bowel cancer.Up to now,the role and mechanism of DENND2D in bladder cancer have not yet been clearly reported,yet it is of great significance to explore the role of DENND2D in bladder cancer and its mechanism.Our group analyzed the clinical and sequencing data of bladder cancer in GEO and TCGA databases with the help of the weighted gene co-expression network in the preliminary research,we surprisedly found that the expression level of DENND2D was strongly related to the invasion and prognosis of bladder cancer.Our ranges of experiments were designed to probe into the role and the specific mechanism of DENND2D in bladder cancer on this basis,which will provide some theoretical basis for taking DENND2D as a therapeutic target for bladder cancer.Method:1.DENND2D,a gene closely related to the invasiveness of bladder cancer,was found by bioinformatics analysis.2.The expression level of DENND2D in immortalized urothelial cell SV-HUC-1,four kinds of bladder cancer T24,5637,UMUC3 and EJ cell lines was detected by q-PCR.3.The bladder cancer cell lines which were overexpressed and knocked down DENND2D was constructed,and the expression level of DENND2D was verified by q-PCR and Western Blot.4.CCK-8 experiment and plate clone formation experiment were used to detect the effect of overexpression or knockdown of DENND2D on the proliferation of bladder cancer cells.5.Cell scratch test and Transwell test were used to detect the effect of DENND2D expression or knock-down on the migration ability of bladder cancer cells.6.Western Blot was used to detect the effect of overexpression or knockdown of DENND2D on the key markers of epithelial-mesenchymal transition.Result:1.DENND2D,a gene closely related to the invasiveness and prognosis of bladder cancer,was found by bioinformatics analysis.2.Compared with human bladder epithelial immortalized cells SV-HUC-1,DENND2D is poorly expressed in bladder cancer T24,5637,UMUC3 and EJ cell lines.3.The proliferation and migration of bladder cancer cells can be inhibited by DENND2D.4.The expression of EMT-related markers in bladder cancer could be restrained by DENND2D.Conclusion:1.DENND2D is considered to be closely related to the prognosis of bladder cancer.2.The proliferation and migration of bladder cancer cells can be inhibited by DENND2D.3.It is possibly DENND2D that inhibits EMT of bladder cancer so as to repress the invasion ability of bladder cancer.