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@#[摘 要] 目的：探讨circFBXL5通过靶向miR-515-5p影响膀胱癌T24细胞的增殖、迁移、侵袭及其分子机制。方法： 收集2020年4月至2020年6月间在苏州市中西医结合医院手术切除的41例膀胱癌组织及其癌旁组织，采用qPCR法检测circFBXL5、miR-515-5p的表达；双荧光素酶报告实验验证circFBXL5与miR-515-5p之间的靶向关系，体外培养人膀胱癌T24细胞，实验分为si-NC组、si-circFBXL5组、anti-miR-NC+si-circFBXL5组和si-circFBXL5+anti-miR-515-5p组；MTT法、细胞克隆形成实验、FCM、Transwell实验和WB法分别检测转染后T24细胞的增殖、细胞克隆形成、迁移、侵袭和凋亡及BAX、Bcl-2蛋白水平。结果：膀胱癌组织中circFBXL5呈高表达，miR-515-5p呈低表达（均P<0.05）；circFBXL5靶向且负向调控miR-515-5p的表达；敲减circFBXL5后T24细胞的增殖抑制率、凋亡率和BAX蛋白水平均显著增高（均P<0.05），细胞克隆形成数和迁移、侵袭细胞数均显著减少（均P<0.05），Bcl-2蛋白水平显著降低（P<0.05）；同时敲减circFBXL5和miR-515-5p可部分逆转敲减circFBXL5对T24细胞增殖的抑制作用。结论：circFBXL5通过调控miR-515-5p表达影响膀胱癌T24细胞的增殖、迁移、侵袭，circFBXL5和miR-515-5p可能膀胱癌治疗的潜在分子靶标。
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@#[Abstract] Objective: To observe the expression of long-chain non-coding RNA (lncRNA) TPTEP1 in bladder cancer tissues and cells, and to observe its effect on the proliferation and invasion of bladder cancer cells and its molecular mechanism. Methods: From August 2017 to October 2019, 43 cases of bladder cancer tissues and paracancer tissues from the patients treated by surgery in the Department Urology, People's Hospital of Dongxihu Distric of Wuhan City. Real-time fluorescence quantitative polymerase chain reaction (qPCR) was used to detect the expression of lncRNA TPTEP1 in bladder cancer tissues and bladder cancer cell lines (T24, BIU-87, 5637, J82, UM-UC-3). The bladder cancer cells with the lowest lncRNA TPTEP1 expression were selected as the experimental object, and transfected with the negative control plasmid (the control group) and lncRNA TPTEP1 over-expression plasmid (the experimental group), respectively. The effect of lncRNA TPTEP1 upregulation on cell proliferation and invasion was detected by MTT method and Transwell experiment. Bioinformatics techniques were used to predict the possible target molecules of lncRNA TPTEP1. qPCR and WB were used to detect the expression levels of lncRNA TPTEP1 downstream molecules. Results: Compared with adjacent tissues, the expression of lncRNA TPTEP1 in bladder cancer tissues was down-regulated (P<0.01). Compared with normal bladder epithelial cells, the expression of lncRNA TPTEP1 in bladder cancer cell lines was down-regulated (P<0.05), and its expression in T24 cells was the lowest (P<0.01). Up-regulation of lncRNA TPTEP1 could inhibit the proliferation (P<0.05) and invasion (P<0.01) of T24 cells. Bioinformatics technology showed that lncRNA TPTEP1 could bind with miR-129-5p, and miR-129-5p could bind with EMP3; up-regulating lncRNA TPTEP1 could inhibit the expression of miR-129-5p in T24 cells (P<0.01), and indirectly promote the mRNA and protein expressions of EMP3 (P<0.01) in T24 cells. The expression of MAPK/ERK signaling pathway related proteins such as p-MEK, p-ERK1/2, p-AKT and p-PI3K decreased (P<0.01). Conclusion: Up-regulating the low-expressed lncRNA TPTEP1 in bladder cancer cell lines can inhibit the proliferation and invasion of bladder cancer T24 cells, and its mechanism is related to indirect promotion of EMP3 gene expression by down-regulating the expression of miR-129-5p.
