人类UHRF1基因肿瘤生物学功能的研究
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摘要
目的:研究hUHRF1基因表达水平改变对乳癌细胞生长、增殖、侵袭、转移以及放射治疗敏感性的影响,及其生物学功能的作用机制,为研究乳癌的发生、发展和治疗提供了新的研究方向和理论基础,为将来hUHRF1基因在肿瘤临床治疗应用提供理论基础和实验依据。
方法:
①采用Western Blot法,检测多种人肿瘤细胞和正常细胞中hUHRF1的蛋白表达情况;Northern Blot法对临床乳癌组织样本和正常乳腺组织中hUHRF1的RNA表达水平进行检测;
②根据GeneBank中hUHRF1的基因序列,自行设计扩增hUHRF1基因全长cDNA的引物一对,构建hUHRF1的真核表达载体和腺病毒表达载体;
③采用阳离子脂质体Lipofactamin2000介导的方法,进行hUHRF1基因转染,在含G418的培养基中筛选阳性克隆,采用RT-PCR和Western Blot法检测阳性克隆中hUHRF1的RNA和蛋白表达水平;
④采用MTT法观察hUHRF1基因转染,对体外培养的不同p53基因表型、不同组织来源的乳癌细胞生长、增殖的影响;皮下瘤液接种法,制备乳癌动物模型,观察hUHRF1基因在体内对乳癌生长的影响;
⑤细胞接受不同剂量的χ射线、UV辐照和γ射线照射后的克隆形成实验,观察细胞辐射敏感性的变化;
⑥划痕实验和Boyden小室法,观察细胞侵袭、转移能力的变化;
⑦流式细胞术分析细胞周期进程和细胞凋亡变化;免疫组化法观察动物肿瘤组织中新生血管的形成;染色体畸变分析观察基因组稳定性变化;Western Blot法检测调控细胞周期、凋亡、DNA损伤修复相关蛋白、细胞侵袭、转移相关基因的蛋白表达水平变化。
结果
①肿瘤细胞中hUHRF1的蛋白表达水平明显高于正常细胞;临床乳癌组织中hUHRF1的RNA水平较正常乳腺组织的表达水平高;
②构建的hUHRF1真核表达载体,经过限制性核酸内切酶BamH I和Xho I酶切后电泳和扩增片断测序,证实载体构建成功;
③G418筛选得到的阳性克隆,经RT-PCR和Western Blot证实,hUHRF1基因在乳癌细胞中高表达,表明高hUHRF1表达的细胞株筛选成功;
④MTT的结果表明:hUHRF1可以促进p53突变型乳癌细胞MDA-MB-231和BT-549的生长,而对p53野生型乳癌细胞MCF-7的生长未见明显影响;而且,hUHRF1的高水平表达,可以通过诱导肿瘤血管形成,在体内促进乳癌细胞的生长;
⑤流式细胞术分析结果表明:hUHRF1基因转染后,p53突变型乳癌细胞MDA-MB-231的细胞周期进程中,G0/G1比例由69.157%降至49.854%,subG1%由16.31%降至9.32%,差异有统计意义;而p53野生型乳癌细胞MCF-7的细胞周期进程的G0/G1比例由73.874%降至72.812%,subG1%由14.88%降至8.08%,差异没有统计意义,即外源性hUHRF1的高表达,可明显缩短p53突变型乳癌细胞MDA-MB-231的细胞周期G0/G1期进程,抑制细胞凋亡的发生,而对p53野生型乳癌细胞MCF-7的生长未产生明显影响。Western Blot法检测细胞周期G1期调控因子cyclinD1和凋亡相关因子Bax在MDA-MB-231细胞中的表达,结果表明,hUHRF1基因转染可诱导cyclinD1的表达水平增强,而抑制促凋亡蛋白Bax的表达,与我们流式细胞术的分析结果一致;
⑥集落形成实验的结果显示,hUHRF1高水平表达可以降低乳癌细胞MDA-MB-231对χ射线、UV辐照,HeLa细胞对γ射线的辐射敏感性;采用siRNA,“敲除”hUHRF1基因,可增加乳癌细胞MDA-MB-231的辐射敏感性;
⑦hUHRF1的高水平表达,可以诱导辐射后的细胞发生明显的G2期阻滞;抑制细胞凋亡;抑制辐照后的细胞中cyclinB1和Bax的表达,而Bcl-2的表达水平影响不明显。
⑧Western Blot法检测DNA损伤修复过程中的调节因素XRCC4,Ku70,Ku80蛋白表达水平变化,结果显示:在相同剂量的χ射线照射下,hUHRF1基因的高表达,可以诱导乳癌细胞MDA-MB-231中Ku70,Ku80的表达增强;染色体的畸变率降低;但在腺病毒感染的HeLa细胞中,Ad5-hUHRF1仅使XRCC4的表达水平增加,采用siRNA“敲除”HeLa细胞中XRCC4的表达,改变了hUHRF1调节的辐射敏感性。
⑨划痕实验和Boyden小室法的结果显示:hUHRF1高水平表达,可以增强乳癌细胞MDA-MB-231的侵袭和转移能力;但是,Western Blot法检测结果发现,hUHRF1基因高水平表达并不改变PTEN和maspin的表达水平,但是降低细胞的PTEN和maspin表达水平则影响了hUHRF1对细胞的侵袭和转移能力。
结论:
①hUHRF1基因高表达,在体外通过缩短细胞周期进程,抑制细胞凋亡,促进p53突变型乳癌细胞的生长,而对p53野生型乳癌细胞的生长无明显影响。
②提高hUHRF1基因表达,通过促进肿瘤血管形成,加速动物体内乳腺肿瘤的生长、增殖和转移。
③提高hUHRF1基因表达通过调控细胞周期,抑制凋亡,促进DNA损伤修复,维持基因组稳定性,降低乳癌细胞的辐射敏感性。
④提高hUHRF1基因表达增强乳癌细胞的侵袭和转移能力,PTEN和maspin可能起着一定的作用。
