CN114177274A

CN114177274A - Application of TDRD10 protein and coding gene thereof in preparation of tumor treatment medicines and tumor treatment medicines

Info

Publication number: CN114177274A
Application number: CN202111522207.1A
Authority: CN
Inventors: 鹿文葆
Original assignee: Institute of Microcirculation of CAMS
Current assignee: Institute of Microcirculation of CAMS
Priority date: 2021-12-13
Filing date: 2021-12-13
Publication date: 2022-03-15

Abstract

The invention relates to a new application of TDRD10 protein and TDRD10 gene in tumor treatment. Specifically, the invention relates to a novel effect of TDRD10 protein and TDRD10 gene in preparation of tumor treatment drugs, a novel application of TDRD10 protein and TDRD10 gene in research and development of anti-tumor drugs as molecular targets, and tumor treatment drugs. The TDRD10 gene is a tumor cell cycle inhibiting gene, and the over-expression of TDRD10 gene in tumor cells can obviously inhibit the proliferation and metastasis of tumor cells.

Description

Application of TDRD10 protein and coding gene thereof in preparation of tumor treatment medicines and tumor treatment medicines

Technical Field

The invention relates to the fields of oncology, genetics and molecular biology, in particular to a novel application of TDRD10 protein and TDRD10 gene, more particularly to a novel application of TDRD10 protein and TDRD10 gene in preparing tumor treatment medicines and a novel application of TDRD10 protein and TDRD10 gene in research and development of anti-tumor medicines as target medicines.

Background

One of the common features of cancer is uncontrolled cell cycle progression (Williams GH and Stoeber k.2012) and leads to unlimited proliferation of cancer cells (Xiong Y, et al.2018), which also accounts for the resistance of tumor cells to traditional therapeutic approaches. Although numerous studies have expanded our understanding of the regulation of cancer cell cycle progression, the mechanisms of post-transcriptional regulation of cell cycle-associated genes in cancer cells, particularly through molecules such as RNA-binding proteins, remain unclear.

Abnormal expression of cell cycle-related genes is one of the root causes of uncontrolled progression of the cancer cell cycle (Fischer M et al 2017). Cell cycle-related genes can be classified into cell cycle-promoting genes (including CDKs, E2F1, CCNDs, CCNES, etc.) and cell cycle-inhibiting genes (including p21, p27, TP53, RB1, etc.) according to gene functions. The balance between them determines the cycle progression of cancer cells (Malumbres M et al, 2001). Most tumors have uncontrolled cell cycle either due to increased expression of cell cycle promoting genes or due to restricted expression of cell cycle inhibiting genes (Malumbres M et al, 2009). Also, the expression levels of many cell cycle-related genes were significantly correlated with the prognosis of tumor patients (Orega SM et al, 2002; Yu D et al, 2006; Leonard JP et al, 2012; Shepard KE et al, 2013; Sawai CM et al, 2012). Thus, current clinical tumor therapy is primarily to suppress cell cycle promoting gene expression to restore sensitivity of cancer cells to cell cycle regulatory therapies.

TDRD10, an RNA binding protein encoded by TDRD10 gene. To date, the role of TDRD10 molecules in human cancer progression is unclear.

Disclosure of Invention

The invention aims to provide a novel application of TDRD10 protein and TDRD10 gene, and more particularly relates to a novel application of TDRD10 protein and TDRD10 gene in preparing a tumor treatment drug and a novel application of TDRD10 gene in research and development of an anti-tumor drug as a target.

The invention firstly protects the application of TDRD10 protein in preparing tumor treatment medicines.

The invention also protects the application of the TDRD10 gene or the biological material with the TDRD10 gene in preparing tumor treatment medicines.

The invention also protects the application of the substance capable of increasing the abundance of TDRD10 protein in organisms in preparing medicaments for treating tumors. The substance capable of up-regulating the abundance of TDRD10 protein in an organism can be TDRD10 protein per se, other proteins which are positioned at the upstream of TDRD10 protein in the organism and can promote the production of TDRD10 protein, other proteins which are positioned at the downstream of TDRD10 protein in the organism and can reduce the degradation of TDRD10 protein, compounds or other small molecules which can promote the increase of TDRD10 protein level in the organism.

The invention also protects the application of the substance capable of up-regulating the abundance of TDRD10 gene in organisms in preparing tumor treatment medicines. The substance capable of up-regulating the abundance of the TDRD10 gene in the organism can be the TDRD10 gene per se, other protein or nucleic acid molecules which are positioned at the upstream of the TDRD10 gene in the organism and can promote the TDRD10 gene to express, other protein or nucleic acid molecules which are positioned at the downstream of the TDRD10 gene in the organism and can reduce the TDRD10 gene to degrade, and compounds or other small molecules which can promote the TDRD10 gene to express in the organism.

The invention also protects the application of the TDRD10 protein or TDRD10 gene as a target in the research and development of anti-tumor drugs. The TDRD10 protein serving as a target can be up-regulated by taking TDRD10 protein as a target. The substance capable of up-regulating TDRD10 protein in an organism can be TDRD10 protein per se, other proteins which are positioned in the organism and upstream of TDRD10 protein and can promote TDRD10 protein production, other proteins which are positioned in the organism and downstream of TDRD10 protein and can reduce TDRD10 protein degradation, compounds or other small molecules which can promote TDRD10 protein level increase in the organism. The TDRD10 gene as a target can be specifically TDRD10 gene as a target to be up-regulated. The substance capable of up-regulating TDRD10 gene in organism may be TDRD10 gene itself, or other protein or nucleic acid molecule capable of promoting TDRD10 gene expression at upstream of TDRD10 gene in organism, or other protein or nucleic acid molecule capable of reducing TDRD10 gene degradation at downstream of TDRD10 gene in organism, or compound or other small molecule capable of promoting TDRD10 gene expression in organism.