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@#[Abstract] Objective: To investigate the expression of miR144-3p in bladder cancer tissues and cells and its effect on the proliferation and invasion of T24 cells. Methods: A total of 36 cases of bladder cancer tissue specimens and 10 cases of normal bladder epithelial tissue specimens were collected from Tangdu Hospital of Air Force Medical University during February 2018 and December 2018. In addition, bladder cancer T24 cell line and normal urothelial cell line SV-HUC-1 were also collected for this study. The levels of miR144-3p in bladder cancer tissues and cells were detected by qPCR methods. The miR-144-3p mimics and miR-NC were transfected into T24 cells by LipofectamineTM 2000, respectively. The proliferation, cell cycle distribution and invasion abilities were detected by MTT, Flow cytometry and Transwell chamber methods, respectively. TargetScan software was used to predict the binding site between miR-144-3p and E2F3 (E2F transcription factor 3); Dual luciferase reporter gene assay was used to verify the relationship between miR-144-3p and E2F3; and WB was used to detect the expression levels of miR-144-3p and E2F3 in cells. Results: The expression of miR-144-3p was downregulated in bladder cancer tissues and cells (all P<0.01). In addition, the expression level of miR-144-3p in muscular invasive bladder cancer tissues was significantly lower than that in non-muscular invasive bladder cancer tissues (P<0.05). Dual luciferase reporter gene assay confirmed that there was a targeted relationship between miR-144-3p and E2F3. Overexpression of miR-144-3p inhibited the proliferation and invasion of T24 cells (all P<0.01) and downregulated the expression of E2F3 (P<0.01); upregulation of E2F3 could reverse the inhibitory effect of miR-144-3p overexpression on proliferation and invasion of T24 cells. Conclusion: miR-144-3p has low expression level in bladder cancer tissues. It inhibits proliferation and invasion of bladder cancer cells by downregulating E2F3.
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@#Aims: Urinary tract infection (UTI) is a common infection caused by many virulent bacteria. Multidrug resistance (MDR) by bacteria represents a major therapeutic challenge worldwide. MRD bacteria have different mechanisms to avoid antibiotics; one of them is horizontal gene transfer. Such genes, encoding antimicrobial resistance, are easily transferred from one bacterium to another. Magnesium and calcium chloride (MgCl2 and CaCl2) have an effect on the permeability of bacterial cell membrane. We aimed these chemical materials could increase the antibiotics efficiency on multidrug resistance bacteria. 250 UTI specimens were collected to isolate multidrug resistant bacteria. Depending on antibiotics resistance, we selected three species of virulent bacteria: Escherichia coli, Staphylococcus aureus and Proteus mirabilis. Then, we tested the effect of MgCl2 and CaCl2 on their antibiotics resistance. Methodology and results: The results showed that percentage of E. coli in UTI infection is the highest (45%), while Enterococcus faecalis is the lowest (3%). The effect of MgCl2 and CaCl2 on bacterial antibiotics resistance has been tested using different types of antibiotics. The findings showed that MgCl2 has significant effect to aid antibiotics against bacteria. In particular, nalidixic acid has shown more efficiency against E. coli and S. aureus but not P. mirabilis. Using different concentrations of CaCl2 increased the efficiency of gentamycin, amoxicillin and trimethoprim against S. aureus, while has increased the efficiency of ampicillin and nalidixic acid against E. coli. However, CaCl2 has no effect on the efficiency of antibiotics against P. mirabilis. In addition, MgCl2, and CaCl2 had no toxic effects in both T24 and 5637 urinary bladder cell lines. Finally, plasmids were isolated from these species to detect any antimicrobial resistance gene such as qnr-A. Conclusion, significance and impact of study: MDR distribution in the worldwide was increased, we highly recommend the avoidance of the random antibiotic usages. The salts CaCl2 and/or MgCl2 can be used at specific concentration to enhance the antibiotics permeability and therefore to decrease the antibiotic resistance.