UHRF1 (ubiquitin-like protein containing PHD and RING domain 1) was first cloned and isolated in 1998, also named NP95 as a 95-kDa nuclear protein consisting of 782 amino acids (18 exons, spanning 60 kb) and is assigned to chromosome 19p13.3. The hUHRF1 open reading frame contains an unusual N-terminal domain that bears a striking resemblance to ubiqutin, a leucine zipper motif, a zinc finger motif, a potential ATP/GTP binding site, a putative cyclin A/E-cdk2 phosphorylation site, retinoblastoma protein (Rb)-binding motifs (331LMCDE335 and 725LCCQE729), a ring finger domain and the SRA-YDG domain.It has been found that mouse UHRF1 is strongly expressed in the testis, spleen, thymus, and lung tissues, but not in the brain, liver, or skeletal muscles. Although biological functions of hUHRF1 are unknown, previous studies have suggested that (i) hUHRF1 does not take a direct part in DNA replication as part of the DNA synthesizing machinery, like PCNA, but is presumably involved in other DNA replication-linked nuclear events; (ii) hUHRF1 may be a growth-regulated gene, since its expression was regulated during the cell cycle, required for the G1/S transition, and specifically induced by E1A which can force postmitotic cells to proliferate, and it is a chromatin-associated ubiquitin ligase; (iii) hUHRF1 mRNA was also increased in the transformed BALB/3T3 cells, suggesting it may participate in the maintenance of the transformed phenotype; (iv) hUHRF1 might help recruit DNA-cytosine-5-methyltransferase 1 (DNMT1) to hemimethylated DNA to facilitate faithful maintenance of DNA methylation; and (v) mouse Np95-null (Np95-/-)embryonic stem cells are more sensitive toχrays, UV light, N-methyl-N′-nitro-N-nitrosoguanidine and hydroxyurea versus wild-type (Np95+/+) or Np95+/? embryonic stem cells. Furthermore, several stable transformants from HEK293 and WI-38 cells that had been transfected with the antisense human UHRF1 cDNA were more sensitive toχrays, UV light and hydroxyurea than the corresponding parental cells. Additionally, there was no significant redistribution of hUHRF1 foci shortly after DNA damage byγ-irradiation, but nodular hUHRF1 foci characteristically seen in G2 phase were also detected in G2-arrested cells followingγ-irradiation. These results indicate that hUHRF1 may play an important role in regulation of radiosensitivity. Taken together, the available studies indicate that hUHRF1 may be a putative oncogene, which will become a new target for cancer treatment.
