KR100654949B1 - Screening method of the genes involved in cancers using fission yeast S.pombe system - Google Patents

Abstract

The present invention relates to a method for analyzing a function of an unidentified gene. Specifically, an expression vector of a cleavage yeast S.pombe into which an unidentified gene is inserted is prepared and introduced into the cleavage yeast, and overexpressed by observing trait changes of the cleavage yeast. It relates to a method for analyzing the function of the gene. The method of the present invention can be usefully used for the analysis of biological functions of genes whose function is not fully known and for the study of the biological pathways in which the genes are involved.

Cleavage yeast (S.pombe), gene function analysis method, lethality test

Description

Screening method of the genes involved in cancers using fission yeast S.pombe system}

1 is a cleavage map of pSLF173-thiamine inducible plasmid (pSLF173), a S. pombe expression vector used as a base vector,

Figure 2 is a schematic diagram showing the manufacturing process of pDES173, which is a S. pombe destination vector that can easily carry human genes,

3 is a schematic diagram showing a process of cloning a human gene into the prepared S. pombe destination vector,

4 is a result of a lethality test when gastric cancer or liver cancer-related human genes are overexpressed in S. pombe .

FIG. 5 is a photograph showing changes in cell shape when gastric cancer or liver cancer-related human genes are overexpressed in S. pombe .

FIG. 6 is a graph showing the results of analysis of cell growth and cell division cycle abnormality when gastric cancer or liver cancer-related human genes are overexpressed in S. pombe . FIG.

7 is a photograph showing the anticancer agent sensitivity measurement of deletion mutants of S.pombe genes showing homology with gastric cancer or liver cancer-related human genes and the recovery of sensitivity by overexpression of human genes.

8 is a result of comparing expression levels of gastric cancer or liver cancer cell lines of hnRNP A1, one of the human genes selected for cancer relevance verification,

Figure 9 is a photograph showing the cell trait change caused after the introduction of hnRNP A1 into NIH3T3 cells, a non-transformed cell line.

The present invention relates to a method for analyzing a function of an unidentified gene. Specifically, an expression vector of a cleavage yeast S.pombe into which an unidentified gene is inserted is prepared and introduced into the cleavage yeast, and overexpressed by observing trait changes of the cleavage yeast. It relates to a method for analyzing the function of the gene.

With the recent completion of the human genome project, all of the sequences of the human genome have been revealed. The estimated number of human genes is about 35,000 ~ 40,000, most of which have only known sequences but no function. There is a lot of research into the function of human genes that do not know their function. Model organisms are used to understand the function of genes. In general, the best way to analyze the biological function of an unknown gene in higher animals such as humans is to use transgenic animals or It is ideal to use knock-out animal models, but animal models are not easy to transform or knock-out, and have a drawback of costly and time-consuming research. There is a limit. Meanwhile, Schizosaccharomyces pombe , a fission yeast, Saccharomyces cerevisiae , a budding yeast, and Caenorhabditis elegans , mice (mouse), Drosophila melanogaster ( Drosophila melanogaster ) has already revealed all the nucleotide sequence of the gene, can be cultured at a low cost, easy genetic research and manipulation, well established biochemical and physiological function analysis system model It can be used as an organism, and can be widely used for biological function analysis of genes whose function is not known, biologic path studies, disease mechanism research and drug efficacy evaluation. Among them, the germinating yeast S. cerevisiae is the simplest model cell of eukaryotic cells. It is widely used as a model cell to reveal the function of genes causing disease (Lum, PK et al. , Cell ., 116: 121-137, 2004).

On the other hand, S.pombe , a split yeast, has recently completed sequencing. S.pombe , like S. cerevisiae , belongs to the ascomycetes, but is not so closely related to evolution. S.pombe (Wood V. et al ., Nature. 45: 871-880, 2002) is a genome base in eukaryotic cells after S. cerevisiae (Goffeau A. et al ., Science , 274: 546-567, 1996). The sixth organism has completed sequencing.

According to the analysis results, S.pombe has an efficient chromosome structure with low functional repeatability of genes among the eukaryotic cells whose sequencing has been determined so far, with 4,824 genes determining the smallest but 43% of the genes are introns. It has been reported to contain.

