泛素特异性肽酶10在胰腺癌吉西他滨耐药中的作用及其机制研究

Effect and mechanism of ubiquitinspecific processing peptidase 10 on gemcitabine resistance of pancreatic cancer

    摘要
  • 摘要: 目的:探讨泛素特异性肽酶10(USP10)在胰腺癌吉西他滨耐药中的作用及机制。
    方法:采用实验研究方法。(1)CCK8检测胰腺癌细胞系BxPC-3、PANC-1及SW1990中USP10的半抑制浓度(IC50)值。(2)Western blot检测胰腺癌细胞系BxPC-3、PANC-1、SW1990、SW1990GEM中USP10蛋白表达水平。(3)细胞分组：将SW1990细胞进行siRNA干扰，转染对照siRNA设为对照组，转染USP10siRNA设为USP10干扰组。(4)CCK8检测对照组和USP10干扰组胰腺癌细胞中吉西他滨的IC50值。(5)克隆形成实验：计数对照组和USP10干扰组胰腺癌细胞形成的细胞克隆数，并计算克隆形成率。(6)流式细胞仪检测细胞凋亡：采用0.30 μg/mL吉西他滨分别干预对照组和USP10干预组细胞48 h。计算凋亡细胞百分比。(7)流式细胞仪检测细胞周期：采用0.3 μg/mL吉西他滨分别干预对照组和USP10干预组细胞48 h，流式细胞仪检测细胞周期。(8)Western blot检测细胞相关蛋白的表达：①采用浓度为0、0.30、0.60、1.20 μg/mL的吉西他滨分别干预对照组和USP10干预组细胞72 h。检测不同浓度下两组细胞中USP10和P21蛋白表达水平。②采用浓度为0.30 μg/mL的吉西他滨分别干预对照组和USP10干预组细胞0、24、48、72 h。检测不同时间点两组细胞中USP10和P21蛋白表达水平。③检测SW1990、SW1990GEM细胞中P53蛋白表达水平。④采用浓度为0、0.30、0.60、1.20 μg/mL的吉西他滨干预SW1990细胞72 h。检测不同浓度下SW1990细胞中P53蛋白表达水平。⑤采用浓度为0.30 μg/mL的吉西他滨分别干预SW1990细胞0、24、48、72 h。检测不同时间点SW1990细胞中P53蛋白表达水平。(9)免疫共沉淀法检测蛋白之间相互作用。计量资料以±s表示，多组间比较采用单因素方差分析，组间比较采用LSD检验。两组比较采用t检验。
    结果:(1)不同胰腺癌细胞系吉西他滨IC50值和 USP10表达水平：①CCK8检测BxPC-3、PANC-1、SW1990胰腺癌细胞采用吉西他滨处理72 h的IC50值分别为(0.070±0.040)μg、(0.120±0.010)μg和(0.350±0.050)μg，3者比较，差异有统计学意义(F=10.765，P<0.05)。SW1990细胞分别与BxPC-3和PANC-1细胞比较，差异均有统计学意义(t=-7.122，-7.011，P<0.05)；BxPC-3和PANC-1细胞比较，差异有统计学意义(t=3.510，P<0.05)。②Western blot方法检测BxPC-3、PANC-1、SW1990胰腺癌细胞中USP10蛋白相对表达量分别为2.280±0.090、1.930±0.030、0.950±0.010，3种细胞系比较，差异有统计学意义(F=12.120，P<0.05)。SW1990细胞分别与BxPC-3和PANC-1细胞比较，差异均有统计学意义(t=14.305，20.455，P<0.05)，BxPC-3和PANC-1细胞比较，差异有统计学意义(t=7.501，P<0.05)。SW1990及SW1990GEM胰腺癌细胞中USP10蛋白相对表达量分别为0.905±0.005、0.350±0.010，两种细胞系比较，差异有统计学意义(t=12.300，P<0.05)。