Downregulation of EB virus miR-BART4 inhibits proliferation and aggressiveness while promoting radiosensitivity of nasopharyngeal carcinoma
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Abstract:
This study aims to explore the role of Epstein-Barr virus (EBV) miR-BART4 in occurrence and progression of nasopharyngeal carcinoma (NPC) and its effect on radiosensitivity.The expressions of EBV and miR-BART4 in 108 cases of NPC tissues and 97 cases of chronic nasopharyngeal inflammation tissues were determined by real time quantitative polymerase chain reaction (PCR), and the relationship between the expression of miR-BART4 and the clinicopathological features of NPC was analyzed. Cell lines, HONEl, CNEl, CNE2, C666-1, 6-10B, and NP-69 were used to compare the expression of miR-BART4, in which the CNE2 cells were selected for further experiments. CNE2 cells were grouped into blank group, negative control (NC) group, miR-BART4 inhibitors group and miR-BART4 mimics group. Cells in above groups were under radiation of 6 Gy X ray for 12 h before grouped into control group, 6 Gy group, NC + 6 Gy group, miR-BART4 inhibitors + 6 Gy group and miR-BART4 mimics + 6 Gy group. Cell proliferation, clone formation ability, cell apoptosis, invasion and migration ability were measured by MTT assay, clone formation assay, flow cytometry (FCM), Transwell assay and scratch test, respectively. Western blot analysis was used to detect the expression of apoptosis-related proteins (cleaved caspase-3, Bax and Bcl-2) and epithelial-mesenchymal transition (EMT) marker protein E-cadherin and Vimentin. mRNA and protein expression of PTEN were detected by qRT-PCR and western blot. Bioinformatics software and luciferase activity experiments were used to verify the targeting relationship between miR-BART4 and PTEN.Positive rate of EBV in NPC tissues (93.5%) was remarkably higher than that in chronic nasopharyngeal inflammation tissues (21.6%). miR-BART4 was highly expressed and mRNA and protein expression of PTEN was lowly expressed in EBV positive NPC tissues compared with EBV negative NPC tissues and chronic nasopharyngeal inflammation tissues. The expression of miR-BART4 was related to the clinical stage, lymph node metastasis and differentiation degree of NPC. Expression of miR-BART4 in CNE2, CNEl, HONEl, C666-1, 6-10B, 5-8F cells was higher than that in NP-69 cells. In CNE2 and C666-1 cell experiments, compared with blank group and NC group, miR-BART4 inhibitors group had decreased miR-BART4 expression, increased mRNA and protein expression of PTEN, cell survival rate, invasion and migration ability and increased cell apoptosis rate, which is totally contrary to the observation in miR-BART4 mimics group. The radiosensitive NPC tissues had higher miR-BART4 expression than that in radio-resistance NPC tissues. In comparison to 6 Gy group and NC + 6 Gy group, cell survival rate and clone number was inhibited, but the cell apoptosis rate was increased in miR-BART4 inhibitors +6 G group, in contrary to the observation in miR-BART4 inhibitors + 6 Gy group. Bioinformatics software and luciferase activity experiments confirmed that miR-BART4 could inhibit the expression of PTEN.EBV may promote development and progression of NPC by up-regulating miR-BART4 expressions, consequently inhibiting its radiosensitivity, whose effect may be related to the targeting inhibition of PTEN expression.Keywords:
Radiosensitivity
clone (Java method)
MTT assay
The dysregulation of microRNA expression in cancer has been associated with the epithelial-mesenchymal transition (EMT) that triggers invasive ability and increases therapeutic resistance. Here, we determined the microRNA expression profile of seven tumor tissues from patients with glioblastoma multiforme (GBM) by use of microRNA array analysis. We discovered that microRNA-7 (miR-7) is consistently downregulated in all tumor samples. Using the microRNA.org algorithm, the T-box 2 gene (TBX2) was identified as a candidate gene targeted by miR-7. In contrast to miR-7, TBX2 had an increased expression in GBM tumors and was linked to poor prognosis. We confirmed that TBX2 mRNA and protein production are significantly repressed by overexpressing miR-7 in GBM cells in vitro. The reporter assay showed that miR-7 significantly represses the signal from luciferase with the 3′ UTR of TBX2. Furthermore, TBX2 overexpression decreased E-cadherin expression and increased Vimentin expression, causing an increasing number of invaded cells in the invasion assay, as well as pulmonary metastasis in vivo. Our findings demonstrated that overexpression of TBX2 in GBM tumors via the downregulation of miR-7 leads to EMT induction and increased cell invasion.
