| Part I Effect of Ku80 re-expression on the proliferation of SMMC7721 hepatoma cellsAims:To explore the effect of Ku80 re-expression on the proliferation of SMMC7721 hepatoma cellsMaterials and methods:1. PcDNA3.1(+)-myc-his-Ku80 and pcDNA3.1(+)-myc-his expressive plasmids were kind gifts from abroad; Transformation, amplification, purification and identification of plasmids:the plasmids were transformed into DH5a bacterium through heat shock, filtrated with Ampicillin on Luria-Bertani medium, then purified with QIAGEN EndoFree Plasmid Maxi Kit, identified with endonuclease BamHI and HindⅢand sequenced.2. PcDNA3.1 (+)-myc-his-Ku80 and pcDNA3.1 (+)-myc-his expressive plasmids were transfected into Ku80 deficient SMMC7721 to generate Ku80-expressing SMMC7721 stable clones through G418 screen. Positive clones that expressed Ku80 protein were identified by western blot.3. Cell proliferation assay and soft agar colony formation assay were employed to investigate cell proliferation and anchorage-independent growth in vitro.4. Xenograft tumor growth and survival analysis of the Ku80-expressing clones and vector-transfected cells were compared in nude mice.Results:1. PcDNA3.1 (+)-myc-his-Ku80 and pcDNA3.1 (+)-myc-his expressive plasmids were successfully gain by various methods of identification.2. The western blot analysis confirmed that Ku80 clones 18,26 and 33 expressed high protein levels of Ku80, whereas the clones with the vector and the parental SMMC7721 cells lacked Ku80 expression.3. Cell proliferation assay indicated that, the cell number of the Ku80-18 (1.715±0.242,×104) and Ku80-26 (1.595±0.303,×104) clones on day 5 were significantly fewer than those of the vector-transfected (4.408±1.486,×104) or the parental SMMC7721 (5.592±0.906,×104) cells (p<0.01), the cell number of the Ku80-18 (2.223±0.481,×104) and Ku80-26 (2.025±0.075,×104) clones on day 7 were significantly fewer than those of the vector-transfected (6.733±0.651,×104) or the parental SMMC7721 (7.03±0.219,×104) cells (p<0.01). Soft agar colony formation assay indicated that, the colony number of Ku80-18 (53±7) and Ku80-26 (50±7) clones on day 5 were significantly fewer than those of the vector-transfected (109±6) or the parental SMMC7721 (117±9) cells (p<0.01).4. Xenograft tumor growth curve of the mice indicated that, Ku80-expressing subcutaneous tumors (Ku80-18 and Ku80-26) grew at significantly slower rates and were smaller at all time points examined since day 24 after injection (p<0.01); when the tumors were removed from the sacrificed mice on day 44 after injection, the mean volumes of the tumors derived from the Ku80-18 (1.66±0.39cm3) and Ku80-26 (1.50±0.31cm3) clones were significantly smaller than those derived from the vector-transfected (3.19±0.39 cmJ) or the parental SMMC7721 (3.04±0.51cm3) cells (p<0.01); the xenograft tumor weight of Ku80-18 (0.1425±0.0712g) and Ku80-26 (0.1300±0.0404g) clones were significantly lighter than those of the vector-transfected (0.5497±0.1273g) or the parental SMMC7721 (0.6071±0.1729g) cells. Nude mice that injected with Ku80-18 cells had slightly longer median survival compared with mice that injected with Vector cells (69.0±2.2 vs 52.0±1.1 days, Kaplan-Meier test:p< 0.01). Part II The potential mechanism of Ku80 re-expession induced growth inhibition in SMMC7721 hepatoma cells.Aims:To investigate the potential mechanism of Ku80 re-xpession induced growth inhibition in SMMC7721 hepatoma cells.Materials and methods:1. The cell cycle distributions of the Ku80-expressing and the vector-transfected SMMC7721 cells were analyzed by FACS.2. The cell apoptosis rates of the Ku80-expressing and the vector-transfected SMMC7721 cells were analyzed by FACS.3. The expression of cell cycle regulators associated with S-phase regulation were analyzed by western blot.4. The expression of cell apoptosis related regulators were analyzed by western blot.5. Cell cycle regulators and Ki67 were analyzed by immunohistochemical staining. Cell apoptosis levels in xenograft tumor tissue were detected by TUNEL assay.6. Three siRNA duplexes targeting p53 were named sip53-1, sip53-2 and sip53-3, and the duplexes targeting p21CIP1/WAF1 were named sip21-1, sip21-2 and sip21-3, respectively. The siRNAs were introduced into the Vector or Ku80-18 cells using Lipofectamine 2000 in serum-free Opti-MEM, according to the manufacturer’s instructions. The expression levels of p53 and p21CIP1/WAF1 proteins were determined by western blot. The transfected cells were grown in complete medium at 37℃and 5% CO2 and harvested at different time points for use in the proliferation assay and cell cycle analysis described previously. Results:1. FACS analysis indicated that, in