Wnt通路抑制剂FrpHE和DKK-1在肿瘤细胞中的诱导表达
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摘要
目的 Wn信号传导途径与肿瘤有着密切的关系，其通路的异常活化参与人类多种癌症的发病过程，因此，抑制或阻断Wnt通路的异常活化有望成为抗肿瘤治疗的新靶点。尽管目前已发现由细胞内分泌的内源性Wnt通路抑制剂能够拮抗Wnt通路，如FrpHE(frizzled-related protein)、DKK-1(Dickkopf-1)等，但有关这些内源性Wnt通路抑制剂的调控机理尚不清楚。本课题的目的在于研究外源性因素对Wnt通路抑制剂FrpHE和DKK-1的诱导表达，我们选择抗癌药物p53及化疗药阿霉素、羟喜树碱、顺铂作为外源性诱导剂，以期弄清Wnt通路及其抑制剂FrpHE和DKK-1是否能够被p53和化疗药这两类药物所调节。
方法 将外源性p53基因导入到p53缺失的人肝癌细胞株(Hep3B)中，通过流式细胞术和RT-PCR观察p53对Wnt通路及抑制剂FrpHE和DKK-1的作用。利用反义寡核苷酸(oligodeoxy nucleotide，ODN)将p53反义ODN导入到人肝癌HepG2(含野生型p53)和人大肠癌Lovo(含野生型p53)细胞株中，观察p53表达受阻后对Wnt通路及FrpHE、DKK-1表达的影响，以进一步明确p53的调节作用。并且在不同p53状态的人肝癌、大肠癌、神经胶质瘤细胞株中(U251,p53突变)加入化疗药物后，以RT-PCR技术检测Wnt通路抑制剂FrpHE mRNA和DKK-1 mRNA的表达水平，明确化疗药对Wnt通路抑制剂FrpHE和DKK-1的调节作用。
结果 1．将转染剂量为5pfu／cell时应用流式细胞术观察Adp53的转基因情况，结果表明Adp53转染24h、48h后96％以上的
第一军医大学博士研究生学位论灰
细胞p53表达呈阳性，平均荧光量增加了约50倍，表明p53基因
转染成功。
2.盯一PeR检测结果(l)p53对F印HE、DKK一1的
表达具有诱导作用:通过加入外源性p53检测结果显示:FrpHE
和DKK一1在人肝癌阴性细胞株(HeP3B，p53缺失)中转染Adp53
后16h、20h、24h、28h、32h、36h、4Oh时，InRNA表达水平20h
起明显增加，32h最高，36h、40h时逐渐回落，但仍高于对照组
水平。FrpHE和DKK一1在人肝癌阴性细胞株转染Adp53剂量为
0 .ospfu/eell，0.spfu/eell，spfu/eell，50pfu/eell时，F印HE
mRNA表达水平在转染剂量为SPfu/cen时最强，在SOPfu/cen
时逐渐回落，但仍高于对照水平;DKK一lmRNA表达水平在转染剂
量为0.05pfu/eell时明显增加，在50pfu/eell时最强。
(2)阻断肿瘤细胞内正常p53的表达，可引起抑制剂FrPHE、
DKK一l表达的下调:通过加入反以寡核昔酸检测结果显示:FrpHE
mRNA表达水平在人肝癌p53阳性细胞株(HePG2)中24h时表达水
平降低，48h和72h时呈现显著的抑制作用，在人大肠癌p53阳
性细胞株(Lovo)中未有表达;DKK一lmRNA表达水平在人肝
癌p53阳性细胞株(HePG2)和大肠癌p53阳性细胞株中24h时表
达水平降低，48h和72h呈现显著的抑制作用;
(3)化疗药对抑制剂FrpHE、DKK一1的表达具有上调作用，
加入化疗药(经喜树碱、阿霉素、顺铂)结果显示:FrpHE mRNA
表达水平在人肝癌细胞株(HePGZ，HeP3B)中与对照组相比表达
水平增加，在人大肠癌和神经胶质瘤细胞中没有表达;DKK一
lmRNA表达水平在人肝癌(HePGZ，HeP3B，)、大肠癌细胞、神
经胶质瘤细胞株中与对照组相比表达水平增加;
第一军医大学博士研究健李位伦失
3.p53对Wnt通路关键因子p一catenin具有明显抑
制作用，流式细胞术检测结果显示:转染Adp53后，在肝癌
(Hep3B)细胞中Wnt通路关键靶基因p一catenin蛋白的表达水
平下降;加入p53反义。DN抑制p53表达后，在肝癌(HePG2)
和大肠癌(Lovo)细胞中p53基因的表达水平明显下降，p
一catenin蛋白的表达升高;加入化疗药后，在不同的肿瘤细胞
中，p一catenin蛋白的表达下降;
结论抑癌基因p53和化疗药经喜树碱、顺铂、阿霉素能够诱导
Wnt通路抑制剂FrPHE和DKK一的表达，进而产生抑制Wnt通路
的作用。抑制剂FrpHE在人大肠癌株(Lovo)和神经胶质瘤细胞
株(u251)种没有表达。证明FrpHE在某些肿瘤细胞中存在缺失现
象。
Objective Wnt signaling pathway is closely related to tumor, its inappropriate activation involving in episode process of human multicancer, therefore, it may be a new target to restrain or block its inappropriate activation for anticancer therapies. Endogenous antagonist has been found at present to be able to antagonize Wnt signaling pathway, but endogenous regulated mechanism remains yet unclear. This study aims at the expression of Wnt signaling pathway inhibitor FrpHE and DKK-1 induced by ectogenous factor. In the study, we used p53, adriamycin, hydrocamptothecin, cisplatin as the ectogenous factors to detect their capability of regulating wnt signaling pathway as well as its inhabitors FrpHE, and DKK-1.