The invention also protects a TDRD10 protein truncated body, namely an RRM functional region of TDRD10 protein.

The functional domain of RRM of TDRD10 protein is amino acid residues 34-107 of TDRD10 protein.

The gene for coding the TDRD10 protein truncated body also belongs to the protection scope of the invention.

The invention also protects the application of the TDRD10 protein truncation in preparing tumor treatment medicines.

The invention also protects the application of the gene coding the TDRD10 protein truncation or the biological material with the gene coding the TDRD10 protein truncation in preparing tumor treatment medicines.

The invention also protects the application of the TDRD10 protein truncated body or the gene for coding the TDRD10 protein truncated body as a target in the research and development of anti-tumor drugs. The TDRD10 protein truncated body is used as a target object, and particularly, the TDRD10 protein truncated body is used as a target object to be up-regulated. The substance capable of up-regulating the TDRD10 protein truncation in the organism can be the TDRD10 protein truncation per se, and other proteins, peptide fragments, compounds or other small molecules capable of promoting the increase of the TDRD10 protein truncation level in the organism. The gene encoding the TDRD10 protein truncation is used as a target, and particularly, the gene encoding the TDRD10 protein truncation is up-regulated as the target. The substance capable of up-regulating the gene encoding the truncated TDRD10 protein in the organism may be the gene encoding the truncated TDRD10 protein itself, or other proteins, polypeptides, nucleic acid molecules, compounds or other small molecules that promote the expression of the gene encoding the truncated TDRD10 protein.

The invention also provides a tumor treatment drug which realizes the drug function by up-regulating the abundance of at least one of TDRD10 protein, TDRD10 gene, TDRD10 protein truncation and gene encoding TDRD10 protein truncation in an organism.

The drug functions as (a1) and/or (a2) and/or (a3) and/or (a4) as follows: (a1) treating tumors; (a2) inhibiting tumor growth and/or metastasis; (a3) inhibiting tumor cell proliferation; (a4) preventing the cycle progression of tumor cells

The function of any one of the medicines is as follows (a1) and/or (a2) and/or (a3) and/or (a 4): (a1) treating tumors; (a2) inhibiting tumor growth and/or metastasis; (a3) inhibiting tumor cell proliferation; (a4) prevent the cycle progression of tumor cells (G1/S phase progression).

Illustratively, any of the TDRD10 proteins described above may be a human TDRD10 protein.

The TDRD10 protein may be (b1), (b2), (b3) or (b4) as follows:

(b1) protein shown as a sequence 1 in a sequence table;

(b2) a fusion protein obtained by attaching a tag to the amino terminus or the carboxy terminus of (b 1);

(b3) a protein obtained by substituting and/or deleting and/or adding one or more amino acid residues in (b1) and having any one of functions (a1) to (a 4);

(b4) a protein derived from a human being, having 98% or more identity to (b1), and having any one of the functions (a1) to (a 4).

The labels are specifically shown in table 1.

TABLE 1 sequences of tags

The TDRD10 protein can also be homologous protein in other species with any function of (a1) to (a 4). Such other species include, but are not limited to, mice, rats, rabbits, dogs, monkeys, orangutans, apes, cows, sheep, pigs, horses, sheep, goats, cats, and the like.

The TDRD10 gene is the gene coding TDRD10 protein or TDRD10 protein.

The TDRD10 gene may be (c1), (c2) or (c 3):

(c1) the coding region is a DNA molecule shown as a sequence 2 in a sequence table;

(c2) a DNA molecule derived from human and having 95% or more identity to (c1) and encoding said protein;

(c3) a DNA molecule which hybridizes under stringent conditions to the nucleotide sequence defined in (c1) and encodes said protein.

The TDRD10 gene may also be a homologous gene in other species. Such other species include, but are not limited to, mice, rats, rabbits, dogs, monkeys, orangutans, apes, cows, sheep, pigs, horses, sheep, goats, cats, and the like.

The TDRD10 protein truncation may be specifically (d1), (d2), (d3) or (d4) as follows:

(d1) protein shown by amino acid residues at positions 34-107 in a sequence 1 in a sequence table;

(d2) a fusion protein obtained by attaching a tag to the amino terminus or the carboxy terminus of (d 1);

(d3) a protein obtained by substituting and/or deleting and/or adding one or more amino acid residues in (d1) and having any one of the functions of (a1) to (a 4);

(d4) a protein derived from a human being, having 98% or more identity to (d1), and having any one of the functions (a1) to (a 4).

The labels are specifically shown in table 1.

The TDRD10 protein truncation can also be homologous protein in other species with any function of (a1) to (a 4). Such other species include, but are not limited to, mice, rats, rabbits, dogs, monkeys, orangutans, apes, cows, sheep, pigs, horses, sheep, goats, cats, and the like.