S. pombe has very well preserved important genes in eukaryotic cells such as cell cycle regulation, proteolysis, protein phosphorylation and RNA splicing.

In addition, since 31% of S.pombe genes are reported to be different from S.cerevisiae genes, but rather closer to humans, it is an efficient way to study the function of human genes by studying the function of S.pombe genes and comparing them with S.cerevisiae genes. Is considered. In addition, 172 kinds of genes similar to those related to human diseases have been found, and among them, 23 kinds of genes related to cancer have been reported.

In addition, it is estimated that genes similar to those related to human disease are present in S. pombe , and the functions of genes that are directly or indirectly involved in various diseases including cancer by identifying the function of the gene using S. pombe . Biological function analysis will be possible. The present inventors have studied the method for analyzing the function of human genes using S.pombe , and established the analysis method. .

An object of the present invention is to use a cleavage yeast S.pombe system It is to provide a method for analyzing the function of human genes whose function is not clear.

The present invention

 i) preparing a destination vector capable of inducing overexpression of a target gene (unidentified gene) in cleavage yeast S.pombe;

ii) preparing an expression vector into which a target gene is inserted using the target vector of step i);

iii) introducing the expression vector of step ii) into normal cleavage yeast to overexpress the desired gene; And

iv) providing a method for analyzing the function of the gene of interest comprising the step of observing the transformation of the yeast of step iii).

The present invention also provides a vector pDES173 for use in the method.

In addition, the present invention provides a gene screened by the above method.

Hereinafter, the present invention will be described in detail.

The present invention

i) preparing a target vector capable of inducing overexpression of the target gene in cleavage yeast S.pombe;

ii) preparing an expression vector into which a target gene is inserted using the target vector of step i);

iii) introducing the expression vector of step ii) into normal cleavage yeast to overexpress the desired gene; And

iv) providing a method for analyzing the function of the gene of interest comprising the step of observing the transformation of the yeast of step iii).

The analysis method is in addition to the steps i), ii), iii), iv)

i) identifying functional complementation between the human gene and the yeast gene using an anticancer agent; or

ii) verifying cancer relatedness of the gene of interest using the human cancer cell line.

SUMMARY OF THE INVENTION An object of the present invention is to provide a method for analyzing the function of human genes that have been mass screened through a microarray using S.pombe system. S. pombe has bioregulatory pathways similar to humans and higher animals because the function of many genes is preserved in both organisms. As an example, experiments have shown that ROD1 , known to be involved in cell differentiation in humans, and nrd1 , a gene responsible for regulating mating pathways in S. pombe , have a functional complementation. was demonstrated (Biol Yamanoto H, et al Mol Cell, May;..... 19 (5) 3829-3841), which human differentiation (differentiation) form mating / spores mechanisms and S.pombe (mating / sporulation) Mechanisms are preserved, which means that they are similar to each other. In addition, cyclin dependent kinase 4 (CDK4), which is known to be involved in cell cycle regulation and is known to be involved in cancer development, is similar in function to cdc2, the only cyclin-dependent kinase of S.pombe. It has been reported (Wood et al ., Nature, 45: 871-880, 2002). The present inventors selected S. pombe as a model organism to analyze the function of human genes based on the above facts.

