新基因PRR11在细胞增殖和细胞周期中功能的研究
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摘要
PRR11(proline rich 11)是一个定位于染色体17q22的功能未知的新基因。我们研究小组前期通过对50多个恶性肿瘤相关新基因进行半定量PCR挖掘筛选研究发现,在DNA损伤后PRR11在mRNA水平表达显著降低,提示该基因可能为受p53调控的肿瘤相关基因。生物信息学分析表明:(1)该基因的染色体定位区域在肺癌等各种肿瘤上是一个高发的基因扩增区;(2)多个实验室的cDNA微阵列数据也提示该基因在肿瘤组织中表达上调。目前国内外尚无对此基因的研究报道,本论文在部分前期研究的基础上,对PRR11的序列克隆、鉴定及其对细胞生长的调节、细胞周期的影响等各方面进行了相对系统的研究。分述如下:
(1)PRR11的全长cDNA克隆及序列分析:首先应用5′-RACE和3′-RACE技术,以HeLa细胞cDNA为模板,分别扩增得到PRR11的5′端和3′端cDNA序列。其中5′-RACE共得到七个扩增片段,比较明显的有四个,长度分别约为200bp、300bp、400bp、700bp,其中,400bp条带扩增效率最高,提示其可能为主要转录本。3′-RACE得到两个扩增条带,长度约为2kb和1.5kb。测序结果表明,5′端200bp、300bp、400bp序列与已公布PRR11 mRNA参考序列nm018304一致,仅转录起始位点不同,而700bp序列则较原参考序列5′-UTR延长了284bp;3′端1.5kb序列较已公布PRR11 mRNA参考序列nm018304在3′-UTR延长了172bp,2kb序列则较原参考序列在3′-UTR延长了778bp。采用生物信息学技术将5′-RACE主要转录本400bp序列和3′-RACE序列进行全长序列拼接,获得两个主要的转录本,全长分别为1520bp和2091bp,但两个转录本所编码的氨基酸序列一致,都为360个氨基酸。采用ClustalW在线软件,将人与其他多个真核生物的PRR11氨基酸序列进行比对,结果发现,人与其他多个真核生物间的PRR11氨基酸序列同源性较高,提示PRR11是一个进化保守的基因。在SMART数据库进行磷酸化分析发现,该基因第287个氨基酸为MAPK磷酸化位点,另外,该基因还含有cdc2磷酸化位点,提示PRR11可能与细胞增殖有关。
(2)PRR11的组织表达谱和细胞周期表达谱分析:分别采用半定量RT-PCR、实时荧光定量PCR和蛋白质印迹分析等方法,分析PRR11在不同正常组织之间、正常组织与肿瘤组织之间以及不同细胞周期时相的表达差异。发现PRR11在宫颈、胎盘等细胞分裂比较活跃的组织中表达较高,在脑、骨骼肌等细胞处于静止状态的组织中表达量很低。与正常组织相比,PRR11在肿瘤组织中的表达明显升高,且其表达量随着恶性程度增加而升高。采用细胞同步化技术,制备不同细胞周期时相的样品,进行PRR11的细胞周期表达谱分析,发现PRR11在G1/S期表达降低,在G2/M期表达升高。以上结果提示PRR11是一个新的G2/M期基因,可能参与细胞周期进程或调控进而调控细胞增殖,并且可能与肿瘤的发生发展有关。
(3)PRR11在细胞增殖、细胞周期中的作用研究:设计合成四个针对PRR11不同位点的短链siRNA,半定量RT-PCR、实时定量PCR和蛋白质印迹分析实验结果表明,合成的短链PRR11siRNA可以成功抑制内源性PRR11的表达,抑制率约90%。对HeLa细胞进行PRR11siRNA瞬时转染,发现PRR11 siRNA可以使细胞形态发生明显改变。MTT结果显示siRNA介导的PRR11表达抑制可显著抑制细胞生长。FACS分析结果表明,PRR11siRNA可以导致细胞的S期阻滞。细胞同步化技术结合FACS分析结果也表明,PRR11siRNA可以引起细胞的S期阻滞,并延缓细胞周期进程。实时定量PCR结果表明,PRR11的表达受E2F1和MYB转录因子的正向调节,外源转染的E2F1和Myb表达质粒可显著增强PRR11的表达,siRNA介导的E2F1和Myb表达抑制可引起PRR11的转录下调。这些结果表明,PRR11参与细胞增殖和细胞周期的调节过程,可能作为E2F和MYB的下游靶分子参与细胞增殖和细胞周期的调节。
本论文对PRR11的鉴定及其在细胞增殖、细胞周期调控的分子功能的系统研究加深了我们对于PRR11分子功能和分子行为机制的认识,将为进一步深入开展PRR11的基础理论研究、其在肿瘤发生发展中的作用机制的研究以及促进以PRR11为分子靶点的抗癌药物开发研究均具有积极的理论和现实意义。
PRR11 (proline rich 11) is a novel gene located in 17q22, and its character is still unknown. It is found from 50 cancer-related new genes by semi-quantitative PCR screening in our previous studies. We have found that the mRNA expression level of PRR11 is significantly decreased after DNA damage, which suggesting that this gene may be a tumor-related gene regulated by p53. Bioinformatics analysis showed that: (1) Chromosomal location region of PRR11 is an area of high incidence of gene amplification in lung cancer and other tumors; (2) A number of cDNA microarray data also suggested that PRR11 is up-regulated in tumor tissues. However, there is no any report about this gene right now. In the present study, we have systematically investigated the identification of PRR11 and its role in cell cycle and proliferation. The results are summarized as follows.