The gene encoding the TDRD10 protein truncation may be (e1), (e2) or (e 3):

(e1) the DNA molecule of the TDRD10 protein truncated body coded in the DNA molecule shown in the sequence 2 in the sequence table;

(e2) a DNA molecule derived from a human and having 95% or greater identity to (e1) and encoding the TDRD10 protein truncation;

(e3) and (c) a DNA molecule which hybridizes with the nucleotide sequence defined in (e1) under strict conditions and encodes the TDRD10 protein truncation.

The gene encoding the TDRD10 protein truncation may also be a homologous gene in other species. Such other species include, but are not limited to, mice, rats, rabbits, dogs, monkeys, orangutans, apes, cows, sheep, pigs, horses, sheep, goats, cats, and the like.

Any of the above described biomaterials having TDRD10 gene may be an expression vector having TDRD10 gene. Any of the above-described biological materials having a gene encoding a TDRD10 protein truncation may be an expression vector having a gene encoding a TDRD10 protein truncation. The expression vector is capable of carrying a nucleotide sequence and of integrating this sequence into the genome of the cell and of replicating in the cell. "expression vectors" include plasmids, cosmids, viruses (bacteriophages, animal viruses, plant viruses, etc.), and artificial chromosomes (e.g., YACs). Viruses (also called viral vectors) as expression vectors that may be currently suitable for clinical gene therapy are as follows: adenoviral vectors, retroviral vectors, adeno-associated viral vectors, lentiviral vectors, herpesvirus vectors, chimeric viral vectors and other viral vectors.

Any of the above-described tumors include, but are not limited to, breast cancer.

Any of the above tumor cells include, but are not limited to, breast cancer cells.

The inventors of the present invention found that the TDRD10 protein has a significant tumor-inhibiting function and can induce tumor cell cycle arrest, and the mechanism is to specifically inhibit the expression of cell cycle promoting gene transcripts (including CDK2, Cyclin D1, CDKN3, WEE1, etc.). The present inventors found that TDRD10 gene expression is suppressed in breast cancer tissues and cells, which may help tumor cells escape cell cycle regulation. Overexpression of the TDRD10 gene can block the cycle G1/S progression of breast cancer cells, however, further inhibition of TDRD10 gene expression by using shRNA can enhance tumor cell proliferation and cycle progression. Consistent with these in vitro observations, overexpression of TDRD10 gene in vivo significantly inhibited tumor growth and metastasis. By analyzing the human tumor tissue sample database, the inventors found that low expression levels of TDRD10 were strongly associated with poor breast cancer patient survival. Furthermore, the expression level of TDRD10 gene in the tissues of cancer patients is significantly inversely correlated with the expression level of its target genes CDK2, CCND1, CDKN3, WEE1 and MCM 2. These results indicate that TDRD10 is a potential tumor suppressor involved in regulating cell cycle signaling pathways to promote gene expression by inhibiting the cell cycle. Based on this, the tumor suppressor protein TDRD10 has very important application prospect in improving the clinical tumor treatment effect.

The invention identifies that the human TDRD10 gene is a tumor cell cycle inhibiting gene for the first time, and the over-expression TDRD10 gene in tumor cells can obviously inhibit the proliferation and the metastasis of the tumor cells.

Drawings

FIG. 1 shows that TDRD10 inhibits tumor cell proliferation and prevents tumor cell cycle progression. Both MDA-MB-468/TDRD10-GFP cells and MCF7/TDRD10-GFP cells efficiently expressed TDRD10-GFP fusion protein (FIGS. 1A and 1B); overexpression of TDRD10 gene significantly inhibited tumor cell proliferation (fig. 1C, and fig. 1D); overexpression of the TDRD10 gene prevented progression of the cycle G1/S phase of tumor cells (FIG. 1E).

FIG. 2 shows that TDRD10 targets 3' UTRs to specifically inhibit mRNAs expression of cell cycle promoting genes. After overexpression of TDRD10 gene, cell cycle promoting gene mRNAs and protein expression are down-regulated (FIG. 2A, FIG. 2B, FIG. 2C, FIG. 2D, FIG. 2E and FIG. 2F); TDRD10 protein could target mRNAs that bind to cell cycle promoting genes, but not cell cycle suppressing genes (fig. 2G and fig. 2H); TDRD10 protein could target the 3 'UTRs of cell cycle promoting genes to degrade their mRNAs, but not the 3' UTRs of cell cycle suppressing genes (fig. 2I).

FIG. 3 shows that the functional RRM domain of TDRD10 is responsible for degrading mRNAs of cell cycle promoting genes. The half-life test of mRNAs of the cell cycle promoting genes shows that the over-expression of TDRD10 gene remarkably reduces the half-life (figure 3A); a schematic diagram of the functional domains of TDRD10 along with the strategy for making the different mutants and the crystal structure of the functional domain of RRM are shown (fig. 3E); cell cycle promoting gene mRNAs were down-regulated after overexpression of the T1 truncation gene (shown as aa 1-108 in the figure) (FIG. 3C); the T1 truncation can target the 3' UTR associated with cell cycle promoting genes to degrade their mRNAs (fig. 3D); overexpression of the T1 truncation gene prevented progression of the G1/S phase cycle of tumor cells (FIG. 3E).

FIG. 4 shows that the TDRD10 gene is overexpressed to inhibit tumor growth and metastasis in vivo. Overexpression of the TDRD10 gene significantly inhibited tumor growth in nude mice (fig. 4A). The recombinant adenovirus expressing TDRD10 gene can inhibit the growth and metastasis of tumor in nude mouse with tumor (FIG. 4B, FIG. 4C, and FIG. 4D). Clinical breast cancer database analysis showed that TDRD10 expression in breast cancer tissues was significantly negatively correlated with cell cycle promoting gene expression (fig. 4E).