The current study was designed to determine the impact of overexpression of hUHRF1 by stable transfection of pcDNA3-hUHRF1 expression vector or adenoviral-mediated transduction of hUHRF1 (Ad5-UHRF1) on cell growth, radiosensitivity and migration as well as invasion in human breast cancer MDA-MB231 cells and human cervical cancer HeLa cells. The findings are summzied as following:
(1) There is a higher expression of hUHRF1 in breast cancer tissues and breast cancer cell lines compare with normal breast tissues and normal cell lines;
(2) Overexpression of hUHRF1 reduced cell doubling time or promoted cell growth in p53-mutant breast cancer cells lines, including MDA-MB-231 and BT-549, but not in wild-type p53 MCF-7 cells. This promotion of cell growth was associated with the regulation of cell cycle, reduction of apoptosis and reduction of apoptosis promoting protein Bax and cyclin D1 by hUHRF1 overexpression;
(3) Overexpression of hUHRF1 reduced the radiosensitivity to ionzing radiation and UV, while knocking down hUHRF1 expression increased the radiosensitivity in MDA-MB-231 and HeLa cells. This radioresistance caused by hUHRF1 overexpression may be relative to (i) abolishing apoptosis induction by radiation; (ii) reducing Bax expression; (iii) reducing chromosome abbration caused radiation; (iv) affecting Ku70, Ku 80 and XRCC4 expression. Knocking-down XRCC4 expression reduced hUHRF1-mediated radioresistance.
(4) Overexpression of hUHRF1 elevated cellular capability of invasion and migration in MDA-MB-231 cells. Although enhanced expression of hUHRF1 did not affect the expression of PTEN and Maspin, two anti-metastasis proteins. However, reduction of PTEN and Maspin will significantly affected the hUHRF1-mediated invasion .
These results provide the first evidence that hUHRF1 is an important gene as a novel oncogene in cell growth, invasion, migration and radiosensitivity of cancer, at least human breasat cancer and cervical cancer cells.
方法:
①采用Western Blot法,检测多种人肿瘤细胞和正常细胞中hUHRF1的蛋白表达情况;Northern Blot法对临床乳癌组织样本和正常乳腺组织中hUHRF1的RNA表达水平进行检测;
②根据GeneBank中hUHRF1的基因序列,自行设计扩增hUHRF1基因全长cDNA的引物一对,构建hUHRF1的真核表达载体和腺病毒表达载体;
③采用阳离子脂质体Lipofactamin2000介导的方法,进行hUHRF1基因转染,在含G418的培养基中筛选阳性克隆,采用RT-PCR和Western Blot法检测阳性克隆中hUHRF1的RNA和蛋白表达水平;
④采用MTT法观察hUHRF1基因转染,对体外培养的不同p53基因表型、不同组织来源的乳癌细胞生长、增殖的影响;皮下瘤液接种法,制备乳癌动物模型,观察hUHRF1基因在体内对乳癌生长的影响;
⑤细胞接受不同剂量的χ射线、UV辐照和γ射线照射后的克隆形成实验,观察细胞辐射敏感性的变化;
⑥划痕实验和Boyden小室法,观察细胞侵袭、转移能力的变化;
⑦流式细胞术分析细胞周期进程和细胞凋亡变化;免疫组化法观察动物肿瘤组织中新生血管的形成;染色体畸变分析观察基因组稳定性变化;Western Blot法检测调控细胞周期、凋亡、DNA损伤修复相关蛋白、细胞侵袭、转移相关基因的蛋白表达水平变化。
结果
①肿瘤细胞中hUHRF1的蛋白表达水平明显高于正常细胞;临床乳癌组织中hUHRF1的RNA水平较正常乳腺组织的表达水平高;
②构建的hUHRF1真核表达载体,经过限制性核酸内切酶BamH I和Xho I酶切后电泳和扩增片断测序,证实载体构建成功;
③G418筛选得到的阳性克隆,经RT-PCR和Western Blot证实,hUHRF1基因在乳癌细胞中高表达,表明高hUHRF1表达的细胞株筛选成功;
④MTT的结果表明:hUHRF1可以促进p53突变型乳癌细胞MDA-MB-231和BT-549的生长,而对p53野生型乳癌细胞MCF-7的生长未见明显影响;而且,hUHRF1的高水平表达,可以通过诱导肿瘤血管形成,在体内促进乳癌细胞的生长;