(1) Full-length cDNA cloning and sequence analysis of PRR11. At first, the 5 'and 3' end cDNA sequences have been identified by 5 ' -RACE and 3' -RACE. Results showed that there are seven fragments have been cloned in 5 ' end, and four of them are more evident。The length are 200bp, 300bp, 400bp and 700bp,respectively. Among them, the 400bp band is the most evident one, and that means it is the main transcript. There are two fragments have been cloned in 3 ' end and the length are 1.5kb and 2kb,respectively. Sequencing results showed that, in 5 ' end fragments, the sequences of 200bp, 300bp, 400bp are conformity with the published mRNA sequence of PRR11.However, the transcription start sites of these three fragments are different. The 700bp sequence is 284bp extended in 5 '-UTR. Compare with the published mRNA sequence of PRR11, the two fragments cloned in 3 ' end are 172bp and 778bp extended in 3 '-UTR. Splicing the 400bp fragment in 5 ' end with the two fragments in 3 ' end, two major transcripts of the full-length of PRR11 are obtained, whose length are 1520bp and 2091bp. However, the amino acid sequences encoded by these two transcripts are the same, which contain 360 amino acids. To investigate the homology of different spaces, alignment was performed among human and other species using ClustalW online software. The result showed that the PRR11 proteins of other species were highly homologous with human PRR11. This high evolutionary conservation implies that PRR11 may have an important biological role. Phosphorylation analysis showed that PRR11 contains MAPK and cdc2 phosphorylation sites. These results suggest that PRR11 might play an important role in the growth and proliferation in eukaryotic cells.
(2) The expression analysis in tissue and cell cycle of PRR11.
In different normal human tissues, PRR11 is abundantly expressed in the thyroid, cervix, placenta and other active tissues but weakly expressed in heart and brain. The expression level of PRR11 is higher in tumor than normal tissues, and its expression increases along with the malignant stage elevates. Analysis of PRR11 expression profile during cell cycle indicated that PRR11 is a novel G2/M phase gene like Plk1 and which shows a higher expression level during G2/M phase and lower expression level during G1/S phase. These results indicated that PRR11 may involve in cell growth and proliferation, and relates with tumor occurrence and progression.
(3)The role of PRR11 in cell growth and proliferation.