Detailed Description

The principles and features of the present invention are described below in conjunction with the following drawings. The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. Unless otherwise stated, the quantitative tests in the following examples were repeated 3 times or more, and the results were averaged.

pEGFP-N1 vector: clontech, Inc.; the pEGFP-N1 vector expresses the EGFP protein, which is also known as GFP protein. MDA-MB-468 cells (human breast cancer cells):

HTB-132^TM. MCF71 cells (human breast cancer cells):

HTB-22^TM. MDA-MB-231 cells (human breast cancer cells):

HTB-26^TM。

example 1 identification of novel function of TDRD10 to prevent progression of tumor cell cycle

1. The TDRD10 gene (the TDRD10 gene is shown as a sequence 2 in a sequence table, and a stop codon is removed from an insertion sequence) is inserted between Hind III and BamH I enzyme cutting sites of a pEGFP-N1 vector to obtain a recombinant plasmid pEGFP-N1-TDRD 10-GFP. The recombinant plasmid pEGFP-N1-TDRD10-GFP has been subjected to sequencing verification. In the recombinant plasmid pEGFP-N1-TDRD10-GFP, the inserted DNA molecules and EGFP genes in the vector form fusion genes to express TDRD10-EGFP fusion proteins (T10-GFP fusion proteins for short).

2. The recombinant plasmid pEGFP-N1-TDRD10-GFP is introduced into MDA-MB-468 cells to obtain recombinant cells, and the recombinant cells are named MDA-MB-468/T10-GFP cells.

3. The pEGFP-N1 vector was introduced into MDA-MB-468 cells to obtain recombinant cells, which were designated MDA-MB-468/GFP cells.

4. The recombinant plasmid pEGFP-N1-TDRD10-GFP is introduced into MCF7 cells to obtain recombinant cells, and the recombinant cells are named MCF7/T10-GFP cells.

5. The pEGFP-N1 vector was introduced into MCF7 cells to obtain recombinant cells, which were designated MCF7/GFP cells.

6. Taking MDA-MB-468/T10-GFP cells and MDA-MB-468/GFP cells, respectively carrying out the following steps: the cultures were incubated in parallel, after 36 hours the cells were lysed and the lysates were collected for immunoblotting. The results of immunoblotting using the TDRD10 antibody are shown in FIG. 1A and FIG. 1B. Endo-TDRD10 represents the TDRD10 protein endogenous to the cell. In the upper lane of FIG. 1A, Con represents MDA-MB-468/GFP cells, and T10/GFP represents MDA-MB-468/TDRD10-GFP cells.

7. MDA-MB-468 cells (labeled MDA468/Parental in the figure), MDA-MB-468/GFP cells (labeled Empty control in the figure) and MDA-MB-468/TDRD10-GFP cells (labeled TDRD10/GFP in the figure) were taken, and the following steps were performed, respectively: the cells were counted at different culture times (0h, 24h, 48h, 72h) in parallel to examine the effect of over-expression of TDRD10 gene on cell proliferation. The results are shown on FIG. 1C.

8. Taking MCF7 cells (labeled MCF7/Parental in the figure), MVF7/GFP cells (labeled Empty control in the figure) and MCF7/TDRD10-GFP cells (labeled TDRD10/GFP in the figure), respectively, the following steps are carried out: the cells were counted at different culture times (0h, 24h, 48h, 72h) in parallel to examine the effect of over-expression of TDRD10 gene on cell proliferation. The results are shown below in FIG. 1C.

9. MDA-MB-468 cells (labeled MDA468/Parental in the figure), MDA-MB-468/GFP cells (labeled Empty control in the figure) and MDA-MB-468/TDRD10-GFP cells (labeled TDRD10/GFP in the figure) were taken, and the following steps were performed, respectively: the MTT staining was performed at different culture times (0h, 24h, 48h, 72h) in parallel to examine the effect of over-expression of TDRD10 gene on cell proliferation. The results are shown in FIG. 1D.

10. Taking MCF7 cells (marked as MCF7/Parental in the figure), MCF7/GFP cells (marked as Empty control in the figure) and MCF7/TDRD10-GFP cells (marked as TDRD10/GFP in the figure), respectively, the following steps are carried out: the MTT staining was performed at different culture times (0h, 24h, 48h, 72h) in parallel to examine the effect of over-expression of TDRD10 gene on cell proliferation. The results are shown below in FIG. 1D.

11. MCF7/TDRD10-GFP cells (labeled TDRD10-GFP in the figure) and MCF7/GFP cells (labeled Empty control in the figure) were taken, and the following steps were performed, respectively: the cultures were run in parallel and after 36 hours, cells were harvested for flow cytometry analysis to obtain cell ratios at each cell cycle. The results are shown in FIG. 1E.

12. MDA-MB-468/TDRD10-GFP cells (labeled TDRD10-GFP in the figure) and MDA-MB-468/GFP cells (labeled Empty control in the figure) were taken, and the following steps were performed, respectively: the cultures were run in parallel and after 36 hours, cells were harvested for flow cytometry analysis to obtain cell ratios at each cell cycle. The results are shown below in FIG. 1E.