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@#Objective: To investigate the expression of metastasis-associated protein 2 (MTA2) in human bladder cancer tissues and its effect on the malignant biological behaviors of bladder cancer T24 cells, as well as to explore the effect of MTA2 on the progression of bladder cancer. Methods: Sixty-two cases of human bladder cancer tissues and 28 cases of normal bladder tissues (from patients with cystitis, and pathologically confirmed as normal tissue) were collected at People’s Hospital of Hebei Province during December 2012 and December 2014. The expression of MTA2 in bladder cancer tissues and normal bladder tissues was detected by immunohistochemical staining, and the correlation between MTA2 expression and clinicopathological characteristics of patients was also analyzed. The bladder cancer T24 cell line stably expressing MTA2 was constructed. The effects of MTA2 on the proliferation, colony formation, migration and invasion of bladder cancer T24 cells were detected by MTS, clone formation, scratch healing and Transwell assay, respectively. Results: Immunohistochemical staining showed that MTA2 expression was significantly up-regulated in bladder cancer tissues as compared with normal bladder tissues (P<0.01). The high expression of MTA2 in bladder cancer tissues was not related to gender, age and tumor volume (P>0.05), but was associated with higher TNM stage, histological grade, and lymphatic infiltration and metastasis (all P<0.05). After over-expression of MTA2 in bladder cancer T24 cell line, the proliferation activity of the cells was significantly increased (P<0.05), and the colony formation, scratch healing, migration and invasion ability were significantly increased (all P<0.01). Conclusions: MTA2 is up-regulated in human bladder cancer tissues and can promote the proliferation, tumor formation, migration and invasion of T24 cells.
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@# Objective: To investigate the role and mechanism of Krüppel-like factor 4 (KLF4) in regulating epithelial-mesenchymal transition (EMT) and migration of bladder cancer cells. Methods: Bladder cancer 5637 and T24 cell lines that stably over-expressing KLF4 (LV-KLF4, experiment group) were constructed, and the negative control group (LV-NC) was also established; the mRNA and protein expressions of KLF4 were verified by qPCR and WB, respectively. Transwell chamber assay was used to detect the migration ability of cells in LV-KLF4 and LV-NC groups. WB was performed to detect the expression levels of EMT-related markers (E-cadherin, N-cadherin, Vimentin) and Wnt signaling pathway-related proteins. Immunofluorescence technique was used to detect the distribution of β-catenin in cells after over-expression of KLF4. Results: The 5637 and T24 cell lines over-expressing KLF4 gene were successfully constructed. Compared with the LV-NC group, the mRNA and protein expressions of KLF4 increased in LV-KLF4 groups (all P<0.01); the expression of E-cadherin increased (P<0.01), while the expressions of N-cadherin, vimentin, and the expression levels of total β -catenin, nuclear β -catenin, MMP 9 and c-Myc decreased (all P<0.01); moreover, the migration ability of cells decreased significantly (P<0.01); the fluorescence expression of β-catenin in cells also decreased significantly in LV-KLF4 group as compared to LV-NC group. Conclusion: Over-expression of KLF4 gene in bladder cancer cells may inhibit EMT process by regulating Wnt/β-catenin signaling pathway, and further inhibit the migration of bladder cancer 5637 and T24 cells.
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@# Objective: To investigate the effect and mechanism of AMP-activated protein kinase α (AMPKα) over-expression on proliferation, migration, invasion and epithelial mesenchymal transition (EMT) of bladder cancer T24 cells. Methods: A bladder cancer T24 cells over-expressing AMPKα was established and divided into T24 group, pc-DNA group and pc-AMPKα group according to different plasmid transfection. Western blotting was used to verify the over-expression ofAMPKα and detect the expressions of EMT-related proteins and EMT pathway-related molecules. Hoechst staining was used to detect apoptosis of transfected T24 cells. CCK8 assay was used to detect cell proliferation. Cell scratch test was used to detect cell migration. Transwell assay was used to detect cell invasion. Results: The bladder cancer cell line T24 over-expressingAMPKα was successfully constructed. Compared with the T24 group and the pc-DNA group, the level of E-cadherin in the pc-AMPKα group was significantly increased (P<0.01) while the levels of Vimentin and N-cadherin were significantly decreased (all P<0.01), and the activities of P38 and STAT3 which related to EMT pathway were significantly inhibited (all P<0.01); cell proliferation, migration and invasion were significantly decreased while cell apoptosis was obviously enhanced (all P<0.01). Conclusion: Over-expression of AMPKα can inhibit the activity of EMT pathway-related molecules, which leads to obvious apoptosis, limited proliferation, reduced invasion and migration of bladder cancer T24 cells, and accompanied by the reversal of EMT.