⑤流式细胞术分析结果表明:hUHRF1基因转染后,p53突变型乳癌细胞MDA-MB-231的细胞周期进程中,G0/G1比例由69.157%降至49.854%,subG1%由16.31%降至9.32%,差异有统计意义;而p53野生型乳癌细胞MCF-7的细胞周期进程的G0/G1比例由73.874%降至72.812%,subG1%由14.88%降至8.08%,差异没有统计意义,即外源性hUHRF1的高表达,可明显缩短p53突变型乳癌细胞MDA-MB-231的细胞周期G0/G1期进程,抑制细胞凋亡的发生,而对p53野生型乳癌细胞MCF-7的生长未产生明显影响。Western Blot法检测细胞周期G1期调控因子cyclinD1和凋亡相关因子Bax在MDA-MB-231细胞中的表达,结果表明,hUHRF1基因转染可诱导cyclinD1的表达水平增强,而抑制促凋亡蛋白Bax的表达,与我们流式细胞术的分析结果一致;
⑥集落形成实验的结果显示,hUHRF1高水平表达可以降低乳癌细胞MDA-MB-231对χ射线、UV辐照,HeLa细胞对γ射线的辐射敏感性;采用siRNA,“敲除”hUHRF1基因,可增加乳癌细胞MDA-MB-231的辐射敏感性;
⑦hUHRF1的高水平表达,可以诱导辐射后的细胞发生明显的G2期阻滞;抑制细胞凋亡;抑制辐照后的细胞中cyclinB1和Bax的表达,而Bcl-2的表达水平影响不明显。
⑧Western Blot法检测DNA损伤修复过程中的调节因素XRCC4,Ku70,Ku80蛋白表达水平变化,结果显示:在相同剂量的χ射线照射下,hUHRF1基因的高表达,可以诱导乳癌细胞MDA-MB-231中Ku70,Ku80的表达增强;染色体的畸变率降低;但在腺病毒感染的HeLa细胞中,Ad5-hUHRF1仅使XRCC4的表达水平增加,采用siRNA“敲除”HeLa细胞中XRCC4的表达,改变了hUHRF1调节的辐射敏感性。
⑨划痕实验和Boyden小室法的结果显示:hUHRF1高水平表达,可以增强乳癌细胞MDA-MB-231的侵袭和转移能力;但是,Western Blot法检测结果发现,hUHRF1基因高水平表达并不改变PTEN和maspin的表达水平,但是降低细胞的PTEN和maspin表达水平则影响了hUHRF1对细胞的侵袭和转移能力。
结论:
①hUHRF1基因高表达,在体外通过缩短细胞周期进程,抑制细胞凋亡,促进p53突变型乳癌细胞的生长,而对p53野生型乳癌细胞的生长无明显影响。
②提高hUHRF1基因表达,通过促进肿瘤血管形成,加速动物体内乳腺肿瘤的生长、增殖和转移。
③提高hUHRF1基因表达通过调控细胞周期,抑制凋亡,促进DNA损伤修复,维持基因组稳定性,降低乳癌细胞的辐射敏感性。
④提高hUHRF1基因表达增强乳癌细胞的侵袭和转移能力,PTEN和maspin可能起着一定的作用。
UHRF1 (ubiquitin-like protein containing PHD and RING domain 1) was first cloned and isolated in 1998, also named NP95 as a 95-kDa nuclear protein consisting of 782 amino acids (18 exons, spanning 60 kb) and is assigned to chromosome 19p13.3. The hUHRF1 open reading frame contains an unusual N-terminal domain that bears a striking resemblance to ubiqutin, a leucine zipper motif, a zinc finger motif, a potential ATP/GTP binding site, a putative cyclin A/E-cdk2 phosphorylation site, retinoblastoma protein (Rb)-binding motifs (331LMCDE335 and 725LCCQE729), a ring finger domain and the SRA-YDG domain.It has been found that mouse UHRF1 is strongly expressed in the testis, spleen, thymus, and lung tissues, but not in the brain, liver, or skeletal muscles. Although biological functions of hUHRF1 are unknown, previous studies have suggested that (i) hUHRF1 does not take a direct part in DNA replication as part of the DNA synthesizing machinery, like PCNA, but is presumably involved in other DNA replication-linked nuclear events; (ii) hUHRF1 may be a growth-regulated gene, since its expression was regulated during the cell cycle, required for the G1/S transition, and specifically induced by E1A which can force postmitotic cells to proliferate, and it is a chromatin-associated ubiquitin ligase; (iii) hUHRF1 mRNA was also increased in the transformed BALB/3T3 cells, suggesting it may participate in the maintenance of the transformed phenotype; (iv) hUHRF1 might help recruit DNA-cytosine-5-methyltransferase 1 (DNMT1) to hemimethylated DNA to facilitate faithful maintenance of DNA methylation; and (v) mouse Np95-null (Np95-/-)embryonic stem cells are more sensitive toχrays, UV light, N-methyl-N′-nitro-N-nitrosoguanidine and hydroxyurea versus wild-type (Np95+/+) or Np95+/? embryonic stem cells. Furthermore, several stable transformants