The results of fig. 1 show that: both MDA-MB-468/TDRD10-GFP cells and MCF7/TDRD10-GFP cells efficiently expressed TDRD10-GFP fusion protein (FIGS. 1A and 1B); the over-expression of TDRD10 gene significantly inhibits the proliferation of tumor cells (FIG. 1C, FIG. 1D); overexpression of the TDRD10 gene prevented progression of the cycle G1/S phase of tumor cells (FIG. 1E).

The above results show that: the TDRD10 gene is over-expressed so as to increase the TDRD10 protein level, and the TDRD10 gene is over-expressed so as to inhibit the proliferation of tumor cells and prevent the cycle progression of the tumor cells, namely, the TDRD10 gene is over-expressed so as to increase the TDRD10 protein level, so that the TDRD10 protein level has the effect of treating tumors. The above functions of TDRD10 protein/TDRD 10 gene were first discovered by the present inventors.

Example 2 TDRD10 specific degradation of cell cycle promoting genes mRNAs

TDRD10 specific degradation cell cycle promoting gene mRNAs

In order to further examine the mechanism of TDRD10 protein inhibiting tumor cell proliferation and preventing cell cycle progression, tumor cell cycle-related genes were examined.

1. The MDA-MB-468/TDRD10-GFP cells of example 1 were cultured for 36 hours, total RNA was extracted and reverse-transcribed into cDNA, and then RNA-seq was performed, and the results are shown in FIG. 2A.

2. The MCF7/TDRD10-GFP cells of example 1 were cultured for 36 hours, total RNA was extracted and reverse-transcribed into cDNA, and then RNA-seq was performed, and the results are shown in FIG. 2B.

3. The MDA-MB-468/TDRD10-GFP cells of example 1 were cultured for 36 hours, total RNA was extracted and reverse-transcribed into cDNA, and then qPCR was performed to detect the expression level of the target gene. The results are shown in FIG. 2C.

3. MCF7/TDRD10-GFP cells in example 1 were taken, cultured for 36 hours, total RNA was extracted and reverse-transcribed into cDNA, and then qPCR was performed to detect the expression level of the target gene. The results are shown in FIG. 2D. .

4. MCF7/TDRD10-GFP cells are taken and subjected to the following steps: the cultures were performed in parallel, and western blots were performed for different culture times (24h, 48h, 72h) to detect the abundance of each target protein, and the results are shown in the lanes corresponding to each time point in fig. 2E. MCF7/GFP cells were taken and subjected to the following steps: the cells were cultured for 72 hours under parallel conditions, western blots were performed, and the abundance of each target protein was detected, and the results are shown in lanes corresponding to Con in FIG. 2E.

5. MDA-MB-468/TDRD10-GFP cells were taken and subjected to the following steps: the cultures were performed in parallel, and western blots were performed for different culture times (24h, 48h, 72h) to detect the abundance of each target protein, and the results are shown in the lanes corresponding to each time point in fig. 2F. Taking MDA-MB-468/GFP cells, and carrying out the following steps: the cells were cultured for 72 hours under parallel conditions, western blots were performed, and the abundance of each target protein was detected, and the results are shown in lanes corresponding to Con in FIG. 2F.

6. MDA-MB-468/TDRD10-GFP cells were taken and subjected to the following steps: after culturing for 36 hours, carrying out cell lysis, and then collecting lysate; incubating GFP antibody (or isotype IgG) with Protein A/G Beads (Santa Cruz Co.) for 2h, adding the lysate and continuing incubation for 2h, centrifuging at 4 ℃ and 2000rpm, washing the Beads thoroughly, and collecting the total RNA by Trizol; taking the total RNA, and carrying out RT-PCR to detect the target gene. The results are shown in FIGS. 2G and 2H. In FIG. 2G, GAPDH served as a negative control.

7. Luciferase reporter assay

The test plasmid and reporter vector were co-transfected in HEK293 cells, cells were lysed 36 hours after transfection, lysates were collected, and luciferase activity was detected using the Dual-luciferase reporter Assay System (Promega), and the results are shown in FIG. 2I. The test plasmids were: the recombinant plasmid pEGFP-N1-TDRD10-GFP (labeled TDRD10 in the figure) or pEGFP-N1 vector (labeled Control in the figure). The reporter vectors were respectively: a reporter vector with the 3 ' UTR of the β -Actin gene, a reporter vector with the 3 ' UTR of the CDK2 gene, a reporter vector with the 3 ' UTR of the CCND1 gene, a reporter vector with the 3 ' UTR of the CDKN3 gene or a reporter vector with the 3 ' UTR of the WEE1 gene.

8. MDA-MB-468/TDRD10-GFP cells (labeled TDRD10-GFP in the figure) and MDA-MB-468/GFP cells (labeled Empty control in the figure) were taken, and the following steps were performed, respectively, to detect the half-lives of the cell cycle promoting genes mRNAs: treating cells with ActD and DRB (working concentration of ActD is 5 mug/mL, working concentration of DRB is 5 mug/mL), taking cells after 0min, 60min, 120min and 240min respectively, extracting total RNA, and then performing RT-PCR to detect target genes. The results are shown in FIG. 3A.