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Objective To investigate the effect of ERH gene knockdown on the proliferation and apoptosis of human bladder cancer T24 cells. Methods T24 cells infected by lentivirus with interference on ERH gene sequence were cloned to establish stable T24 cells clone in ERH gene suppression. The expression of ERH mRNA gene in bladder cancer was detected by using quantitative real time polymerase chain reaction (qPCR). The effects of ERH knockout on the cell proliferation and apoptosis were examined by using methylthiazolyl tetrazolium (MTT) assay, colony formation assay and flow cytometry. The effect of ERH knockout on the tumorigenic effect of T24 cells in vivo was verified by subcutaneous tumor formation in nude mice. Results After lentiviral transfection, qPCR results showed that the knockdown effect of ERH mRNA in ERH normal group (untreated T24 cells) was better than that in ERH gene knockdown group, and the difference was statistically significant [(1.006±0.126) vs. (0.079±0.007); t=12.72, P=0.0002]. After knocking out ERH gene, MTT assay showed that the proliferation ability of T24 cells in ERH gene knockdown group was weakened compared with ERH normal group, and the difference was statistically significant [A490 value: (0.13±0.00) vs. (0.66±0.01);t=104.61, P<0.0001]. Colony formation assay indicated that the ability of clone in ERH normal group was weakened compared with ERH gene knockdown group [(10.5 ±1.2) vs. (196.4 ±4.0); t= 73.63, P< 0.0001]. Flow cytometry showed that the cell apoptosis rate in ERH gene knockdown group was higher than that in ERH normal group [(11.0 ±0.5) ％ vs. (4.2 ±0.5) ％; t= 16.06, P<0.0001]. Imaging results of subcutaneous tumor formation in nude mice showed that the total fluorescence intensity of the tumor area in ERH gene knockdown group was (4.67 ±0.59) × 1010 μW/cm2, and the corresponding part in ERH normal group was (9.54±4.20) × 1010μW/cm2 (t=3.64, P=0.0051);tumor weight in ERH gene knockdown group was (0.80±0.62) g, and in ERH normal group was (1.79±0.71) g (t=3.33, P=0.0037). Conclusion ERH gene knockout can inhibit the proliferation of human bladder cancer T24 cells, and promote the cell apoptosis.
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OBJECTIVE: To construct a cell model with simultaneous knockout of RNA methyltransferase-like( METTL) 3 and METTL14 using clustered regularly interspaced palindromic repeats( CRISPR)/CRISPR-associated protein 9( Cas9)system in human bladder cancer T24 cells,in order to provide new tools and means for further studying gene intervention in occupational cancers. METHODS: The small guide RNA( sgRNA) oligonucleotide sequences targeting METTL3 and METTL14 were designed,synthesized and constructed into the Lenti-multi-CRISPR vector. The Lenti-iCas9-neo inducible plasmids were transfected into T24 cells by lentivirus system,and the positive cells were sorted by flow cytometry. Finally,the recombinant plasmid Lenti-multi-METTL3-METTL14 was transfected into positively sorted cells and screened to obtain stable cell lines. T24-Lenti-multi-CRISPR cells and T24-KO-METTL3-METTL14 were used as control group and multigene knockout group. The Western blot was used to examine relative expression of METTL3 and METTL14 protein in the two groups. RESULTS: The targeting sgRNA was successfully inserted into the Lenti-multi-CRISPR plasmid and confirmed by sequencing. The flow cytometry was used to obtain a large amount of the positive cells. The stable cell line inducted of doxycycline with simultaneous knockout of METTL3 and METTL14 gene was obtained. The relative expression of METTL3 and METTL14 protein decreased respectively by 52. 08% and 64. 04% after 96 hours of doxycycline treatment in the multigene knockout group compared with the control group( P < 0. 01). CONCLUSION: The stable T24 bladder cancer cell with simultaneous double gene knockout of METTL3 and METTL14 was obtained for the first time,which will be helpful for providing a foundation for mechanistic study for prevention and treatment of candidate tumor targets and bladder cancer.
