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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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@#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 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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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 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 explore the effect of gene hepaCAM transfection on the proliferation in human bladder carcinoma cell line. Methods: Plasmid pEGFP-N2-hepaCAM was transfected into T24 cells by Lipofectamine2000, and the T24 cell stably expressing hepaCAM gene was established by G4l8 selection.The expression of hepaCAM mRNA and protein was detected by RT-PCR and Western blot assays. The effects of hepaCAM on cell proliferation were measured by MTT and colony formation assays. The cell cycle progression were measured by flow cytometry. The expression of CyclinD1 mRNA was detected by RT-PCR. Results:Expression of hepaCAM gene and protein were proved by RT-PCR and Western blot assays. The MTT assay showed cell growth in the T24/hepaCAM-GFP group was significantly slow when compared with non-transfection and mock-vehicle groups whereas the rates of clonal formation were greatly decreased(P
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Aim To investigate the cytotoxic activity of HDAC inhibitor Trichostatin A (TSA)combined with anticancer drugs targeting DNA on T24 bladder cancer cell line.Methods MTT assay was used to detect the inhibitory rate of TSA alone or combined with ADM, MMC and DDP respectively on T24 bladder cancer cell in cancer cell proliferation by administering TSA alone or combined with ADM, MMC and DDP respectively.Jin′s equation was used to evaluate the efficacy of drug combination. Results The growth inhibitory rate of TSA combined with ADM, MMC and DDP respectively was in a concentration-dependent manner. The synergism of TSA combined with MMC was most significant. When administered in lower or moderate concentration, TSA combined with ADM or DDP in lower or moderate concentration demonstrated synergic effect too.Conclusion HDAC inhibitor TSA enhances the cytotoxic activity of anticancer drugs targeting DNA on bladder cancer cells and is promising to be used in chemotherapeutic regimens for advanced bladder cancer.