from HEK293 and WI-38 cells that had been transfected with the antisense human UHRF1 cDNA were more sensitive toχrays, UV light and hydroxyurea than the corresponding parental cells. Additionally, there was no significant redistribution of hUHRF1 foci shortly after DNA damage byγ-irradiation, but nodular hUHRF1 foci characteristically seen in G2 phase were also detected in G2-arrested cells followingγ-irradiation. These results indicate that hUHRF1 may play an important role in regulation of radiosensitivity. Taken together, the available studies indicate that hUHRF1 may be a putative oncogene, which will become a new target for cancer treatment.
The current study was designed to determine the impact of overexpression of hUHRF1 by stable transfection of pcDNA3-hUHRF1 expression vector or adenoviral-mediated transduction of hUHRF1 (Ad5-UHRF1) on cell growth, radiosensitivity and migration as well as invasion in human breast cancer MDA-MB231 cells and human cervical cancer HeLa cells. The findings are summzied as following:
(1) There is a higher expression of hUHRF1 in breast cancer tissues and breast cancer cell lines compare with normal breast tissues and normal cell lines;
(2) Overexpression of hUHRF1 reduced cell doubling time or promoted cell growth in p53-mutant breast cancer cells lines, including MDA-MB-231 and BT-549, but not in wild-type p53 MCF-7 cells. This promotion of cell growth was associated with the regulation of cell cycle, reduction of apoptosis and reduction of apoptosis promoting protein Bax and cyclin D1 by hUHRF1 overexpression;
(3) Overexpression of hUHRF1 reduced the radiosensitivity to ionzing radiation and UV, while knocking down hUHRF1 expression increased the radiosensitivity in MDA-MB-231 and HeLa cells. This radioresistance caused by hUHRF1 overexpression may be relative to (i) abolishing apoptosis induction by radiation; (ii) reducing Bax expression; (iii) reducing chromosome abbration caused radiation; (iv) affecting Ku70, Ku 80 and XRCC4 expression. Knocking-down XRCC4 expression reduced hUHRF1-mediated radioresistance.
(4) Overexpression of hUHRF1 elevated cellular capability of invasion and migration in MDA-MB-231 cells. Although enhanced expression of hUHRF1 did not affect the expression of PTEN and Maspin, two anti-metastasis proteins. However, reduction of PTEN and Maspin will significantly affected the hUHRF1-mediated invasion .
These results provide the first evidence that hUHRF1 is an important gene as a novel oncogene in cell growth, invasion, migration and radiosensitivity of cancer, at least human breasat cancer and cervical cancer cells.
引文
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15. Masahiro Muto,Yasuyoshi Kanari, Eiko Kubo, et al. Targeted disruption of NP95 gene renders murine embryonic stem cells hypersensitive to DNA damaging agents and DNA replication blocks.J Biol Chem, 2002, 277(37): 34549-34555.
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