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Objective:To investigate the role of Baicalein combined with U0126 resisting human bladder cancer T-24 cells in vitro and mechanism.Methods: T-24 cells were dealt with Baicalein combined with U0126,flow cytometry was used to detect cell cycle and cell apoptosis,microscope to count cell number,TUNEL method to detects cell apoptosis index,and Real time quantitative PCR and Western blot to measure extracellular signal regulating kinase 1/2 (ERK1/2), CyclinD1, GSK-3β and AKT RNA level, protein level of T-24 cells respectively.Effect of Baicalein and U0126 on apoptosis and proliferation of bladder cancer cell was analyzed.Results: Cell apoptosis rate was significantly increased after T-24 cells dealt with various concentrations of Baicalein.Cell proportion of G0/G1 phase was significantly increased,while cell percentage of S phase was obviously decreased and cell count was decreased,after T-24 cells were dealt with Baicalein for 24 h.After T-24 cells were dealt with Baicalein combined with U0126 for 24 h,cell proportion of S phase was evidently decreased.T-24 cells were dealt with Baicalein or U0126 obviously promoted cell apoptosis,which was more obvious with Baicalein combined with U0126.Phosphorylation level of GSK-3β,ERK1/2,and AKT was significantly reduced and expression of ERK1/2 and CyclinD1 mRNA was evidently lower after Baicalein or U0126 or Baicalein combined with U0126,and combined application had more remarkable effect.Conclusion: Baicalein and U0126 can induce apoptosis of T-24 cells,increase cell proportion in G0/G1 phase,reduce cell proportion of S phase,and Baicalein combined with U0126 effect has more remarkable effect.
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AIM:To observe the effects of the combination of berberin and epirubicin on the cell cycle of T24 bladder cancer cells and the underlying mechanisms.METHODS:The cancer cells were exposed to epirubicin in the presence or absence of different concentrations of berberin.The viability of the cancer cells was determined by MTT assay.The cell cycle distribution was detected by flow cytometry, and the protein levels of cyclin D1, CDK2, CDK4, P21 and P27 were detected by Western blot.RESULTS:Berberine markedly enhanced the inhibitory effect of epirubicin on the viability of T24 cells and promoted epirubicin-induced cell cycle arrest at G0/G1 phase as compared with the negative control cells.Epirubicin increased the protein expression of P27 and P21, both of which were enhanced by treatment with berberin.In contrast, berberin exposure further decreased the protein expression of cyclin D1, CDK2 and CDK4 in epirubicin-treated T24 cells.CONCLUSION:Berberine significantly promotes epirubicin-induced G0 /G1 phase arrest in human bladder cancer cells by up-regulating P27 and P21 expression and inhibiting the expression of cyclin D1, CDK2 and CDK4.
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Objective To investigate the inhibitory effects of high mobility group chromosomal protein N2 (HMGN2)on growth of human bladder cancer T24 cells and ectopic tumor growth of nude mice. Methods MTT and flow cytometry assay were conducted to detect cell growth of bladder epithelial cells(T24)cells in vitro. The transplantation tumor models in nude mice were constructed by injecting T24 cells in vivo. The para-tumorswere injected with PBS,HMGN2 protein and cisdichlorodiamineplatinum(DDP),respectively. Tumor volume and weight were calculated. The expression of cell proliferation-related proteins was detected by Western blot assay. Results MTT assay proved that HMGN2 could significantly inhibit the growth of T24 cells. Flow cytometry assay verified that HMGN2 could block T24 cells in S stage of the cell cycle. The average tumor volume and weight in the HMGN2 group and DDP positive control group were smaller than those in the PBS group(P<0.05,respectively), with the tumor inhibitory rate of 25% and 23%,respectively. The results of Westernblot showed that HMGN2 could decrease Bcl-2 expression and increase Bax expression in tumor. Conclusion HMGN2 has a significant antitumor effect on T24 cells and bladder carcinoma in nude mice,which may be associated with the induction of the apoptosis of carcinoma cells and the regulation of the cell cycle.
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AIM:To investigate the effect of small interfering RNA ( siRNA)-mediated ABCE1 knockdown on the survival, cell cycle and invasion of human bladder cancer cell line T24.METHODS:The siRNA against ABCE1 was constructed and transfected into the T24 cells with LipofectamineTM 2000.The expression of ABCE1 was detected by RT-PCR and Western blot.Flow cytometry was used to detect the cell cycle.The effects of ABCE1 gene silencing on prolifera-tion, migration and invasion of T24 cells were evaluated by CCK-8 assay, wound-healing assay and Transwell invasion as-say, respectively.RESULTS: Compared with control group and blank group, the mRNA and protein levels of ABCE1 were significantly decreased in experimental group after transfected with ABCE1 siRNA.The cell cycle was arrested at G0/G1 phase and the cell number in S phase was decreased in the T24 cells.Compared with control group and blank group, the proliferation of T24 cells in experimental group was inhibited significantly, and the migration and invasion abilities of T24 cells in experimental group decreased significantly.CONCLUSION:Knockdown of ABCE1 gene may decrease migration, invasion and proliferation abilities in T24 cells.