The result of the first step shows that: upon overexpression of TDRD10 gene, cell cycle promoting genes mRNAs were down-regulated (fig. 2A, 2B, 2C, 2D, 2E and 2F); TDRD10 protein could target mRNAs that bind to cell cycle promoting genes, but not cell cycle suppressing genes (fig. 2G and fig. 2H); TDRD10 protein could target the 3 'UTRs of cell cycle promoting genes to degrade their mRNAs, but could not target the 3' UTRs of cell cycle suppressing genes (fig. 2I); the half-life test of mRNAs of the cell cycle promoting genes shows that the over-expression of TDRD10 gene remarkably reduces the half-life of the mRNAs (figure 3A), which further proves that the TDRD10 protein specifically degrades the mRNAs of the cell cycle promoting genes.

The above results show that: the inventor of the invention discovers a new function of the TDRD10 protein for the first time, namely, selectively degrading cell cycle promoting genes mRNAs, and the new function explains the essential reason that the over-expression of the TDRD10 gene can prevent the cell cycle of a tumor from progressing.

Secondly, the RRM functional region of TDRD10 protein is responsible for preventing the cell cycle progression of the tumor

The domain schematic of the TDRD10 protein is shown in FIG. 3B. The RRM structural domain is the amino acid residues 34-107, and the Tudor structural domain is the amino acid residue 210-317.

1. Several recombinant plasmids were prepared as follows (each recombinant plasmid was sequence verified):

inserting the DNA molecules of the code truncation aa 1-108 in the sequence 2 of the sequence table into a pEGFP-N1 vector to obtain a recombinant plasmid pEGFP-N1-T1-GFP; the recombinant plasmid pEGFP-N1-T1-GFP expresses T1-EGFP fusion protein. Truncation 1 is represented by T1, and is shown as amino acid residues 1-107 in sequence 1 of the sequence table.

Inserting the DNA molecule of the coding truncation aa 108-366 in the sequence 2 of the sequence table into a pEGFP-N1 vector to obtain a recombinant plasmid pEGFP-N1-T2-GFP; the recombinant plasmid pEGFP-N1-T2-GFP expresses T2-EGFP fusion protein. Truncation 2 is represented by T2, and is represented by amino acid residues 108-366 in sequence 1 of the sequence table.

2. The recombinant plasmid pEGFP-N1-T1-GFP is introduced into MDA-MB-468 cells to obtain recombinant cells, and the recombinant cells are named MDA-MB-468/T1-GFP cells. The recombinant plasmid pEGFP-N1-T2-GFP is introduced into MDA-MB-468 cells to obtain recombinant cells, and the recombinant cells are named MDA-MB-468/T2-GFP cells.

3. The MDA-MB-468/TDRD10-GFP cells (labeled TDRD10-GFP in the figure) prepared in the example 1, the MDA-MB-468/T1-GFP cells (labeled aa 1-108 in the figure) and the MDA-MB-468/T2-GFP cells (labeled aa 108-366 in the figure) prepared in the step 2 are taken, and the following steps are respectively carried out: the culture was performed under parallel conditions, and after 36 hours, cells were taken, total RNA was extracted and reverse-transcribed into cDNA, and then qPCR was performed to detect the expression amount of the cell cycle promoting gene. The results are shown in FIG. 3C.

4. Luciferase reporter assay

The test plasmid and reporter vector were co-transfected in HEK293 cells, cells were lysed 36 hours after transfection, lysates were collected, and luciferase activity was detected using the Dual-luciferase reporter Assay System (Promega), and the results are shown in FIG. 3D. The test plasmids were: recombinant plasmid pEGFP-N1-TDRD10-GFP (marked as TDRD10-GFP in the figure) or pEGFP-N1 vector (marked as Empty vector in the figure) or recombinant plasmid pEGFP-N1-T1-GFP (marked as aa 1-108 in the figure) or recombinant plasmid pEGFP-N1-T2-GFP (marked as aa 108-366 in the figure). The reporter vectors were respectively: a reporter vector with the 3 ' UTR of the β -Actin gene, a reporter vector with the 3 ' UTR of the CDK2 gene, a reporter vector with the 3 ' UTR of the CCND1 gene, a reporter vector with the 3 ' UTR of the CDKN3 gene or a reporter vector with the 3 ' UTR of the WEE1 gene.

5. Taking the MDA-MB-468/TDRD10-GFP cells (labeled TDRD10-GFP in the figure) and the MDA-MB-468/GFP cells (labeled Empty Control in the figure) in example 1, the MDA-MB-468/T1-GFP cells (labeled aa 1-108 in the figure) and the MDA-MB-468/T2-GFP cells (labeled aa 108-366 in the figure) prepared in step 2 were taken, and the following steps were respectively carried out: the cultures were incubated in parallel conditions and after 36 hours flow cytometry analysis was performed to obtain the cell ratios at each cell cycle. The results are shown in FIG. 3E.

The result of the second step shows that: cell cycle promoting genes mRNAs were down-regulated following overexpression of the T1 truncation gene (fig. 3C); the T1 truncation can target the 3' UTR associated with cell cycle promoting genes to degrade their mRNAs (fig. 3D); overexpression of the T1 truncation gene prevented progression of the tumor cell' S cycle G1/S phase (FIG. 3E).

The above results show that: the RRM functional region of TDRD10 protein plays an important role in specifically degrading cell cycle promoting genes.

Example 3 overexpression of TDRD10 Gene (increasing TDRD10 protein levels) inhibits tumor growth and metastasis

1. The MDA-MB-468/TDRD10-GFP cells (labeled TDRD10-GFP in the figure) and MDA-MB-468/GFP cells (labeled TDRD10-GFP in the figure) in example 1 were takenDenoted Empty Vector), the following steps are performed, respectively: back subcutaneous injection of BALB/c nude mice (3X 10 injection per mouse)⁶Individual cells/100 μ L PBS buffer), days counted from the start of injection. Tumor volume in situ was measured daily from day 18 to day 56 and the change in tumor volume in situ over time is shown in figure 4A (mean of 5 mice).

