| Bladder cancer is one of the commonest urinary malignant tumors. The incidence of bladder cancer ranks the ninth among the malignant tumors worldwide. The incidence of bladder cancer of male patients ranks the eighth in China. In recent years, the incidence of bladder cancer was reported to increase year by year in some cities of China.AnnexinⅡis a member of annexin family, also known as AnnexinA2, P36 and LipoeortinⅡ, which is a class of calcium-dependent phospholipid binding protein. AnnexinⅡcan be distributed in the cell surface, nucleus and cytoplasm, which have many functions including participation in transduction, DNA synthesis, cell migration and cell proliferation. AnnexinⅡhas the function of promoting the invasion and metastasis of tumors as well as involving antiapoptotic activities etc. Abnormal expression of AnnexinⅡcan be observed in various types of tumors such as gastric cancer, pancreatic ductal adenocarcinoma, hepatocellular carcinoma, colon cancer, lung cancer and other tumors and tumor invasion and metastasis or prognosis has been proved in recent study.Arsenic trioxide (As2O3) is the main component of arsenic, which was used to cure the chronic myeloid leukemia, rheumatism, eczema and non-Hodgkin's disease and other diseases in the earlier days. Its mechanism has been proved that the induc-tion of cell differentiation or promotion of apoptosis are the two main ways to exert its effects through vitro and in vivo experiments. In addition, As2O3 have good th-erapeutic effect on many other malignant tumors, such as gastric cancer, liver cancer, lung cancer, bladder cancer and prostate cancer etc.Antisense oligonucleotide (ASODN) is nucleic acids of short chain (composed by aboutl5-25 nucleotides) which was undergone some chemical modification whose base order complemented with a specific target RNA sequence, after going into the c-ell, it combines with target sequences to form double-stranded structure in accordance with the Watson-Crick base pairing principle.. Antisense nucleic acid technology is currently the most mature and prospective technology among various gene therapies in treating malignant tumors at this stage. The combination of antisense RNA with the target genes can affect the expression through a variety of different mechanisms. Ant-isense oligonucleotides can not only induce apoptosis, but also increase sensitivity of tumor cells to chemotherapeutic drugs, which certainly will be a good choice in gene therapy.In this study, with different concentrations of As2O3 and AnnexinⅡantisense oligonucleotide(ASODN), combined group(As2O3 and AnnexinⅡASODN) and the control group were set up to respectively treat, bladder cancer BIU-87 cells in 24h, 48h and 72h. Protein and mRNA expression of AnnexinⅡin BIU-87 cells of bladder cancer was inspected through immunohistochemistry and situ hybridization.The BIU-87 cell invasion in was detected by Boyden chamber invasion test. Materials and methods1.Bladder cancer BIU-87 cell lines were kindly provided by the Institute of Urology of Peking University.2.BIU-87 bladder cancer cells culture:the recovery bladder cancer BIU-87 cells, RPMI-1640 medium (containing 10% fetal bovine serum, penicillin 100u/ml, streptomycin 100μml),37℃,5% CO2 incubation adherent culture box.3.BIU-87 bladder cancer cell recovery, incubation, and passage group. AS2O3 solitary group:three different concentrations (2,4和6μmol/L) of As2O3 was used for the treatment of BIU-87 cells for 24h,48h,72h; AnnexinⅡantisense oligonucleotide solitary group:three different concentrations (6,12和18μmol/L) of AnnexinⅡantisense oligonucleotides were used for the transfection of bladder cancer BIU-87 cells for 24h,48h,72h; the combined treatment group:As2O3 combined with AnnexinⅡantisense oligonucleotide (18μmol/L of AnnexinⅡantisense oligonucleotides after 24h 6μmol/L of As2O3 and then the role of 24h); the same time a blank control group.4. Protein and mRNA expression of AnnexinⅡof bladder cancer BIU-87 cells treated by immunocytochemistry detection.5. Protein and mRNA expression of AnnexinⅡof bladder cancer BIU-87 cells treated by in situ hybridization.6. To detect the BIU-87 cell invasion through boyden chamber inva-sion.7. Statistical analysis:To apply SPSS 10.0 statistical software for statistical analysis.Results1. Compared with the control group, AnnexinⅡprotein and mRNA expression of three different concentrations of AnnexinⅡantisense oligonucleotide management of bladder cancer BIU-87 cells 24h,48h,72h, decreased, and the differences were statistically significant (P<0.05). In the same period, with the increase of antisense oligonucleotides concentration, the expression of protein and mRNA of AnnexinⅡcells decreased, and both differences were statistically significant (P<0.05). In the same transfection concentrations group, AnnexinⅡprotein and mRNA expression in cells also reduced with the extension of time, and both the differences were statistically significant (P<0.05).2.Compared with the control group, with the treatment of three different concentrations of AS2O3 on bladder cancer BIU-87 cells 24h,48h,72h, the expression of protein and mRNA of AnnexinⅡin each group decreased, and the differences were statistically significant (P<0.05). In the same period, with the increase of AS2O3 concentration, the expression of protein and mRNA of AnnexinⅡcells decreased, and both differences were statistically significant (P<0.05). In the same concentration processing group, AnnexinⅡprotein and mRNA expression in cells also reduced with the extension of time, and both the differences were statistically significant (P<0.05).3. Compared with the control group, AS2O3 combined with AnnexinⅡantisense oligonucleotide for the treatment of bladder cancer BIU-87 cells for 24h,48h,72h, AnnexinⅡprotein and mRNA expression significantly decreased, and the differences were statistically significant (P<0.05); and AnnexinⅡprotein and mRNA expression also decreased with the time,, and the differences were statistically significant (P<0.05). Compared with the solitary treatment for 24h,48h,72h hours, AnnexinⅡprotein and mRNA expression also significantly reduced, and the differences were more statistically significant (P<0.05).4.Compared with the control group, AS2O3 and AnnexinⅡASODNS and the combination group can inhibit the invasion force of bladder cancer BIU-87 cell, and the differences were statistically significant (P<0.05). The inhibition of the combination group was more significant.Conclusions1. AS2O3 can inhibit the expression of AnnexinⅡprotein and mRNA and the invasion force of bladder cancer BIU-87 cell. 2. AnnexinⅡantisense oligonucleotide can inhibit the expression of AnnexinⅡprotein and mRNA and the invasion force of bladder cancer BIU-87 cell.3. As2O3 and AnnexinⅡantisense oligonucleotides have obvious synergies. |
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