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Objective To investigate the effect of cytokine-induced kill cells( CIK)of the cord blood on bladder transitional cell carcinoma T24 in vitro. Methods The cord blood mononuclear cells( CBMC)were induced with different cytokines such as Interleukin 2( IL-2 ),interferon γ( IFN-γ),CD3Mc-Ab into CIK cells. The proliferation ability of CIK cells was measured by calculation in different culturing time,and the phenotype of CIK cells was analyzed by the flow cytometry. Bladder transitional cell carcinoma T24 cells were mixed with CIK cells to detect the mortality of the cancer cells with CCK-8 method in vitro. Results With the cultured time of CIK cells prolong,both the number and killing activity increased. the ratio of CIK cells and T24 cells were 1:2. 5,1:5,1:10 and 1:20 when the kill rate of T24 cells by CIK cells were(18. 98 ± 2. 61)%, (35. 59 ± 4. 74)%,(45. 34 ± 5. 03)% and(69. 24 ± 5. 19)%. Conclusion The CIK cells of the cord blood is proved to be with strong anti-cancer activity against T24 cells in vitro.
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Objective To investigate the effect of over-expression of human ribonuclease inhibitor suppresses invasion and migration of transplanted bladder cancer .Methods The T24 cells were stably transfected with pIRES2-EGFP-RI and pIRES2-EG-FP plasmid respectively .Using the cell transfected with pIRES2-EGFP and untransfected cell as controls .and the positive clones were screened by G418 ,respectively ;Tumor cells of the three groups at 2 × 106 were respectively injected into the back of BALB/C nude mice to establish the xenograft models .Change of micro-blood vessels in tumor tissue and expression of CD31 were detected by Immunohisto-chemical and HE staining .Immunohisto-chemical assay was used to detect the expression of RI ,MMP-2 ,MMP-9 ,E-cadherin ,N-cadherin ,Vimentin ,Snail ,Slug ,Twist in the tumors .Results Animal experiment showed that the T24-RI cells group significantly inhibited the growth of bladder cancer compared with the other two control groups .Compared with the T24 and T24 vector cells groups ,the microvessel density in tumor tissue of T24-RI group was notably reduced and the expressions of MMP-2 , MMP-9 ,N-cadherin ,Vimentin ,Snail ,Slug ,Twist significantly were decreased simultaneously ,while the expressions of RI and E-cadherin were increased .Conclusion up-regulation RI can inhibit the growth of transplanted bladder cancer in nude mice by decrea-sing the expression of invasion protein and EM T protein .
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Objective: To investigate the effects of inhibition of microRNA-221 (miR-221) expression on the proliferation and apoptosis of bladder cancer cells. Methods: Has-miR-221 inhibitor and has-miR-221 inhibitor negative control were synthesized, and then transfected into bladder cancer T24 and J82 cells. The transfection efficiency was observed under a fluorescence microscope 5 h after transfection. The expression levels of miR-221 in T24 and J82 cells were detected by real-time fluorescence quantitative PCR at 24, 48 and 72 h after transfection; the proliferation of T24 and J82 cells was also detected by MTT assay. The expression levels of p53 upregulated modulator of apoptosis (PUMA), Bax and Bcl-2 mRNAs and proteins in T24 and J82 cells 48 h after transfection were measured by RT-PCR and Western blotting, respectively; the apoptosis of T24 and J82 cells was determined by flow cytometry (FCM) and acridine orange (AO)-ethidium bromide (EB) staining. Results: The efficiencies of has-miR-221 inhibitor transfection into T24 and J82 cells were 80% and 90%, respectively. The expression levels of miR-221 in T24 and J82 cells after transfection with has-miR-221 inhibitor were lower than those in the negative control group and the blank control group (only adding liposome) (P < 0.05). The proliferative inhibition rates of T24 and J82 cells after transfection with has-miR-221 inhibitor were higher than those in the negative control group and the blank control group (P < 0.05), and this effect was in a time-dependent manner. The expression levels of PUMA and Bax mRNAs and proteins in T24 and J82 cells after transfection with has-miR-221 inhibitor were higher than those in the negative control group and the blank control group (P < 0.05), and the expression levels of Bcl-2 mRNA and protein were opposite (P < 0.05). The apoptosis rates of T24 and J82 cells after transfection with has-miR-221 inhibitor were higher than those in the negative control group and the blank control group (P < 0.05). Conclusion: Inhibition of miR-221 expression can suppress the proliferation of bladder cancer cells and induce apoptosis.