2. MDA-MB-231 cells were taken and injected subcutaneously into BALB/c nude mice (3X 10 injection per mouse)⁶Cells/100 μ L PBS buffer). Days were counted from the injection of MDA-MB-231 cells. Mean tumor in situ diameter of each mouse at day 40>3 mm. Recombinant adenovirus was injected every other day starting on day 40 (1X 10 per mouse)¹⁰pfu), 5 total injections. Two treatment groups were set, and a recombinant adenovirus expressing TDRD10 gene (TDRD10 gene is shown in sequence 2 of the sequence table, and a recombinant adenovirus expressing TDRD10 gene is shown by Ad-TDRD 10) or a control adenovirus (shown by Ad-Con, and compared with a recombinant adenovirus expressing TDRD10 gene, the control adenovirus is different only in that the TDRD10 gene is not present) was administered. Tumor volume in situ was measured daily from day 20 to day 60 and the change in tumor volume in situ over time is shown in figure 4B (mean of 5 mice). The photograph of the mouse orthotopic tumor after 60 days is shown in FIG. 4C. Mice were sacrificed and dissected on day 36, and whole lung tissue was taken for HE staining to detect lung metastasis. The results are shown in FIG. 4D, left panel 1 and left panel 2. The number of white nodules in lung tissue was counted and shown in the right 1 panel of fig. 4D (average of 5 mice). The linear correlation between TDRD10 and cell cycle genes in the TCGA breast cancer database was analyzed and the results are shown in fig. 4E.

The TDRD10 can obviously inhibit the growth of the tumor and inhibit the lung metastasis of the tumor cells in vivo, and the research finds that the TDRD10 is obviously related to clinical data. The experiment results show that the over-expression of the TDRD10 gene (the TDRD10 protein level is increased) can obviously inhibit the tumor growth and the tumor metastasis.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Sequence listing

<110> institute of microcirculation of Chinese academy of medical sciences

<120> TDRD10 protein and application of coding gene thereof in preparation of tumor treatment medicines, and tumor treatment medicines