In the present invention, an analysis system using cleavage yeast S.pombe for secondary screening and verification of genes showing differences in expression levels in the tissues of gastric cancer and liver cancer compared to normal tissues by microarray technique.
More specifically, in the present invention, the ' S. pombe system ' is intended to easily transfer a large amount of human genes by modifying the S. pombe basic expression vector, and converting cassette into pSLF73 (Invitrogen) which is an induced expression vector of S. pombe . Cancer-related human gene screening system using S. pombe object vector (pDES173) prepared by the introduction and the cleavage yeast ( S. pombe ) transformed by the object vector 'means.
In addition, in the present invention, ' secondary screening ' iii) 'screening process (primary)' for selecting cancer-related candidate genes by observing cell type changes of cleavage yeast containing the S. pombe target vector and ii) Functional complementarity between human and S. pombe genes was confirmed by identifying anticancer drug sensitivity and lethality recovery using a split yeast heterozygous deletion mutant lacking the S. pombe genes with sequence homology with the selected cancer-related candidate genes. It means 'screening process (secondary)' to confirm.
The present inventors used the Gateway ^TM system (Invitrogen, USA) to easily express hundreds of human genes in yeast in order to construct an analysis system using the cleavage yeast S.pombe . Specifically, after digesting the SmaI site of the pSLF173 base vector (see FIG. 1 ), which is an S. pombe induced expression vector, the conversion cassette was linked and cloned, and the completed plasmid was named pDES173 ( FIG. 2 ). The plasmid was transformed into E. coli and deposited with the Biotechnology Research Institute Gene Bank on January 6, 2005 (Accession No .: KCTC 10757BP). Production of S. pombe expression clone using the conversion cassette, when overexpression of several kinds of human genes in S. pombe , each of the gene cDNA constructs are re-obtained and cloned individually for S.pombe expression vector system This has the advantage of reducing the hassle of doing. Final gene expression clones were prepared to fuse candidate human genes related to gastric or liver cancer cloned into entry vectors to the completed pDES173 vector (see FIG. 3 ).

As described above, the candidate genes related to gastric cancer and liver cancer were cloned into the cleavage yeast expression vector, and then cell trait changes and the extent of cell growth inhibition which occurred upon its overexpression were confirmed. Transformed with the lithium acetate (lithium acetate) method of stomach cancer, liver cancer-related candidate human genes for the ED665 (h-) normal haploid (haploid) strains were S.pombe (transformation). In order to express cancer related candidate genes in ED665 (h-), a minimal medium (EMM + AL + thia) and a thiamine-free medium (EMM + AL) with 2 μM of thiamine were used. As a control, pDES173 vector was expressed without the cancer related candidate genes. By comparing the number and growth of colonies grown in EMM + AL solid medium and EMM + AL + thia solid medium, the traits showing abnormalities in cell growth or completely inhibited cell growth during human gene overexpression were confirmed ( FIG. 4 and FIG . 4) . 5 ).

In addition, in order to confirm the change in cell transformation, transformed cells were inoculated in EMM + AL liquid medium and EMM + AL + thia (2uM) liquid medium, and the cells obtained after the culture were observed under an optical microscope. Cell trait changes that occur when human genes are overexpressed were compared with those of normal cells, and the expression of human genes was confirmed by Western blot using antibodies that recognize HA-tags in the N-terminal region of the expressed protein. It was. Overexpression of human genes and abnormality in the cell cycle are analyzed using FACScan ^™ (Becton, Dickinson and company), which analyzes DNA content after staining chromosomal DNA (PI) by treating PI (propidium iodide) in cells. (See FIG. 6 ).

Next, by selecting the genes in which the trait change occurs in the above experiment, a cleavage yeast deletion mutant in which the genes showing homology with these selected genes in S. pombe was deleted.

In general, a method for preparing a pool of deletion mutants is known as an effective research method using S.pombe as a model organism, and it is a high-utility method in various aspects such as functional analysis of human genes, drug screening, and oligo microchips. to be.

As an example of a variant preparation method, the targeted mutagenesis method must have prior information about the gene sequence to be mutated, be a living organism capable of homologous recombination, and in the preparation of the deletion mutant. Although it takes a long time and has a disadvantage in that the manufacturing cost is higher than that of random mutagenesis method, it is possible to prepare a deletion mutant for all genes at the genome level, and based on the sequence information of previously obtained genes Accurate deletion mutants can be produced and have the advantage of being capable of quantitative analysis in large quantities (Daniel D. et al. , Nat. Genet. , 14: 450-456, 1996).

