葡萄糖调节蛋白78对胃癌SGC7901细胞转移的影响及机制
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摘要
目的:探讨钙离子螯合剂BAPTA-AM和钙离子载体A23187对胃癌SGC7901细胞GRP78表达水平的影响,分析GRP78表达改变对胃癌细胞侵袭转移能力的影响,并初步探讨其可能的机制。
方法:体外培养人胃癌SGC7901细胞株,并将其分为BAPTA-AM处理组、对照组和A23187处理组,采用RT-PCR和Western-blot检测各组细胞GRP78在mRNA和蛋白水平的表达,经体外侵袭实验和伤口愈合实验检测各组细胞侵袭和转移能力的变化,运用免疫细胞化学、Western-blot检测各组细胞E-钙粘蛋白和N-钙粘蛋白的表达,分析GRP78在胃癌细胞转移中的作用机制。
结果:
1. A23187组、对照组、20μmol/LBAPTA-AM组、40μmol/LBAPTA -AM组以及60μmol/LBAPTA-AM组GRP78-mRNA与内参β-actin的灰度值之比分别为:1.070±0.005、1.011±0.012、0.701±0.009、0.543±0.009以及0.348±0.013,GRP78-mRNA在A23187组的表达量较对照组升高(P<0.01),在3个BAPTA-AM组的表达量较对照组降低(P<0.01);A23187组、对照组及40μmol/LBAPTA-AM组GRP78蛋白与内参β-actin的灰度值之比分别为:0.911±0.004、0.823±0.007、0.647±0.015,GRP78蛋白在A23187组的表达量较对照组升高(P<0.01),在BAPTA-AM组的表达量较对照组降低(P <0.01)。
2. A23187组48小时划痕愈合程度较对照组升高(79.68%±0.91% vs 57.32%±0.80%,P<0.01),每视野细胞平均数较对照组增加(89±2 vs 59±2,P<0.01);BAPTA-AM组48小时划痕愈合程度较对照组降低(39.92%±0.79% vs 57.32%±0.80%,P<0.01),每视野细胞平均数较对照组减少(43±1 vs 59±2,P<0.01)。A23187组侵袭转移能力较对照组增强,BAPTA-AM组侵袭转移能力较对照组减弱。
3.免疫细胞化学、Western blot实验结果显示,A23187组E-钙粘蛋白的表达水平较对照组降低(0.106±0.003 vs 0.122±0.003,P<0.01; 0.495±0.010 vs 0.709±0.008,P<0.01),BAPTA-AM组E-钙粘蛋白的表达水平较对照组升高(0.133±0.003 vs 0.122±0.003,P<0.01;0.915±0.017 vs 0.709±0.008,P<0.01);A23187组N-钙粘蛋白的表达水平较对照组升高(0.143±0.003 vs 0.131±0.003,P<0.01;0.541±0.009 vs 0.344±0.006,P<0.01 ), BAPTA-AM组N-钙粘蛋白的表达水平较对照组降低(0.104±0.004 vs 0.131±0.003,P<0.01;0.210±0.004 vs 0.344±0.006,P<0.01)。
结论:BAPTA-AM可下调胃癌细胞SGC7901中GRP78的表达,而A23187则可诱导其表达。GRP78的表达水平可影响胃癌细胞的侵袭和转移能力,其机制可能与GRP78改变了E-钙粘蛋白和N-钙粘蛋白的表达从而影响了肿瘤的上皮间叶转化水平有关。
Objective: To study the effect of BAPTA-AM and A23187 on GRP78 expression of gastric carcinoma SGC-7901 cells in vitro, examine the role of GRP78 in invasion and metastasis of these tumor cells, and investigate their mechanism initially.
Methods: The SGC7901 cells were cultured in medium in vitro and divided into three groups: BAPTA-AM-treated group, control group and A23187-treated group. RT-PCR and Western blot were used to assess the expression of GRP78 at both mRNA and protein levels. The invasion and metastasis levels of SGC7901 cells were evaluated by matrigel invasion assay and a scratch wound assay. Immuno-cytochemistry and Western blot were performed to test the expression of E-cadherin and N-cadherin.