The siRNA-mediated PRR11 knockdown caused a significant morphogenesis changes and growth inhibition in HeLa cells. In addition, PRR11 depletion also induced the S phase arrest during cell cycle. Furthermore, results suggested that PRR11 can be regulated by transcription factor E2F family and MYB, and this might be the mechanism that PRR11 involve in cell proliferation and cell cycle.
Taken together, those results indicated that PRR11 plays an important role in in cell growth, cell proliferation and cell cycle, and it may be used as an attractive target to develop novel therapeutic chemosensitizers and gene therapeutic drugs against cancer.
(1)PRR11的全长cDNA克隆及序列分析:首先应用5′-RACE和3′-RACE技术,以HeLa细胞cDNA为模板,分别扩增得到PRR11的5′端和3′端cDNA序列。其中5′-RACE共得到七个扩增片段,比较明显的有四个,长度分别约为200bp、300bp、400bp、700bp,其中,400bp条带扩增效率最高,提示其可能为主要转录本。3′-RACE得到两个扩增条带,长度约为2kb和1.5kb。测序结果表明,5′端200bp、300bp、400bp序列与已公布PRR11 mRNA参考序列nm018304一致,仅转录起始位点不同,而700bp序列则较原参考序列5′-UTR延长了284bp;3′端1.5kb序列较已公布PRR11 mRNA参考序列nm018304在3′-UTR延长了172bp,2kb序列则较原参考序列在3′-UTR延长了778bp。采用生物信息学技术将5′-RACE主要转录本400bp序列和3′-RACE序列进行全长序列拼接,获得两个主要的转录本,全长分别为1520bp和2091bp,但两个转录本所编码的氨基酸序列一致,都为360个氨基酸。采用ClustalW在线软件,将人与其他多个真核生物的PRR11氨基酸序列进行比对,结果发现,人与其他多个真核生物间的PRR11氨基酸序列同源性较高,提示PRR11是一个进化保守的基因。在SMART数据库进行磷酸化分析发现,该基因第287个氨基酸为MAPK磷酸化位点,另外,该基因还含有cdc2磷酸化位点,提示PRR11可能与细胞增殖有关。
(2)PRR11的组织表达谱和细胞周期表达谱分析:分别采用半定量RT-PCR、实时荧光定量PCR和蛋白质印迹分析等方法,分析PRR11在不同正常组织之间、正常组织与肿瘤组织之间以及不同细胞周期时相的表达差异。发现PRR11在宫颈、胎盘等细胞分裂比较活跃的组织中表达较高,在脑、骨骼肌等细胞处于静止状态的组织中表达量很低。与正常组织相比,PRR11在肿瘤组织中的表达明显升高,且其表达量随着恶性程度增加而升高。采用细胞同步化技术,制备不同细胞周期时相的样品,进行PRR11的细胞周期表达谱分析,发现PRR11在G1/S期表达降低,在G2/M期表达升高。以上结果提示PRR11是一个新的G2/M期基因,可能参与细胞周期进程或调控进而调控细胞增殖,并且可能与肿瘤的发生发展有关。
(3)PRR11在细胞增殖、细胞周期中的作用研究:设计合成四个针对PRR11不同位点的短链siRNA,半定量RT-PCR、实时定量PCR和蛋白质印迹分析实验结果表明,合成的短链PRR11siRNA可以成功抑制内源性PRR11的表达,抑制率约90%。对HeLa细胞进行PRR11siRNA瞬时转染,发现PRR11 siRNA可以使细胞形态发生明显改变。MTT结果显示siRNA介导的PRR11表达抑制可显著抑制细胞生长。FACS分析结果表明,PRR11siRNA可以导致细胞的S期阻滞。细胞同步化技术结合FACS分析结果也表明,PRR11siRNA可以引起细胞的S期阻滞,并延缓细胞周期进程。实时定量PCR结果表明,PRR11的表达受E2F1和MYB转录因子的正向调节,外源转染的E2F1和Myb表达质粒可显著增强PRR11的表达,siRNA介导的E2F1和Myb表达抑制可引起PRR11的转录下调。这些结果表明,PRR11参与细胞增殖和细胞周期的调节过程,可能作为E2F和MYB的下游靶分子参与细胞增殖和细胞周期的调节。
本论文对PRR11的鉴定及其在细胞增殖、细胞周期调控的分子功能的系统研究加深了我们对于PRR11分子功能和分子行为机制的认识,将为进一步深入开展PRR11的基础理论研究、其在肿瘤发生发展中的作用机制的研究以及促进以PRR11为分子靶点的抗癌药物开发研究均具有积极的理论和现实意义。
PRR11 (proline rich 11) is a novel gene located in 17q22, and its character is still unknown. It is found from 50 cancer-related new genes by semi-quantitative PCR screening in our previous studies. We have found that the mRNA expression level of PRR11 is significantly decreased after DNA damage, which suggesting that this gene may be a tumor-related gene regulated by p53. Bioinformatics analysis showed that: (1) Chromosomal location region of PRR11 is an area of high incidence of gene amplification in lung cancer and other tumors; (2) A number of cDNA microarray data also suggested that PRR11 is up-regulated in tumor tissues. However, there is no any report about this gene right now. In the present study, we have systematically investigated the identification of PRR11 and its role in cell cycle and proliferation. The results are summarized as follows.