The present inventors have identified heterozygote mutants in which genes having homology with human genes have been deleted, and then doxorubicin, Kule C. et al. , Mol. Pharmacol. 46: 1234-1240, 1994), rapamycin (Ai, W. et al. , Mol. Cell. , 10: 1295-1305, 2002), camptothecin, Thomas CJ et al. , Bioorg.Med Chem., April 1; 12 (7): 1585-1604, 2004), hydrazinocurcumin (Hizazinocurcumin, Shim et al. , Bioorg.Med . Chem. , 10: 2439-2444, 2002) and the like. Eight anticancer drugs were treated to screen for sensitivity to each drug. Then, the sensitivity of the treated drug was confirmed by artificial lethal (synthetic lethal). Each drug was treated based on IC ₅₀ values. Sensitivity to Anti-cancer Drugs in S.pombe Heterozygous Mutants Mutants are selected by lethality to overexpress human genes homologous to the genes deleted from the deletion mutants. The functional complementation of the human gene to the S.pombe gene was confirmed by confirming disappearance (see FIG. 7 ). That is, it was confirmed that the sensitivity was restored by introducing and expressing a human gene having homology with the deleted gene to the S.pombe deletion mutant indicating the sensitivity to the drug, and introduced with the deleted S.pombe gene. It can be seen that the function of human genes is complementary to each other.

In order to find out whether the second screened cancer-related candidate human gene is actually associated with cancer through the overexpression experiment using the cleavage yeast system and the anticancer drug effect verification, the present inventors and the like have not yet confirmed the function of the hnRNP A1 gene. Was selected. Northern blot and RT-PCR were analyzed in human gastric cancer, liver cancer cell lines and normal cells to confirm that hnRNP A1 specifically increased expression in gastric cancer and liver cancer cell lines (see FIG. 8 ). When the hnRNP A1 gene was overexpressed using the CMV promoter in NIH3T3, a mammalian cell line, the morphological changes were observed (see FIG. 9 ).

Therefore, it was confirmed that the functional analysis method of the target gene using S.pombe of the present invention can be actually useful for the functional analysis of the unknown gene.

The present invention also provides a vector pDES173 (Accession No .: KCTC 10757BP) used in the above method.

In the present invention, a functional analysis system was constructed by using a cleavage yeast S.pombe genes showing differences in expression levels in the tissues of gastric cancer and liver cancer compared to normal tissues through a microarray technique.

To this end, the Gateway ^TM system (Invitrogen) was used to easily express hundreds of human genes in yeast in large quantities. Specifically, S.pombe induction The SmaI site of the expression vector pSLF173 base vector (see Figure 1 ) was cleaved and cloned by concatenation of a conversion cassette and the completed plasmid was named pDES173 (see Figure 2 ). The plasmid was transformed into Escherichia coli and deposited with the Biotechnology Research Institute Gene Bank on January 6, 2005 (Accession No .: KCTC 10757BP).

Production of S.pombe expression clones using the conversion cassette, when overexpressing several types of human genes in S.pombe , re-acquired each gene cDNA construct and cloned individually to the S.pombe expression vector system. This has the advantage of reducing the hassle of doing so can be useful.

In addition, the present invention provides a gene screened by the above method.

To determine whether cancer-related candidate human genes screened secondarily using the cleavage yeast system were actually associated with cancer, the expression level of the hnRNP A1 gene, whose function has not yet been clearly identified, was determined in human gastric cancer, liver cancer cell lines and normal cells. As a result of verifying the association with cancer by analyzing Northern blot and RT-PCR, hnRNP A1, one of the genes screened through the S.pombe system of the present invention, is specifically expressed in gastric cancer and liver cancer cell lines. (See FIG. 8 ), the morphological changes were observed as expected when overexpressed using the CMV promoter in the non-transformed cell line NIH3T3 (see FIG. 9 ).

Genes screened using the S.pombe system of the present invention can be used for cancer specific molecular markers or screening methods for new drug design.