Results:
1. The ratios of gray values in A23187 group, control group, 20μmol/LBAPTA-AM group, 40μmol/LBAPTA-AM group, 60μmol/LBA -PTA-AM group were as follow: 1.070±0.005,1.011±0.012,0.701±0.009, 0.543±0.009,0.348±0.013, the expression of GRP78-mRNA was elevated in A23187 group and reduced in BAPTA-AM groups compared with that in control group(P<0.01); The ratios of GRP78 protein gray values andβ-actin gray values in A23187 group, control group, 40μmol/L BAPTA- -AM group were as follow: 0.911±0.004、0.823±0.007、0.647±0.015, the expression of GRP78 protein was elevated in A23187 group and reduced in BAPTA-AM groups compared with that in control group(P<0.01).
2. In A23187 group, the degree of healing was higher than that in control group(79.68%±0.91% vs 57.32%±0.80%,P<0.01) and the mean value of the invasive cells in one filed was more than that in control group(89±2 vs 59±2,P<0.01). In BAPTA-AM group, the degree of healing was lower than that in control group (39.92%±0.79% vs 57.32%±0.80%, P<0.01)and the mean value of the invasive cells in one filed was less than that in control group(43±1 vs 59±2,P<0.01). The invasion and migration levels were increased in A23187 group and decreased in BAPTA-AM group compared with that in control group.
3. Immuno-cytochemistry, Western blot showed the expression level of E-cadherin protein was decreased in A23187 group(0.106±0.003 vs 0.122±0.003,P<0.01; 0.495±0.010 vs 0.709±0.008,P<0.01)and increased in BAPTA-AM group compared with that in control group(0.133±0.003 vs 0.122±0.003,P<0.01; 0.915±0.017 vs 0.709±0.008,P<0.01); The expression level of N-cadherin protein was elevated in A23187 group(0.143±0.003 vs 0.131±0.003,P<0.01;0.541±0.009 vs 0.344±0.006,P<0.01)and reduced in BAPTA-AM group compared with that in control group(0.104±0.004 vs 0.131±0.003,P<0.01;0.210±0.004 vs 0.344±0.006,P<0.01).
Conclusion: BAPTA-AM was an inhibitor of GRP78 expression in gastric carcinoma SGC7901 cells, and A23187 could induce expression of GRP78. Moreover, the regulation of GRP78 could affect the invasion and metastasis levels of tumor cells in vitro and it may be resulted from the change of N-cadherin expression and E-cadherin expression, which play an important role in EMT.
方法:体外培养人胃癌SGC7901细胞株,并将其分为BAPTA-AM处理组、对照组和A23187处理组,采用RT-PCR和Western-blot检测各组细胞GRP78在mRNA和蛋白水平的表达,经体外侵袭实验和伤口愈合实验检测各组细胞侵袭和转移能力的变化,运用免疫细胞化学、Western-blot检测各组细胞E-钙粘蛋白和N-钙粘蛋白的表达,分析GRP78在胃癌细胞转移中的作用机制。
结果:
1. A23187组、对照组、20μmol/LBAPTA-AM组、40μmol/LBAPTA -AM组以及60μmol/LBAPTA-AM组GRP78-mRNA与内参β-actin的灰度值之比分别为:1.070±0.005、1.011±0.012、0.701±0.009、0.543±0.009以及0.348±0.013,GRP78-mRNA在A23187组的表达量较对照组升高(P<0.01),在3个BAPTA-AM组的表达量较对照组降低(P<0.01);A23187组、对照组及40μmol/LBAPTA-AM组GRP78蛋白与内参β-actin的灰度值之比分别为:0.911±0.004、0.823±0.007、0.647±0.015,GRP78蛋白在A23187组的表达量较对照组升高(P<0.01),在BAPTA-AM组的表达量较对照组降低(P <0.01)。
2. A23187组48小时划痕愈合程度较对照组升高(79.68%±0.91% vs 57.32%±0.80%,P<0.01),每视野细胞平均数较对照组增加(89±2 vs 59±2,P<0.01);BAPTA-AM组48小时划痕愈合程度较对照组降低(39.92%±0.79% vs 57.32%±0.80%,P<0.01),每视野细胞平均数较对照组减少(43±1 vs 59±2,P<0.01)。A23187组侵袭转移能力较对照组增强,BAPTA-AM组侵袭转移能力较对照组减弱。
3.免疫细胞化学、Western blot实验结果显示,A23187组E-钙粘蛋白的表达水平较对照组降低(0.106±0.003 vs 0.122±0.003,P<0.01; 0.495±0.010 vs 0.709±0.008,P<0.01),BAPTA-AM组E-钙粘蛋白的表达水平较对照组升高(0.133±0.003 vs 0.122±0.003,P<0.01;0.915±0.017 vs 0.709±0.008,P<0.01);A23187组N-钙粘蛋白的表达水平较对照组升高(0.143±0.003 vs 0.131±0.003,P<0.01;0.541±0.009 vs 0.344±0.006,P<0.01 ), BAPTA-AM组N-钙粘蛋白的表达水平较对照组降低(0.104±0.004 vs 0.131±0.003,P<0.01;0.210±0.004 vs 0.344±0.006,P<0.01)。
结论:BAPTA-AM可下调胃癌细胞SGC7901中GRP78的表达,而A23187则可诱导其表达。GRP78的表达水平可影响胃癌细胞的侵袭和转移能力,其机制可能与GRP78改变了E-钙粘蛋白和N-钙粘蛋白的表达从而影响了肿瘤的上皮间叶转化水平有关。
Objective: To study the effect of BAPTA-AM and A23187 on GRP78 expression of gastric carcinoma SGC-7901 cells in vitro, examine the role of GRP78 in invasion and metastasis of these tumor cells, and investigate their mechanism initially.