Methods Human hepatocarcinom cells lines (HepSB) were transfected with an ectogenesis p53 (Adp53) to observe the action of p53 on wnt signaling pathway by fluorescence-activated cell sorting (FACS) and RT-PCR.HepG2 and Lovo cell lines (wild p53) were transfected with p53 antisense ODN to detect the expression of wnt signaling pathway and wnt inhibitor FrpHE and DKK-1 after p53 were blocked. In various p53 status cell lines Hep3B,Lovo,U251 cells were treated with chemoagents, FrpHE, DKK-1 gene mRNA expression levels were detected by RT-PCR.
Results l.The study revealed that p53 protein expression emerged in more than 96% of Hep3B cells 24h and 48h after Adp53
transfection, with its average fluorescent intensity increased by 50 times by FACS.
2. The RT-PCR results: (1) FrpHE and DKK-1 were induced by p53. Our results demonstrated obvious increase of mRNA 20h after the Hep3B cells were transfected with ectogenetic Adp53, FrpHE and DKK-1 mRNA, the peak 32h after the transfection and then decline expression (which, however was higher than that in the controls) 36, 40h after the transfection. On the other hand, FrpHE mRNA had the highest expression when FrpHE and DKK-1 was transfected with Adp53 at the dose of 5pfu/cell and the expression began to decline at the dosage of 50pfu/cell even though it was higher than that in the control group, while DKK-1 mRNA had the apparently growing express at the dose of 0.05pfu/cell and then the highest expression at the dose of 50pfu/cell.
(2) FrpHE and DKK-1 were down-regulated by blocked normal p53 in tumor cells. The results demonstrated that FrpHE mRNA expression in HepG2 cells was declined in 24h and remained markedly unchanged in 48h and 72h, and it was not expressed in Lovo cells, though. DKK-1 mRNA expressions in HepG2, Lovo cells were declined in 24h and remained markedly unchanged in 48h and 72h in 48h and 72h.
(3) FrpHE and DKK-1 were up-regulated by chemoagents. Our study showed that FrpHE mRNA expressions in HepG2 and Hep3B cells were increased compared with the control groups, but nil in Lovo and U251 cells. DKK-1 mRNA expressions in HepG2, Hep3B, Lovo and U251 cells were increased compare with control groups.
3. 3 -catenin, the key factor of Wnt signaling pathway was sharply inhibited by p53. The FACS results demonstrated that the expression of P -catenin was declined in Hep3B cells after transfection with Adp53. (3 -catenin expression hoisted but p53 gene expression was declined in HepG2 and Lovo cells after transfection with antisense p53 ODN. p53 expression hoisted in HepG2, Lovo and U251 cells by chemotherapy.