Hereinafter, the present invention will be described in detail by examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 Gateway ^TM  Cloning of Human Genes Using the System

S. pombe target vector pDES173 was prepared by introducing a conversion cassette into pSLF173 (Invitrogen), an induction expression vector of S. pombe . Specifically, pSLF173 was reacted with a restriction enzyme SmaI at 25 ° C. for 1 to 2 hours, followed by electrophoresis to determine a suitable size in an agarose gel. 10 ng of the conversion cassette DNA and 20-50 ng of the SmaI- treated pSLF173 DNA were ligated with a T4 ligase at 16 ° C. for 16 hours. E. coli by electrophoration Introduced into DB3.1 strain. After transfection into LB medium (LB + amp) to which ampicillin was added (LB + amp), cultured at 37 ° C. for 16 hours to obtain transformant colonies, which were cultured in 2-3 ml of LB + amp liquid medium at 37 ° C. for 16 hours. Then, plasmid DNA was extracted using plasmid extraction kit (Bioneer, Korea). Conversion was confirmed whether the cloning and cloning location using a site in the BamHⅠ SalⅠ and pSLF173 in the cassette, the cloned plasmid was confirmed the final decision by checking the base sequence in S. pombe object vector (destination vector) (pDES173) . The plasmid was transformed into Escherichia coli and deposited with the Biotechnology Research Institute Gene Bank on January 6, 2005 (Accession No .: KCTC 10757BP). The identified vector was co-integrated with an entry clone containing cancer-related human genes using the Gateway ^TM LR Clonase Enzyme Mix (Invitrogen), and the resulting vector was cloned in S. pombe . Constructs that can be expressed were prepared. Clones prepared were identified after specific restriction enzymes and finally selected ( FIGS. 2 and 3 ).

Example 2 Confirmation of Overexpression Effect of Human Gene

Human genes constructed in the S. pombe expression vector (pDES173) prepared in Example 1 were transformed into a lithium acetate method of ED665 (h-) (ATCC, USA), a haploid strain of S. pombe . As a selection medium for identification of transformants, a medium in which adenine and leucine were added to the EMM medium (hereinafter referred to as EMM + AL) was used in consideration of the marker characteristics of the plasmid pSLF173 and the nutritional requirements of the strain.

In order to express cancer-related human genes in ED665 (h-), EMM + AL + thia medium with 2 μM of thiamine and EMM + AL medium without TAM were used, respectively. As a control, pDES173 without human genes was expressed together. After incubation for 3 days at 30 ℃, by comparing the colonies grown in EMM + AL solid medium and EMM + AL + thia solid medium, it was confirmed that the abnormality in cell growth or completely inhibited cell growth.

In addition, in order to confirm cell trait changes, colonies grown in EMM + AL + thia solid medium were incubated in 10 ml of EMM + AL + thia liquid medium at 30 ° C. for about 16 hours. In order to compare the effects on the cells with and without induction, the cultured cells were washed three times with sterile distilled water, completely removed with thiamin, and then EMM + AL liquid medium and EMM + AL + thia (2uM) liquid medium, respectively. 10 ml again was inoculated so that the OD ₆₀₀ value is 0.3. After incubation at 30 ° C. for 6 to 8 hours, the OD ₆₀₀ was diluted to 0.1 in the same medium, and then incubated for 16 hours.

The cells obtained above were observed under an optical microscope to confirm cell trait changes that occur when human genes were overexpressed in S. pombe , and whether or not human genes were properly expressed was confirmed by western blot. A monoclonal antibody against the HA motif tagged at the N-terminal part of the Western blosi gene was used.

As a result, it was confirmed that not only the trait change but also the cell growth that appeared when each human gene was overexpressed in S. pombe . Of the 500 genes, 145 genes showed changes in cell traits, and 76 genes affected cell growth (see FIGS. 4 , 5 and 6 ). Human genes showing cellular trait changes and human genes that inhibited cell growth may be involved in the regulation of cell division cycle or may play an important role in cell growth and survival.

Example 3 Homologous to Human Genes S. pombe Investigation of sensitivity of anticancer drugs using variants lacking genes and inhibition of artificial lethality through overexpression of human genes

Variant strains were incubated for 16 hours at 30 ° C. in 3 ml of EMM + UL liquid medium containing uracil and leucin, and then incubated for 8-9 hours in 3 ml of the same liquid medium with an initial OD ₆₀₀ of 0.3. The final OD ₆₀₀ was adjusted to 1.0 and diluted six times at a ratio of 1: 4 to spot the EMM + UL medium containing the anticancer agent and the medium containing the anticancer agent, respectively. After spotting, the cells were incubated at 30 ° C. for 3 to 4 days and screened for variants showing sensitivity or resistance to anticancer drugs. In the case of anticancer drugs, whether the growth inhibitory variants can restore sensitivity or lethality by introducing human genes homologous to the genes deleted from each variant, ie whether functional complementation is possible. It was confirmed.