Methods: The SGC7901 cells were cultured in medium in vitro and divided into three groups: BAPTA-AM-treated group, control group and A23187-treated group. RT-PCR and Western blot were used to assess the expression of GRP78 at both mRNA and protein levels. The invasion and metastasis levels of SGC7901 cells were evaluated by matrigel invasion assay and a scratch wound assay. Immuno-cytochemistry and Western blot were performed to test the expression of E-cadherin and N-cadherin.
Results:
1. The ratios of gray values in A23187 group, control group, 20μmol/LBAPTA-AM group, 40μmol/LBAPTA-AM group, 60μmol/LBA -PTA-AM group were as follow: 1.070±0.005,1.011±0.012,0.701±0.009, 0.543±0.009,0.348±0.013, the expression of GRP78-mRNA was elevated in A23187 group and reduced in BAPTA-AM groups compared with that in control group(P<0.01); The ratios of GRP78 protein gray values andβ-actin gray values in A23187 group, control group, 40μmol/L BAPTA- -AM group were as follow: 0.911±0.004、0.823±0.007、0.647±0.015, the expression of GRP78 protein was elevated in A23187 group and reduced in BAPTA-AM groups compared with that in control group(P<0.01).
2. In A23187 group, the degree of healing was higher than that in control group(79.68%±0.91% vs 57.32%±0.80%,P<0.01) and the mean value of the invasive cells in one filed was more than that in control group(89±2 vs 59±2,P<0.01). In BAPTA-AM group, the degree of healing was lower than that in control group (39.92%±0.79% vs 57.32%±0.80%, P<0.01)and the mean value of the invasive cells in one filed was less than that in control group(43±1 vs 59±2,P<0.01). The invasion and migration levels were increased in A23187 group and decreased in BAPTA-AM group compared with that in control group.
3. Immuno-cytochemistry, Western blot showed the expression level of E-cadherin protein was decreased in A23187 group(0.106±0.003 vs 0.122±0.003,P<0.01; 0.495±0.010 vs 0.709±0.008,P<0.01)and increased in BAPTA-AM group compared with that in control group(0.133±0.003 vs 0.122±0.003,P<0.01; 0.915±0.017 vs 0.709±0.008,P<0.01); The expression level of N-cadherin protein was elevated in A23187 group(0.143±0.003 vs 0.131±0.003,P<0.01;0.541±0.009 vs 0.344±0.006,P<0.01)and reduced in BAPTA-AM group compared with that in control group(0.104±0.004 vs 0.131±0.003,P<0.01;0.210±0.004 vs 0.344±0.006,P<0.01).
Conclusion: BAPTA-AM was an inhibitor of GRP78 expression in gastric carcinoma SGC7901 cells, and A23187 could induce expression of GRP78. Moreover, the regulation of GRP78 could affect the invasion and metastasis levels of tumor cells in vitro and it may be resulted from the change of N-cadherin expression and E-cadherin expression, which play an important role in EMT.
引文
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[2]刘荣华,王世宣,马湘一,等.葡萄糖调节蛋白78在乳腺癌细胞侵袭及转移中的作用[J].广东医学,2008,29(10):1617-1619.
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