Conclusion Wnt signaling pathway inhibitor FrpHE and DKK-1, induced by anticancer p53 and chemoagents like adriamycin, hydroxycamptothecin, cisplatin, antagonized Wnt signaling pathway. No inhibitor FrpHE was expressed in Lovo and U251, indicating it varied with some tumor cells inhibitor FrpHE.
方法 将外源性p53基因导入到p53缺失的人肝癌细胞株(Hep3B)中，通过流式细胞术和RT-PCR观察p53对Wnt通路及抑制剂FrpHE和DKK-1的作用。利用反义寡核苷酸(oligodeoxy nucleotide，ODN)将p53反义ODN导入到人肝癌HepG2(含野生型p53)和人大肠癌Lovo(含野生型p53)细胞株中，观察p53表达受阻后对Wnt通路及FrpHE、DKK-1表达的影响，以进一步明确p53的调节作用。并且在不同p53状态的人肝癌、大肠癌、神经胶质瘤细胞株中(U251,p53突变)加入化疗药物后，以RT-PCR技术检测Wnt通路抑制剂FrpHE mRNA和DKK-1 mRNA的表达水平，明确化疗药对Wnt通路抑制剂FrpHE和DKK-1的调节作用。
结果 1．将转染剂量为5pfu／cell时应用流式细胞术观察Adp53的转基因情况，结果表明Adp53转染24h、48h后96％以上的
第一军医大学博士研究生学位论灰
细胞p53表达呈阳性，平均荧光量增加了约50倍，表明p53基因
转染成功。
2.盯一PeR检测结果(l)p53对F印HE、DKK一1的
表达具有诱导作用:通过加入外源性p53检测结果显示:FrpHE
和DKK一1在人肝癌阴性细胞株(HeP3B，p53缺失)中转染Adp53
后16h、20h、24h、28h、32h、36h、4Oh时，InRNA表达水平20h
起明显增加，32h最高，36h、40h时逐渐回落，但仍高于对照组
水平。FrpHE和DKK一1在人肝癌阴性细胞株转染Adp53剂量为
0 .ospfu/eell，0.spfu/eell，spfu/eell，50pfu/eell时，F印HE
mRNA表达水平在转染剂量为SPfu/cen时最强，在SOPfu/cen
时逐渐回落，但仍高于对照水平;DKK一lmRNA表达水平在转染剂
量为0.05pfu/eell时明显增加，在50pfu/eell时最强。
(2)阻断肿瘤细胞内正常p53的表达，可引起抑制剂FrPHE、
DKK一l表达的下调:通过加入反以寡核昔酸检测结果显示:FrpHE
mRNA表达水平在人肝癌p53阳性细胞株(HePG2)中24h时表达水
平降低，48h和72h时呈现显著的抑制作用，在人大肠癌p53阳
性细胞株(Lovo)中未有表达;DKK一lmRNA表达水平在人肝
癌p53阳性细胞株(HePG2)和大肠癌p53阳性细胞株中24h时表
达水平降低，48h和72h呈现显著的抑制作用;
(3)化疗药对抑制剂FrpHE、DKK一1的表达具有上调作用，
加入化疗药(经喜树碱、阿霉素、顺铂)结果显示:FrpHE mRNA
表达水平在人肝癌细胞株(HePGZ，HeP3B)中与对照组相比表达
水平增加，在人大肠癌和神经胶质瘤细胞中没有表达;DKK一
lmRNA表达水平在人肝癌(HePGZ，HeP3B，)、大肠癌细胞、神
经胶质瘤细胞株中与对照组相比表达水平增加;
第一军医大学博士研究健李位伦失
3.p53对Wnt通路关键因子p一catenin具有明显抑
制作用，流式细胞术检测结果显示:转染Adp53后，在肝癌
(Hep3B)细胞中Wnt通路关键靶基因p一catenin蛋白的表达水
平下降;加入p53反义。DN抑制p53表达后，在肝癌(HePG2)
和大肠癌(Lovo)细胞中p53基因的表达水平明显下降，p
一catenin蛋白的表达升高;加入化疗药后，在不同的肿瘤细胞
中，p一catenin蛋白的表达下降;
结论抑癌基因p53和化疗药经喜树碱、顺铂、阿霉素能够诱导
Wnt通路抑制剂FrPHE和DKK一的表达，进而产生抑制Wnt通路
的作用。抑制剂FrpHE在人大肠癌株(Lovo)和神经胶质瘤细胞
株(u251)种没有表达。证明FrpHE在某些肿瘤细胞中存在缺失现
象。
Objective Wnt signaling pathway is closely related to tumor, its inappropriate activation involving in episode process of human multicancer, therefore, it may be a new target to restrain or block its inappropriate activation for anticancer therapies. Endogenous antagonist has been found at present to be able to antagonize Wnt signaling pathway, but endogenous regulated mechanism remains yet unclear. This study aims at the expression of Wnt signaling pathway inhibitor FrpHE and DKK-1 induced by ectogenous factor. In the study, we used p53, adriamycin, hydrocamptothecin, cisplatin as the ectogenous factors to detect their capability of regulating wnt signaling pathway as well as its inhabitors FrpHE, and DKK-1.