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Objective To assess the relationship between radiosensitive heterogeneity and the apoptosis of human nasopharyngeal carcinoma (NPC) cell line CNE-2Z. Methods Flow cytometry, fluorescence microscopy and Western-blot were used to detect the apoptosis and apoptosis-related protein expression in subtypes H5 and S1 of NPC CNE-2Z cell line after radiation. Results The apoptosis rate of H5 and S1 cells increased with extension of after-irradiation time, but H5 had a higher apoptosisrate than S1 and the difference was of statistical significance (P0.05).Conclusion The radiosensitive heterogeneity of human NPC cell line CNZ-2Z is positively correlated to the apoptosis of cells after irradiation.
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Objective To investigate the effect of COX-2 inhibitor celecoxib on radiosensitity of irradiation-resistant cell line CNE-2R of nasopharyngeal carcinoma and the potential mechanism. Methods Via exposing to a series of X-ray( 2,4,6,8Gy,3 times for each dose),radio-resistant cell subline CNE-2R was established from human nasopharyngeal carcinoma cell CNE-2. Radiosensitivity was detected by clone formation assay. CNE-2R and CNE-2 cell lines were exposed to 25,50,75 μmol·L-1celecoxib,respectively. Western blotting was used to detect the protein expression of COX-2. Clone formation assay was performed to measure the survival fraction of CNE-2 and CNE-2R after radiotherapy alone or radiotherapy combined with 30 μmol·L-1celecoxib treatment. Flow cytometry was used to measure influence of radiotherapy alone or radiotherapy combined with 30 μmol·L-1celecoxib treatment on cell apoptosis. Number of residual γ-H2 AX foci was observed by immunofluorescence assay. Results The colony forming assay demonstrated that the values of SF2,D0,Dq,and N of CNE-2R cell subline [( 0. 81±0. 05),( 2. 15±0. 07) Gy,( 2. 94±0. 08) Gy,( 3. 91±0. 07),respectively]was significant higher than those of CNE-2 cell line [( 0. 61±0. 08),( 1. 47±0.06) Gy,( 1. 68±0. 10) Gy,( 3. 13 ±0. 05),respectively]. The expression of COX-2 protein was significantly downregulated with increasing celecoxib concentration. Surviving fraction was decreased in both CNE-2 and CNE-2R cell lines after irradiation. After radiotherapy combined with celecoxib,apoptosis rates of CNE-2 and CNE-2R cell lines [( 13. 10±0. 63) %,( 5. 30 ±0. 75) %]were higher than those of the corresponding control groups [( 4. 90 ± 0. 71) %,( 1. 82± 0. 82) %]. Celecoxib increased radiosensitivity in nasopharyngeal carcinoma CNE-2R and CNE-2 cell lines. The number of residual γ-H2 AX foci after irradiation was increased by celecoxib pretreatment. The difference was statistically significant( P 0. 05). Conclusion Celecoxib can enhance radiosensitivity of radio-resistant cell subline CNE-2R of human nasopharyngeal carcinoma in vitro.
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Objective To study the effect of diallyl disulfide on CNE1. Methods The growth inhibition of CNE1 cells was measured by MTT.The effect of DADS on induction of apoptosis of CNE1 cells was studied by light microscopy and flow cytometry,respectively.Bcl-2,Bax were determined using immunohistochemical.Western blot analysis was performed to detect the expression of Caspase-3 protein. Results MTT assay showed that DADS inhibited the growth of CNE1 cells.Partial cells presented characteristic morphological changes of apoptosis.Flow cytometry analysis showed that treating CNE1 cells with DADS increased the percentage of apoptosis cells.Immunohistochemistry revealed that the Bax expression was increased while Bcl-2 expressed was decreased.Western Blot analysis showed that Caspase-3 expression were increased.Conclusion DADS could significantly induce apoptosis of CNE1,Apoptosis of tumor cells is closely associated with down-regulation of the ratio of bcl-2/bax.then activation of Caspase-3.