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Objective To investigate the effect of aurora kinase inhibitor VX-680 on cell apoptosis and Bcl-2 expressions in human bladder cancer T24 cells. Methods T24 cells were cultured and treated with VX-680 at various concentrations and time points in vitro. VX-680-induced apoptosis of T24 cells was calculated by flow cytometry. The morphological change of treated cells was observed by microscopy;Bcl-2 mRNA and protein expression in T24 cells was detected by RT-PCR and Western blot assay , respectively. Results The apoptosis rate of VX-680-induced T24 cells increased in a dose-and time-dependent manner. The increase of apoptosis rate and decrease of Bcl-2 mRNA and protein expression in VX-680-induced T24 cells were in a dose-dependent manner. Conclusion VX-680 can significantly induce the apoptosis of T24 cells by down-regulating Bcl-2expression in a dose-dependent manner.
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Objective To investigate the regulating function of p120-catenin (p120ctn) on proliferation and invasion of human bladder cancer cell T24.Methods Special siRNA was used to silence the expression of p120ctn in T24 cells.MTT assay was used to examine the T24 cell growth rate.The invasion of T24 cells and the control cells were measured by transwell chamber assay.Results The silencing of p120ctn could improve the proliferation and the invasiveness of T24 tumor cells.Transwell chamber assay showed that the in vitro invasion function of T24 cells was significant increase after p120ctn-siRNA.Conclusion p120ctn inhibited the bladder cancer proliferation and invasiveness of bladder carcinoma cells.
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Objective To observe the effects of brazilin on proliferation and apoptosis in T24 cells.Methods Trypan blue exclusion test was performed to detect the inhibition of brazilin on the growth of T24 cell lines in vitro cultured within different time.After exposure to different concentrations of brazilin,homogeneous bioluminescence assay was used to detect the inhibitory action of brazilin,Caspase-3 and Caspase-9 activity on T24 cells.Cellular apoptosis was measured by flow cytometry (FCM) and observed by laser scanning confocal microscope.Results Brazilin could significantly inhibit the proliferation of T24 cells after 8 hours,the inhibitory rates of the brazilin at concentration of 25,50,100,200 μg/ml against T24 cells respectively were 43.19 ％,60.73 ％,86.38 ％ and 93.89 ％ (P ＜ 0.05).After exposured to 50 μg/ml of brazilin,the inhibition ration to T24 cells increased with time prolonging (52.72 ％ in 4 h,60.73 ％ in 8 h,91.77 ％ in 24 h,96.41％ in 48 h) (P ＜ 0.05).The activity of Caspase-3 and Caspase-9 increased slightly when brazilin was at 25 μg/ml,but there was no statistical differences compared with that in the control group (P ＞ 0.05).When cells were treated with an increase of the concentration of brazilin from range of 7.5-60 μg/ml for 16 hours,the apoptosis ratio in turn showed a upward trend of 0.15 ％,1.35 ％,2.91％,34.76 ％.It could be seen by laser scanning confocal microscope that the apoptosis occurred in the cells.Conclusion Brazilin can effectively inhibit the proliferation of T24 cells and induce apoptosis in a dose and time dependent manner.
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Objective: To investigate the effects of silencing regenerating islet-derived 1 alpha (REG 1A) gene by small interfering RNA (siRNA) on the cell proliferation and the cell cycle of the human bladder cancer cell line T24. Methods: Three siRNAs targeting human REG1A were synthesized, which were transfected into T24 cells using a liposome approach. The expressions of REG1A mRNA and protein in T24 cells after transfection with REG1A siRNA were determined by real-time fluorescence quantitative-PCR (RFQ-PCR) and Western blotting, respectively. The cell proliferation and cell cycle were detected by cell counting kit-8 (CCK-8) method and flow cytometry (FCM), respectively. Results: As compared with the blank control (untransfected with siRNA), the expression levels of REG1A mRNA and protein were decreased (83.10±0.06)% and (55.81±0.16)% after transfection with REG1A siRNA, respectively (P <0.05). The cell proliferation was inhibited (P <0.05), the cell cycle was arrested at G0/G1 phase (P <0.05), and the proliferation index was decreased (P <0.05). Conclusion: REG1A siRNA can effectively suppress the expressions of REG1A mRNA and protein in bladder cancer cell line T24, and inhibit the cell proliferation in vitro. Copyright© 2012 by TUMOR.