Methods Human hepatocarcinom cells lines (HepSB) were transfected with an ectogenesis p53 (Adp53) to observe the action of p53 on wnt signaling pathway by fluorescence-activated cell sorting (FACS) and RT-PCR.HepG2 and Lovo cell lines (wild p53) were transfected with p53 antisense ODN to detect the expression of wnt signaling pathway and wnt inhibitor FrpHE and DKK-1 after p53 were blocked. In various p53 status cell lines Hep3B,Lovo,U251 cells were treated with chemoagents, FrpHE, DKK-1 gene mRNA expression levels were detected by RT-PCR.
Results l.The study revealed that p53 protein expression emerged in more than 96% of Hep3B cells 24h and 48h after Adp53
transfection, with its average fluorescent intensity increased by 50 times by FACS.
2. The RT-PCR results: (1) FrpHE and DKK-1 were induced by p53. Our results demonstrated obvious increase of mRNA 20h after the Hep3B cells were transfected with ectogenetic Adp53, FrpHE and DKK-1 mRNA, the peak 32h after the transfection and then decline expression (which, however was higher than that in the controls) 36, 40h after the transfection. On the other hand, FrpHE mRNA had the highest expression when FrpHE and DKK-1 was transfected with Adp53 at the dose of 5pfu/cell and the expression began to decline at the dosage of 50pfu/cell even though it was higher than that in the control group, while DKK-1 mRNA had the apparently growing express at the dose of 0.05pfu/cell and then the highest expression at the dose of 50pfu/cell.
(2) FrpHE and DKK-1 were down-regulated by blocked normal p53 in tumor cells. The results demonstrated that FrpHE mRNA expression in HepG2 cells was declined in 24h and remained markedly unchanged in 48h and 72h, and it was not expressed in Lovo cells, though. DKK-1 mRNA expressions in HepG2, Lovo cells were declined in 24h and remained markedly unchanged in 48h and 72h in 48h and 72h.
(3) FrpHE and DKK-1 were up-regulated by chemoagents. Our study showed that FrpHE mRNA expressions in HepG2 and Hep3B cells were increased compared with the control groups, but nil in Lovo and U251 cells. DKK-1 mRNA expressions in HepG2, Hep3B, Lovo and U251 cells were increased compare with control groups.
3. 3 -catenin, the key factor of Wnt signaling pathway was sharply inhibited by p53. The FACS results demonstrated that the expression of P -catenin was declined in Hep3B cells after transfection with Adp53. (3 -catenin expression hoisted but p53 gene expression was declined in HepG2 and Lovo cells after transfection with antisense p53 ODN. p53 expression hoisted in HepG2, Lovo and U251 cells by chemotherapy.
Conclusion Wnt signaling pathway inhibitor FrpHE and DKK-1, induced by anticancer p53 and chemoagents like adriamycin, hydroxycamptothecin, cisplatin, antagonized Wnt signaling pathway. No inhibitor FrpHE was expressed in Lovo and U251, indicating it varied with some tumor cells inhibitor FrpHE.
引文
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