(2)CCK8检测对照组和USP10干扰组胰腺癌细胞采用吉西他滨处理72 h的IC50值分别为(0.410±0.040)μg、(0.950±0.070)μg，两组比较，差异有统计学意义(t=54.792，P<0.05)。(3)克隆形成实验结果显示：对照组和USP10干扰组胰腺癌细胞形成的细胞克隆数分别为(1 200±84)个和(2 300±105)个，两组比较，差异有统计学意义(t=28.977，P<0.05)。克隆形成率分别为24%±3%和46%±5%，两组比较，差异有统计学意义(t=10.200，P<0.05)。(4)流式细胞仪检测对照组和USP10干扰组胰腺癌细胞的细胞凋亡率分别为21%±4%、53%±3%，两组比较，差异有统计学意义(t=7.133，P<0.05)。(5)流式细胞仪检测对照组和USP10干扰组胰腺癌细胞的G0/G1期细胞比例分别为69%±5%和46%±4%，S期分别为21%±3%和48%±3%，G2/M期细胞比例分别为9%±4%和11%±3%，两组上述指标比较，差异均有统计学意义(t=9.392，11.308，7.152，P<0.05)。(6)Western blot检测胰腺癌细胞耐药机制相关蛋白的表达：①在0、0.30、0.60、1.20 μg/mL吉西他滨处理下，Western blot检测对照组胰腺癌细胞中USP10和P21蛋白表达量随药物浓度增加而增加，分别为0.960±0.050～2.205±0.150和0.840±0.010～2.505±0.150，具有浓度依赖性(F=10.108，23.650，P<0.05)；USP10干扰组胰腺癌细胞中USP10和P21蛋白表达量不随药物浓度增加而变化，分别为0.120±0.010～0.905±0.050和0.520±0.010～0.435±0.010，不具有浓度依赖性(F=4.288，16.807，P>0.05)。而P21表达基础值USP10干扰组也明显低于对照组，差异有统计学意义(t=3.765，P<0.05)。②采用0.3 μg/mL吉西他滨分别干预0、24、48、72 h后，Western blot检测对照组胰腺癌细胞中USP10和P21蛋白表达量随干预时间增加而增加，分别为0.840±0.050～2.120±0.050和0.130±0.010～2.605±0.030，具有时间依赖性(F=9.754，16.431，P<0.05)；USP10干扰组胰腺癌细胞中USP10和P21蛋白表达量不随干预时间增加而变化，分别为0.210±0.010～0.575±0.010和0.120±0.010～0.330±0.010，不具有时间依赖性(F=8.390，12.756，P>0.05)。③Western blot检测胰腺癌细胞系SW1990及SW1990GEM中P53蛋白的相对表达量分别为2.650±0.050和1.450±0.060，两者比较，差异有统计学意义(t=19.075，P<0.05)。对照组和USP10干扰组胰腺癌细胞中P53蛋白的相对表达量分别为3.250±0.050和1.550±0.050，两组比较，差异有统计学意义(t=9.240，P<0.05)。④在0、0.30、0.60、1.20 μg/mL吉西他滨干预72 h，Western blot检测SW1990胰腺癌细胞中P53蛋白相对表达量分别为0.590±0.050、1.015±0.050、2.050±0.050、2.850±0.050，呈现浓度依赖性(F=34.088，P<0.05)。⑤采用0.3 μg/mL吉西他滨分别干预0、24、48、 72 h后，Western blot检测SW1990胰腺癌细胞中P53蛋白相对表达量分别为0.890±0.050、1.225±0.030、2.180±0.150、3.030±0.150，呈现时间依赖性(F=29.650，P<0.05)。(7)免疫共沉淀实验结果显示： SW1990细胞蛋白中，P53蛋白复合物中存在USP10蛋白的表达；USP10蛋白复合物中存在大量P53蛋白表达。
    结论:胰腺癌中去泛素化酶USP10可能通过P53依赖途径参与了P21聚集诱发的吉西他滨原发及获得性耐药。