Diallyl disulfide
MTT assay
Cytometry
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Objective: To investigate the effect of KLT combined with DDP on the proliferation and apoptosis of SiHa cell. Methods: Different concentrations of KLT injection(1, 2, 4, 6, 8 mg/ml)and DDP(1.5, 3, 6, 9, 12 μg/mL)were solo applied to SiHa cells. Cell proliferation was measured by MTT, cell apoptosis was detected by flow cytometry. The appropriate drug concentration(KLT 6 mg/ml,DDP 3 μg/mL)were selected. Then, KLT combined with DDP injection was applied to SiHa cells. Cell proliferation was measured by MTT, cell apoptosis was detected by flow cytometry. Results: ① Cell proliferation were dramatically inhibited by KLT injection(25,50,100,150,200 μg/mL)and DDP(1.5, 3, 6, 9, 12 μg/mL)at 24 h,48 h(P0.05). In addition, Cell proliferation were dramatically inhibited by injected KLT combined with DDP. ②Combination therapy, apoptosis rate and inhibition rate of proliferation in SiHa cells treated by KLT combined with DDP were higher than the monotherapy. Conclusion: DDP, KLT and DDP combined with KLT can inhibit the proliferation, accelerate the apoptosis in SiHa cells. What's more, DDP combined with KLT have synergistic effect on the apoptosis in SiHa cells.
MTT assay
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Objective
To explore whether exendin-4 inhibits AR42J cells and its mechanism.
Methods
AR42J cells were treated with exendin-4 under multiple concentrations(1, 5, 10 pmol/L) at 24, 48, 72, 96, 120 h to assess its cell viability by MTT assay and got the IC-50 and time points. Then checking whether exendin-4 could induce the AR42Jcells apoptosis by setting normal control (NC) group, exendin-4 (Ex-4) group and Ex-4+ z-VAD-fmk (apoptosis inhibitor) group, and exploring whether 3-MA which is autophagy inhibitor could inhibit the AR42J cells apoptosis induced by exendin-4 by setting NC group, Ex-4 group and Ex-4+ 3-MA group. Cell viability was analyzed by MTT and the cells apoptosis was detected by flow cytometry and the protein levels of caspase-3, LC3 and p62 were studied by Western blot.
Results
Concentration of 10 pmol/L exendin-4 and and time point 72 h were selected for the further study . z-VAD-fmk pretreatment can significantly inhibit the cell viability of exendin-4 by (81.2±3.3)% vs. (49.4±3.0)% (P<0.05). Flow cytometry showed that exendin-4 could induce the AR42J cells apoptosis by (28.2±1.4)% vs. (3.6±0.8)%, and increased the caspase-3 level by Western blot, which both can be reversed by (79.1±2.3)% vs. (49.8±2.5)% (P<0.05) when the cells were treated for 72 h, as was apoptosis ratio by (14.5±2.1)% vs. (29.2±3.2)%. Western blot showed that exendin-4 can upregulate protein levels of LC3B-II, p62和caspase-3 and 3-MA, and pretreatment can inhibit the upregulation of LC3B-II and caspase-3 but further increased the upregulation of p62 induced by exendin-4.
Conclusions
Exendin-4 can induce AR42J cells apoptosis and 3-MA pretreating can inhibit exendin-4 cytotoxicity through downregulating autophagy. So autophagy inhibitor 3-MA could potentially extenuate the cytotoxicity of exendin-4 in pancreatic acinar cells.