response to serum-stimulation for 0h,12h,24h,36h and 48h,the percent of vector cells in S phase were 37.1±3.83,34.56±2.29,34.89±3.46,39.8±1.68 and 29.72±3.11,respectively; the corresponding readouts of Ku80 overexpressers were equal to 51.62±2.29,71.88±5.29,74.14±3.7,85.46±4.08 and 77.84±4.15, respectively. There was a significant difference in cell percentages in S phase between the vector-transfected and the Ku80-expressing cells (p<0.01).2. FACS analysis indicated that, the apoptosis rates in SMMC7721, vector-transfected cells, Ku80-18 and Ku80-26 clones were 1.81±0.15%, 1.83±0.25%,9.44±1.52% and 9.26±1.72%, respectively. There was a significant difference in cell apoptosis rate between Ku80-18 clones and SMMC7721 or Vector-trasfected cells (p<0.01); there was a significant difference in cell apoptosis rate between Ku80-26 clones and SMMC7721 or Vector-trasfected cells (p<0.01). While there was no significant difference in cell apoptosis rate between Ku80-18 and Ku80-26 clones (p>0.05), and there was no significant difference in cell apoptosis rate between SMMC7721 and Vector-trasfected cells (p>0.05).3. Western blot showed that the expression levels of p53 and p21CIP1/WAF1 significantly increased in the Ku80-expressing cells, whereas the expression levels of cdk2 and cyclin A significantly decreased. The expression level of cyclin E remained unchanged.4. Western blot showed that levels of cleaved PARP, active caspase-3 and caspase-8 significantly increased in the Ku80-expressing cells compared with SMMC7721 or Vector-transfected cells, whereas the expression levels of Bcl-2 decreased. The expression level of Bax remained unchanged.5. Immohistochemical staining showed that positive cell percentage for ku80, p53 and p21 increased in Ku80 transfected cell forming tumor tissue compared with that of vector transfected(p<0.01), while cyclinA and cdk2 significantly decreased(p<0.01). The positive percent of cyclin E remained unchanged(p>0.05). TUNEL assay showed that, the cell apoptosis rates in tumor tissue derived from SMMC7721, vector-transfected cells, Ku80-18 and Ku80-26 clones were 1.81±0.15%,1.83±0.25%,9.44±1.52% and 9.26±1.72%, respectively. There was a significant difference in cell apoptosis rate between Ku80-18 clones group and SMMC7721 or Vector-trasfected cells group (p<0.01); there was a significant difference in cell apoptosis rate between Ku80-26 clones group and SMMC7721 or Vector-trasfected cells group (p<0.01). While there was no significant difference in cell apoptosis rate between Ku80-18 and Ku80-26 clones group (p>0.05), and there was no significant difference in cell apoptosis rate between SMMC7721 and Vector-trasfected cells group (p>0.05).6. The cell cycle analysis indicated that S-phase arrest was overcome in the sip53-3-or sip21-1-transfected Ku80-18 cells because the percentages of these cells in S phase decreased to the same levels as that of the vector cells after 72 hours of culture. In addition, there was no significant difference between the percentages of mock and sip53-3- or sip21-1-transfected vector cells in S phase at all time points after transfection(p>0.05). The cell proliferation assay further showed that the knockdown of p53 or p21 by sip53-3 or sip21-1 overcame the growth suppression induced by Ku80 in the Ku80-18 clone cells after day 5 of culture. Compared with the number of mock-transfected Ku80-18 cells, an average increase by 253% or 245% in the number of Ku80-18 cells transfected with sip21-1 or sip53-3 was observed on day 5 of culture (p<0.01).There was no significant difference in cell numbers among the mock-, sip21-1- and sip53-3-transfected vector cells at all time points after transfection.Statistical analysisAll results represent the average from triplicate experiments, and all results are expressed as the mean±standard derivation. The associations between categorical variables were assessed using the chi-square test or the Fisher exact test. Analysis of variance (ANOVA) was performed to determine the statistical significance among the groups. Analyses of survival were carried out with the Kaplan and Meier method. And a value of p<0.05 was considered statistically significant.Conclusion1. Re-expression of Ku80 in human HCC cells significantly suppressed cell proliferation and anchorage-independent growth in vitro as well as xenograft tumor growth in nude mice.2. Ku80 re-expression causes S-phase arrest and cell apoptosis in SMMC7721 cells.3. Ku80 re-expression induced the upregulation of p53 and p21CIP1/WAF1 in the SMMC7721 cells.4. The Ku80-induced growth suppression and cell cycle arrest depend on the p53-p21 pathway activation. |
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