     

    Abstract: Objective:To investigate the effect and mechanism of ubiquitinspecific processing peptidase 10 (USP10) on gemcitabine resistance of pancreatic cancer.
    Methods:(1) The 50% inhibiting concentration (IC50) of USP10 in the PANC-1, BxPC-3 and SW1990 cell lines of pancreatic cancer was detected by CCK8 assay. (2) The expression of USP10 in the BxPC-3, PANC-1, SW1990 and SW1990GEM cell lines of pancreatic cancer was detected by Western blot. (3) Small interfering RNA (siRNA) of SW1900 cell lines was conducted, siRNA and USP10siRNA were transected and then were respectively allocated into the control group and USP10 group. (4) IC50 of gemcitabine in the cancer cell lines between the 2 groups was detected by CCK8 assay. (5) Colonyforming unit assay: number of cloned cells was counted and colonyforming efficiency (CFE) was calculated. (6) Cells in the 2 groups were interfered with 0.3 μg/mL gemcitabine for 48 hours, and cell apoptosis was detected by flow cytometry and percentage of apoptosis was calculated. (7) Cells in the 2 groups were interfered with 0.3 μg/mL gemcitabine for 48 hours, and cell cycle was detected by flow cytometry. (8) The relative expressions of proteins in the 2 groups were detected by Western blot: ① the relative expressions of USP10 and P21 of cell lines interfered with 0, 0.30, 0.60, 1.20 μg/mL gemcitabine for 72 hours in the 2 groups were detected. ② The relative expressions of USP10 and P21 of cell lines interfered with 0.30 μg/mL gemcitabine at 0, 24, 48,72 hours in the 2 groups were detected. ③ The relative expression of P53 in SW1990 and SW1990GEM cell lines was detected. ④ The relative expression of P53 in SW1990 cell line interfered with 0, 0.30, 0.60, 1.20 μg/mL gemcitabine for 72 hours was detected. ⑤ The relative expression of P53 in SW1990 cell line interfered with 0.30 μg/mL gemcitabine at 0, 24, 48,72 hours was detected. (9) The interaction between the proteins was detected by coimmunoprecipitation. Measurement data was presented as ±s. The comparisons among groups were evaluated with the oneway ANOVA, and parirwise comparison was analyzed by the LSD test. Comparison between groups was analyzed by t test.
    Results:(1) The IC50 of gemcitabine and relative expression of USP10 in the cancer cell lines: ① the IC50 of BxPC-3, PANC-1 and SW1990 cell lines interfered with gemcitabine for 72 hours which was detected using CCK8 assay was (0.070±0.040)μg, (0.120±0.010)μg and (0.350±0.050)μg, with a statistically significant difference among them (F=10.765, P<0.05), and with statistically significant differences between SW1990 cell line and BxPC-3 or PANC-1 cell lines and between BxPC-3 and PANC-1 cell lines (t=-7.122,-7.011, 3.510, P<0.05). ② The relative expression of USP10 in the BxPC-3, PANC-1 and SW1990 cell lines which was detected using Western blot were respectively 2.280±0.090, 1.930±0.030 and 0.950±0.010, with a statistically significant difference among them (F=12.120, P<0.05), and with statistically significant differences between SW1990 cell line and BxPC-3 or PANC-1 cell line and between BxPC-3 and PANC-1 cell lines (t=14.305, 20.455, 7.501, P<0.05). The relative expression of USP10 in SW1990 and SW1990GEM cell lines were 0.905±0.005 and 0.350±0.010, with a statistically significant difference (t=12.300, P<0.05). (2) The IC50 of gemcitabine in the cancer cell lines which was detected by CCK8 assay was (0.410±0.040)μg in the control group and (0.950±0.070)μg in the USP10 group, showing a statistically significant difference between the 2 groups (t=54.792, P<0.05). (3) The result of colonyforming unit assay showed that number of cloned cells and CFE were 1200±84, 24%±3% in the control group and 2 300±105, 46%±5% in the USP10 group, showing statistically significant differences between the 2 groups (t=28.977, 10.200, P<0.05). (4) Percentage of cell apoptosis which was detected by flow cytometry was 21%±4% in the control group and 53%±3% in the USP10 group, showing a statistically significant difference (t=7.133, P<0.05). (5) The cell ratios of G0/G1 stage, S stage and G2/M stage which were detected by flow cytometry was 69%±5%, 21%±3%, 9%±4% in the control group and 46%±4%, 48%±3%, 11%±3% in the USP10 group, showing statistically significant differences between the 2 groups (t=9.392, 11.308, 7.152, P<0.05). (6) The relative expressions of resistancerelated proteins were detected by Western blot: ① the relative expressions of USP10 and P21 of cancer cell lines interfered with 0, 0.30, 0.60, 1.20 μg/mL gemcitabine for 72 hours were respectively 0.960±0.050-2.205±0.150 and 0.840±0.010-2.505±0.150 in the control group, 0.120±0.010-0.905±0.050 and 0.520±0.010-0.435±0.010 in the USP10 group, with significant concentrationdependence in the control group (F=10.108, 23.650, P<0.05) and with no concentrationdependence in the USP10 group (F=4.288, 16.807, P>0.05), and there was a statistically significant difference in the expression of P21 between the 2 groups (t=3.765, P<0.05). ② The relative expressions of USP10 and P21 of cancer cell lines interfered with 0.30 μg/mL gemcitabine at 0, 24, 48, 72 hours were respectively 0.840±0.050-2.120±0.050, 0.130±0.010-2.605±0.030 in the control group and 0.210±0.010-0.575±0.010, 0.120±0.010-0.330±0.010 in the USP10 group, with significant time dependence in the control group (F=9.754, 16.431, P<0.05) and with no time dependence in the USP10 group (F=8.390, 12.756, P>0.05). ③ The relative expressions of P53 in SW1990 and SW1990GEM cell lines were 2.650±0.050 and 1.450±0.060, respectively, showing a statistically significant difference (t=19.075, P<0.05). The relative expressions of P53 in the control and USP10 groups were 3.250±0.050 and 1.550±0.050, respectively, showing a statistically significant difference (t=9.240, P<0.05). ④ The relative expressions of P53 in SW1990 cell line interfered with 0, 0.30, 0.60, 1.20 μg/mL gemcitabine for 72 hours were 0.590±0.050, 1.015±0.050, 2.050±0.050 and 2.850±0.050, with a significant concentrationdependence (F=34.088, P<0.05). ⑤ The relative expressions of P53 in SW1990 cell line interfered with 0.30 μg/mL gemcitabine at 0, 24, 48, 72 hours were 0.890±0.050, 1.225±0.030, 2.180±0.150 and 3.030±0.150, with a significant time dependence (F=29.650, P<0.05). (7) The results of coimmunoprecipitation: there was expression of USP10 protein in P53 protein complexes and expression of P53 protein in USP10 protein complexes.
    Conclusion:USP10 promotes primary and acquired resistance of gemcitabine in pancreatic cancer via activation of P53/P21 pathway.

     

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