| Background and Objective:Bladder cancer is the most common malignant tumor of the urinary tract, accounting for 90% of the urogenital malignancy and 1%-2% of all malignant tumors and it is a direct threat to the survival of patients. Urothelial carcinoma comprises most of the pathological types of bladder cancer. According to the depth of invasion, it divides into non-muscle invasive bladder cancer and muscle invasive bladder cancer. 70%-80% of the newly diagnosed patients possess non-muscle invasive type and treatment of this type is dominated by transurethral resection of bladder tumor combined with intravesical chemotherapy. The overall prognosis is not bad, and only 10% patients progress to muscle invasive bladder cancer, yet a quite high rate of recurrence occurs, which consumes high and brings huge economic burden for patients with constantly monitor. However, muscle invasive bladder cancer possesses high degree of malignancy. Radical cystectomy is considered as the standard therapy and in the meanwhile systemic chemotherapy is required. Nevertheless 50% patients with invasive bladder cancer present recurrence and metastasis after surgery in two years, and the 5-year survival rate is only 27%-50%. Along with toxic and side effects of long-term chemotherapy, it shows a very poor prognosis. Due to the malignancy and poor outcomes,new methods for diagnose and therapy are urgently needed. With the development of molecular biology technology people have confirmed that many factors such as oncogenes, tumor suppressor genes, small RNA etc are involved in the formation and progression of bladder cancer. Despite this, gene-targeting therapy sometimes tends to yield poor results because of the non-specific markers. So people are eager to find new specific target for diagnosis and treatment.Mechanisms of the occurrence of bladder cancer are very complex, including activation of oncogenes such as c-Myc, H-ras, EGFR, HER2 and inactivation of tumor suppressor genes such as p21, Rb, p16ink4a/p19ink4 b, p53. Recently, people also find that mitochondrial DNA and proteins involved in mitochondrial energy metabolism can participate in the occurrence and evolution of tumors, such as mitochondrial protein translation elongation factor Tu(TUFM), cytochrome B(Cyt B), mitochondrial DNA subunit ND6 gene etc,which bring great attention for the study between mitochondria and tumor. Meanwhile, researches also confirm that mitochondrial dysfunction is an important characteristic of malignant tumor, which is not only due to the regulation of oncogenes and tumor suppressor genes, but also the hypoxic microenvironment within tumors. Hypoxia is a universal phenomenon existing within solid tumors and even hematologic malignancies and it could impact both of structure and function of mitochondria, thus leading to changes including mitochondrial functional gene mutation and energy metabolism and the biological behavior of tumor cells. Mitochondrial ribosomal protein S5(MRPS5) belongs to the eukaryotic mitochondrial ribosomal 28 S small subunit member and locates on chromosome 2p11.2-q11.2. It contains 430 amino acids and with a molecular mass of 48 KDa. MRPS5 participates mainly in synthesis of 13 mitochondrial derived proteins which are responsible for mitochondrial oxidative phosphorylation. A recent study showed that MRPS5 regulated the lifespan of nematodes and mice through the mitochondrial energy metabolism dysfunction pathway. As we know, aging is a predisposing factor of tumor occurrence and bladder cancer incidence rate increases with age; in addition, longevity regulation related gene Sir2, daf-2, lin2, dct2, nnt-1 etc are all involved in the occurrence and development of tumors. In the meantime, as mentioned earlier, mitochondrial structure abnormality and dysfunction are important features of tumor,so we speculate that MRPS5 which is a structural component of mitochondrion and involved in the regulation of lifespan by way of mitochondrial dysfunction may be associated closely with tumor biological characteristics. There exsist no reports at home and abroad on MRPS5 which concentrate on the biological behavior effects of bladder cancer. So we carry out the preliminary study of the phenomenon with interest. In this study, we firstly focused on the expression of MRPS5 in bladder cancer and adjacent normal tissues and explored whether MRPS5 was regulated by hypoxia. Based on this, we then penetrated through the phenomenon and exploited lentiviral vector technology to interfere the expression of MRPS5 