Key words:
Exendin-4; Pancreatic acinar cell; Apoptosis; Autophagy inhibitor, 3-MA
Viability assay
MTT assay
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Objective:To research the effects of flavonoids of Astragalus complanatus(FAC) on proliferation,apoptosis and expressions of p53,c-My and NF-кB in human breast cancer MCF-7 cells.Methods:The inhibit rate of MCF-7 cells treated by different concentration of FAC was studied by MTT at different times.The apoptosis rate of MCF-7 cell was essayed by flow cytometry after treated with effective concentration of FAC and time points according to MTT.The expressions of p53,c-Myc and NF-кB were quantified by fluorescence immunoassay and Western blot.Results:According to MTT,0.0625,0.125,0,25,0.5 mg/ml of FAC treated for 24 h,48 h,72 h markedly inhibited proliferation of MCF-7 (P 0.05,vs control).According to flow cytometry,MCF-7 was induced to apoptosis by FAC.After 72 h treatment with FAC(0.0625,0.125,0.25,0.5 mg /ml),the apoptosis rates were 6.5%,18.3%,49.6% and 57.9%,respectively.The expression of p53 was up-regulated,while c-Myc and NF-κB were markedly reduced detected by fluorescence immunoassay and Western blot.Conclusion:FAC could inhibit proliferation and induce apoptosis of MCF-7.Its role may be related to decreased expression of NF-кB and c-Myc,increased expression of p53.
MCF-7
MTT assay
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Objective:To study the effect of Crotonis Fructus alkaloid(CA) on apoptosis and the expression of Bcl-2 and Bax in human hepatoma SMMC-7721 cells.Method:After treatment with CA for 24-48 h,MTT assay was used to determine the cell growth inhibitory rate in vitro,flow cytometry was used to detect the apoptosis of SMMC-7721 cells before and after CA treatment.Western blot was used to detect the expression of Bcl-2 and Bax.Result:CA inhibited the growth of SMMC-7721 cells in a dose-and time-dependent manner.After treatment with 50-200 mg·L-1 CA for 24 h,the number of apoptosis cells was increased dose-dependently from(0.26±0.14)% in the control group to(5.85±0.47)%,(17.70±1.23)%,(33.25±2.08)% and(49.52±1.85)% in CA groups,respectively.By Western blot analysis,we found the level of Bax was up-regulated,whereas,the level of Bcl-2 down-regulated.Conclusion:CA can efficiently inhibit the proliferation and induce apoptosis in SMMC-7721 cells.Its mechanism is likely related to the down-regulation of Bcl-2 and up-regulation of Bax.
MTT assay
BAX Protein
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Objective To study the effect of quercetin on proliferation and apoptosis of HO-8910 ovarian cancer cell in vitro.Methods The proliferation of cells was detected by MTT method.Cell cycle and apoptotic percentage were analyzed by flow Cytometry(FCM).The expression of p53 and Bcl-2 were analyzed by FCM.Results Quercetin could inhibit the proliferation of HO-8910 cell as direct relation of concentrations and times.The flow cytometry showed that the proportion of G1 phase and the apoptotic rate were increased significantly in quercetin groups,the proportion of S phase and G2/M phase was reduced.In the FCM quercetin induced upregulation expression of p53 and downregulation expression of Bcl-2.Conclusions Quercetin could inhibit growth and induce apoptosis of ovarian cancer HO-8910 cell.Cell cycle was controlled in G1/G0.Meanwhile,quercetin could induce upregulation expression of p53 and downregulation expression of Bcl-2,while may be the mechanism of apoptosis of ovarian cancer by quercetin.
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This study was conducted to investigate the effect of nitidine chloride (NC) on the proliferation and apoptosis of nasopharyngeal carcinoma cell line CNE1, CNE2, TWO3, and C666-1, and to explore its antitumor mechanism.NC was dissolved in IMDM medium and cultured with nasopharyngeal carcinoma cell line CNE1, CNE2, TWO3 and C666-1. Cell morphology, cell proliferation, cell apoptosis, p53 mRNA and p53 protein levels were assessed.After incubation with NC for 24 h, typical apoptotic morphology was observed. NC inhibited the proliferation and induced apoptosis of all 4 cell lines in a time-dose dependent manner. p53 mRNA and p53 protein levels were significantly increased.NC inhibited proliferation and induced apoptosis of nasopharyngeal carcinoma cells with upregulation of p53 gene.
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