<130> 1

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 366

<212> PRT

<213> TDRD10

<400> 1

Met Ser Trp Asn Ile Ser His Pro Gln Leu Ser Asp Lys Leu Phe Gly

1 5 10 15

Lys Asn Gly Val Leu Glu Glu Gln Lys Ser Pro Gly Phe Lys Lys Arg

20 25 30

Glu Thr Glu Val Tyr Val Gly Asn Leu Pro Leu Asp Ile Ser Lys Glu

35 40 45

Glu Ile Leu Tyr Leu Leu Lys Asp Phe Asn Pro Leu Asp Val His Lys

50 55 60

Ile Gln Asn Gly Cys Lys Cys Phe Ala Phe Val Asp Leu Gly Ser Met

65 70 75 80

Gln Lys Val Thr Leu Ala Ile Gln Glu Leu Asn Gly Lys Leu Phe His

85 90 95

Lys Arg Lys Leu Phe Val Asn Thr Ser Lys Arg Pro Pro Lys Arg Thr

100 105 110

Pro Asp Met Ile Gln Gln Pro Arg Ala Pro Leu Val Leu Glu Lys Ala

115 120 125

Ser Gly Glu Gly Phe Gly Lys Thr Ala Ala Ile Ile Gln Leu Ala Pro

130 135 140

Lys Ala Pro Val Asp Leu Cys Glu Thr Glu Lys Leu Arg Ala Ala Phe

145 150 155 160

Phe Ala Val Pro Leu Glu Met Arg Gly Ser Phe Leu Val Leu Leu Leu

165 170 175

Arg Glu Cys Phe Arg Asp Leu Ser Trp Leu Ala Leu Ile His Ser Val

180 185 190

Arg Gly Glu Ala Gly Leu Leu Val Thr Ser Ile Val Pro Lys Thr Pro

195 200 205

Phe Phe Trp Ala Met His Val Thr Glu Ala Leu His Gln Asn Met Gln

210 215 220

Ala Leu Phe Ser Thr Leu Ala Gln Ala Glu Glu Gln Gln Pro Tyr Leu

225 230 235 240

Glu Gly Ser Thr Val Met Arg Gly Thr Arg Cys Leu Ala Glu Tyr His

245 250 255

Leu Gly Asp Tyr Gly His Ala Trp Asn Arg Cys Trp Val Leu Asp Arg

260 265 270

Val Asp Thr Trp Ala Val Val Met Phe Ile Asp Phe Gly Gln Leu Ala

275 280 285

Thr Ile Pro Val Gln Ser Leu Arg Ser Leu Asp Ser Asp Asp Phe Trp

290 295 300

Thr Ile Pro Pro Leu Thr Gln Pro Phe Met Leu Glu Lys Asp Ile Leu

305 310 315 320

Ser Ser Tyr Glu Val Val His Arg Ile Leu Lys Gly Lys Ile Thr Gly

325 330 335

Ala Leu Asn Ser Ala Val Thr Ala Pro Ala Ser Asn Leu Ala Val Val

340 345 350

Pro Pro Leu Leu Pro Leu Gly Cys Leu Gln Gln Ala Ala Ala

355 360 365

<210> 2

<211> 1101

<212> DNA

<213> TDRD10

<400> 2

atgtcctgga acattagtca cccccaactc tctgataaac tgtttgggaa gaatggagtg 60

ttggaggagc agaaatctcc aggattcaag aaaagagaga cagaggtgta tgttggcaat 120

cttccactgg atatttctaa ggaggaaatt ctgtaccttc taaaggactt caaccctctt 180

gatgtccaca aaatccagaa tggctgcaaa tgctttgcat ttgtagatct gggctccatg 240

cagaaagtga cacttgcaat ccaggagctg aatggtaaac tcttccacaa gcgaaaactg 300

ttcgtgaata caagcaaaag gccccccaag aggacccctg atatgatcca gcagcctcgg 360

gccccgctgg tgttggagaa ggcttctggt gaaggatttg gcaaaaccgc cgctattata 420

cagctcgctc ctaaagctcc tgttgacctg tgtgagacag agaaactgag ggcagccttc 480

tttgcagtcc cgttggaaat gagagggtcc ttcctggtgc tgctcctgag ggaatgcttc 540

cgagacctga gctggctggc actcatccat agcgtccgtg gggaggcggg gctgctggtg 600

acgagtatcg tcccgaagac cccgtttttc tgggctatgc acgtcactga ggctctgcac 660

cagaacatgc aggctctgtt tagcaccctg gctcaggcgg aggagcagca gccctacctg 720

gagggctcca ccgttatgcg cgggactcgc tgtctggcag agtaccacct gggggattat 780

ggacacgcct ggaacaggtg ttgggtgctg gacagggtgg acacctgggc tgtggtcatg 840

ttcattgatt ttggacagtt ggccaccatc cctgtgcagt ctctgcgcag cctagacagc 900

gacgacttct ggaccatccc acccctgact cagccattca tgctggagaa agacattttg 960

agttcgtatg aggttgtcca tcgaatcctc aaagggaaaa tcactggtgc tttgaactcg 1020

gcggtaactg ctcctgcatc taacttggct gttgtccctc cactcctgcc cttggggtgt 1080

ctgcagcagg ctgctgccta g 1101

Claims

The application of TDRD10 protein in preparing tumor treating medicine; the function of the medicine is as follows (a1) and/or (a2) and/or (a3) and/or (a 4): (a1) treating tumors; (a2) inhibiting tumor growth and/or metastasis; (a3) inhibiting tumor cell proliferation; (a4) preventing the cycle progression of tumor cells.
The application of TDRD10 gene or biological material with TDRD10 gene in preparing tumor treating medicine; the function of the medicine is as follows (a1) and/or (a2) and/or (a3) and/or (a 4): (a1) treating tumors; (a2) inhibiting tumor growth and/or metastasis; (a3) inhibiting tumor cell proliferation; (a4) preventing the cycle progression of tumor cells.
3. The application of the substance capable of increasing the abundance of TDRD10 protein in organisms in preparing medicaments for treating tumors; the function of the medicine is as follows (a1) and/or (a2) and/or (a3) and/or (a 4): (a1) treating tumors; (a2) inhibiting tumor growth and/or metastasis; (a3) inhibiting tumor cell proliferation; (a4) preventing the cycle progression of tumor cells.
4. The application of the substance capable of increasing the abundance of TDRD10 gene in organisms in preparing tumor treatment medicines; the function of the medicine is as follows (a1) and/or (a2) and/or (a3) and/or (a 4): (a1) treating tumors; (a2) inhibiting tumor growth and/or metastasis; (a3) inhibiting tumor cell proliferation; (a4) preventing the cycle progression of tumor cells.
5. A truncated TDRD10 protein, namely the RRM functional region of TDRD10 protein.
6. A gene encoding the truncated TDRD10 protein of claim 5.
7. Use of the truncated TDRD10 protein of claim 5 for the preparation of a medicament for the treatment of tumors; the function of the medicine is as follows (a1) and/or (a2) and/or (a3) and/or (a 4): (a1) treating tumors; (a2) inhibiting tumor growth and/or metastasis; (a3) inhibiting tumor cell proliferation; (a4) preventing the cycle progression of tumor cells.
8. Use of a gene encoding the TDRD10 protein truncation of claim 5 or a biological material having a gene encoding the TDRD10 protein truncation of claim 5 in the preparation of a medicament for the treatment of a tumor; the function of the medicine is as follows (a1) and/or (a2) and/or (a3) and/or (a 4): (a1) treating tumors; (a2) inhibiting tumor growth and/or metastasis; (a3) inhibiting tumor cell proliferation; (a4) preventing the cycle progression of tumor cells.
The use of TDRD10 protein, TDRD10 gene, TDRD10 protein truncation described in claim 5, or gene encoding TDRD10 protein truncation as a target in the development of antitumor drugs.
10. A medicament for treating tumors, which is characterized in that: the drug achieves a drug function by up-regulating the abundance of at least one of the TDRD10 protein, TDRD10 gene, TDRD10 protein truncate of claim 5, and gene encoding the TDRD10 protein truncate in an organism;

the drug functions as (a1) and/or (a2) and/or (a3) and/or (a4) as follows: (a1) treating tumors; (a2) inhibiting tumor growth and/or metastasis; (a3) inhibiting tumor cell proliferation; (a4) preventing the cycle progression of tumor cells.