(1) Full-length cDNA cloning and sequence analysis of PRR11. At first, the 5 'and 3' end cDNA sequences have been identified by 5 ' -RACE and 3' -RACE. Results showed that there are seven fragments have been cloned in 5 ' end, and four of them are more evident。The length are 200bp, 300bp, 400bp and 700bp,respectively. Among them, the 400bp band is the most evident one, and that means it is the main transcript. There are two fragments have been cloned in 3 ' end and the length are 1.5kb and 2kb,respectively. Sequencing results showed that, in 5 ' end fragments, the sequences of 200bp, 300bp, 400bp are conformity with the published mRNA sequence of PRR11.However, the transcription start sites of these three fragments are different. The 700bp sequence is 284bp extended in 5 '-UTR. Compare with the published mRNA sequence of PRR11, the two fragments cloned in 3 ' end are 172bp and 778bp extended in 3 '-UTR. Splicing the 400bp fragment in 5 ' end with the two fragments in 3 ' end, two major transcripts of the full-length of PRR11 are obtained, whose length are 1520bp and 2091bp. However, the amino acid sequences encoded by these two transcripts are the same, which contain 360 amino acids. To investigate the homology of different spaces, alignment was performed among human and other species using ClustalW online software. The result showed that the PRR11 proteins of other species were highly homologous with human PRR11. This high evolutionary conservation implies that PRR11 may have an important biological role. Phosphorylation analysis showed that PRR11 contains MAPK and cdc2 phosphorylation sites. These results suggest that PRR11 might play an important role in the growth and proliferation in eukaryotic cells.
(2) The expression analysis in tissue and cell cycle of PRR11.
In different normal human tissues, PRR11 is abundantly expressed in the thyroid, cervix, placenta and other active tissues but weakly expressed in heart and brain. The expression level of PRR11 is higher in tumor than normal tissues, and its expression increases along with the malignant stage elevates. Analysis of PRR11 expression profile during cell cycle indicated that PRR11 is a novel G2/M phase gene like Plk1 and which shows a higher expression level during G2/M phase and lower expression level during G1/S phase. These results indicated that PRR11 may involve in cell growth and proliferation, and relates with tumor occurrence and progression.
(3)The role of PRR11 in cell growth and proliferation.
The siRNA-mediated PRR11 knockdown caused a significant morphogenesis changes and growth inhibition in HeLa cells. In addition, PRR11 depletion also induced the S phase arrest during cell cycle. Furthermore, results suggested that PRR11 can be regulated by transcription factor E2F family and MYB, and this might be the mechanism that PRR11 involve in cell proliferation and cell cycle.
Taken together, those results indicated that PRR11 plays an important role in in cell growth, cell proliferation and cell cycle, and it may be used as an attractive target to develop novel therapeutic chemosensitizers and gene therapeutic drugs against cancer.
引文
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