in T24 cell and observed effects of MRPS5 on bladder cancer cells’ biological behaviours in normoxia and hypoxia, for the purpose of providing new treatment targets for the diagnosis and treatment of bladder cancer.Methods and Contents:1. Detection of MRPS5 within bladder cancer tissues and hypoxic bladder cancer cell line T24Bladder cancer and the adjacent normal tissues were collected from South West Hospital of Third Military Medical University Department of urology operation for bladder cancer patients, with ethics committee approval and informed consent was obtained. Expression of MRPS5 within 12 bladder cancer tissues and paired adjacent normal bladder tissues was measured by Western Blot;in the meantime, bladder cancer cell T24 was treated in normoxia(21% O2) and hypoxia(1% O2) circumstances for 24 hours and then proteins were collected to determine the expression of MRPS5 by Western Blot.2. Effects of silencing MRPS5 on the biological behaviors of T24 cells in normoxic and hypoxic backgroundIn order to investigate the effect of MRPS5 on the biological behavior of T24 cells, we constructed the lentivirus vector Lv-sh MRPS5, and a prepared Lv-shScramble was used as control. Then T24 cell was infected by the lentivirus for 72 hours, subsequently Western Blot was exploited to detect expression of MRPS5 to confirm effectiveness of the vector. Then T24 cells were divided into experimental group(Lv-shMRPS5) and control group(Lv-shScramble) and cultured in normal oxygen(21% O2) and hypoxic oxygen(1% O2) concentration environment respectively. 72 hours after infection by respective lentivirus, glycolytic related proteins HIF-1, PDK1, PKM2 were detected by Western Blot. CellTiter-Blue Cell Viability Assay was used to reflex cell viability and expression of proliferating cell nuclear antigen PCNA was detected by Western Blot. Flow cytometry was used to detect the rate of apoptosis and cell cycle distribution. Transwell chamber was used to detect the cell migration ability. EMT(Epithelial Mesenchymal Transition)-associated proteins ZEB1, Slug, vimentin were reflected by Western Blot. Sphere forming ability was observed by sphere formation experiment in ultra low adhesion of 96 hole plate. Expression of cancer stem cell related transcription factors Nanog, Oct4, c-Myc and Sox2 protein were detected by western blot; NOD/SCID mice subcutaneous transplantation tumor experiment was used to reflect in-vivo tumorigenic ability.Results:The expression of MRPS5 protein within bladder cancer tissues was higher than adjacent normal tissues in majority and hypoxia(1% O2) could stimulate its expression in T24 cell. Constructed interference lentiviral vector Lv-shMRPS5 was effective in decreasing the expression of MRPS5 in T24 cell. With the silence of MRPS5 in T24 cells in normoxia and hypoxia, expression of glycolytic related proteins HIF-1, PDK1, PKM2 were lower, cell proliferation viability decreased(P<0.05) and proliferation marker PCNA showed a significantly lower expression;Morover, the cell cycle was arrested in S phase and early apoptosis rate increased. Transwell experiment showed there existed no difference between two groups in normoxia and EMT-associated proteins ZEB1, Slug, vimentin remained unchanged as well; Sphere formation experiment indicated that sphere formation efficiency was not changed(P>0.05) but sphere sizes were significantly decreased when MRPS5 was silenced and the expression of Nanog, Oct4, c-Myc and Sox2 protein were all thereupon decreased in normoxia and hypoxia as well. Mice subcutaneous transplantation tumor experiment result showed that the time of tumor formation delayed and the average weight was lighter(P<0.05) and average volume was smaller(P<0.05) after 6 weeks when MRPS5 was silenced partly.Conclusions:The expression of MRPS5 protein within bladder cancer tissues was high. When MRPS5 was interfered in T24 cell,glycolytic related proteins showed decreased expression and T24 cells possessed reductive proliferation ability and the stem cells within T24 were inhibited, meanwhile, the in-vivo tumorigenic ability of immune-deficiency mice decreased. However MRPS5 did not affect cell migration ability. Hypoxia could induce the expression of MRPS5 and with interference of MRPS5 the HIF-1 expression in hypoxic T24 cells decreased, indicating MRPS5 may have relationship with hypoxia.However, the influence on biological behavior of T24 cell in hypoxia are similar with that in normoxia when MRPS5 was interfered. |
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