As a result of analyzing the S. pombe variant having homology with human genes for this experiment, it was confirmed that 131 genes (about 28%) out of a total of 500 human genes were homologous. By introducing a human gene having homology with the deleted gene to a deletion mutant showing drug sensitivity, it was confirmed that the drug sensitivity of each variant was reduced ( see FIG. 7 ). Thus, drug sensitivity using the S. pombe deletion mutant showed that the human gene and the S. pombe gene have mutual complementarity not only in nucleotide sequence but also in function.

<Example 4> S. pombe  Validation of Cancer Relevance of Selected Human Genes Through Secondary Screening in the System

In order to verify the cancer relevance of human genes selected through secondary screening in S. pombe system, the expression levels of these genes in various gastric and liver cancer cell lines were analyzed using Northern blot and RT-PCR. For example, RT-PCR and Northern blot analysis using hnRNP A1 gene among 171 selected genes showed that hnRNP A1 significantly increased its RNA level in most human gastric and liver cancer cell lines compared to normal cell lines. It could be confirmed ( FIG. 8 ).

In addition, when the hnRNP A1 was overexpressed in a non-transformed normal cell line NIH3T3 (mouse embryonicfibroblast cell line), it was observed that the cell proliferation increased and changed into transformed traits that changed in shape as they aggregated between cells ( FIG. 9 ) . See ). Therefore, 171 human genes screened and screened in S. pombe system, including hnRNP A1, have significantly increased expression levels in human gastric cancer and liver cancer. Therefore, it can function as a specific molecular marker of Korean human gastric cancer and liver cancer.

As described above, in the present invention, by constructing a system that can easily transfer a large amount of human genes by modifying the S. pombe basic expression vector, it is easy to analyze the effects of the overexpression of human genes to facilitate gene function analysis We built a way to do it. This system is useful as a good model screening system for screening genes related to generous cancers obtained by simple microarray analysis of gastric and liver cancer tissues, and can be applied to the study of the function of human genes whose function and biological pathway are not well known. have.

Claims (11)

Iii) preparing a target vector capable of inducing overexpression of an unidentified target gene in a split yeast S. pombe ; Ii) preparing an expression vector into which the target gene is inserted using the target vector of step iii); Iii ) overexpressing the gene of interest by introducing the expression vector of step ii) into normal cleavage yeast ( S. pombe ); And Iii) observing cell trait changes due to cell growth or cell cycle changes occurring in the yeast of step iii). According to claim 1, After the step iii), to confirm the functional complementation (functional complementation) between cancer-related genes and yeast genes, Iii) preparing a S. pombe heterozygous deletion mutant in which the S. pombe gene having sequence homology with the cancer-related gene selected in step iii) is deleted; Iii) measuring the sensitivity to the drug by treating the fission yeast ( S. pombe ) deletion mutant with an anticancer agent; And Iii) introducing the cancer-related gene having sequence homology with the deleted gene into a S. pombe deletion mutant which shows the sensitivity to the drug of step iii) to confirm whether the sensitivity is recovered. Screening method comprising the additional. The cancer of the selected cancer-related genes according to claim 1, wherein the degree of expression of the selected gene after step iii) is compared between the cancer cell line and the normal cell line or the expression of the selected gene is overexpressed in the normal cell line to observe the phenotypic change. Verifying the relevance. The method of claim 1, wherein the target vector of step i) is pDES173 (Accession Number: KCTC 10757BP). delete The cancer of the selected cancer-related genes according to claim 2, wherein the degree of expression of the selected gene after step iii) is compared between the cancer cell line and the normal cell line or the expression of the selected gene is overexpressed in the normal cell line to observe phenotypic changes. Verifying the relevance. 7. The method of claim 3 or 6, wherein the cancer cell line is a gastric or liver cancer cell line. delete Purpose vector pDES173 (Accession Number: KCTC 10757BP) used in the method of claim 1 delete delete

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