Application of CPEB4 protein and coding gene thereof in preparation of tumor treatment drug, and tumor treatment drug

Abstract

The invention relates to a novel application of a CPEB4 protein and a CPEB4 gene in tumor treatment. Specifically, the invention relates to a novel effect of a CPEB4 protein and a CPEB4 gene in preparation of tumor treatment drugs. The invention has important theoretical value for mechanism research of tumor cell proliferation, tumor cell cycle progress, tumor growth and metastasis. The invention has very important application value for tumor treatment.

Description

Technical Field

[0001] The present invention relates to the fields of oncology, genetics, and molecular biology. Specifically, the present invention relates to a new use of CPEB4 protein and CPEB4 gene. More specifically, the present invention relates to a new use of CPEB4 protein and CPEB4 gene in preparing tumor therapeutic drugs. Background Art

[0002] An important reason for the unlimited proliferation of tumor cells is the uncontrolled cell cycle progression (Xiong Y, et al. 2018), which is also one of the reasons why clinical targeted cell cycle therapy for tumors is not effective. Although many studies have enriched people's understanding of the regulation of tumor cell cycle progression, the post-transcriptional regulation of cell cycle-related gene expression in tumor cells, especially the regulatory role and mechanism of molecules such as RNA binding proteins, remains unclear.

[0003] Abnormal expression of cell cycle-related genes is a fundamental reason for the uncontrolled progression of cancer cell cycle (Fischer et al. 2017). According to gene function, cell cycle-related genes can be divided into cell cycle promoting genes (including CDKs, E2F1, CCNDs, CCNEs, etc.) and cell cycle inhibitory genes (including p21, p27, TP53, RB1, etc.). The balance between them determines the cycle progression of cancer cells (Malumbres M et al., 2001). Most tumor cell cycle out of control is either due to increased expression of cell cycle promoting genes or limited expression of cell cycle inhibitory genes (Malumbres M et al., 2009). In cancer, the expression levels of many cell cycle-related genes are significantly correlated with the prognosis of tumor patients (Leonard JP et al., 2012; Sawai CM et al., 2012; Sheppard KE et al., 2013). Therefore, clinical targeted cell cycle therapy for tumors mainly inhibits the expression of cell cycle promoting genes or limits the activity of molecules through targeted drugs, thereby preventing the cycle progression and unlimited proliferation of cancer cells.

[0004] CPEB4 (Cytoplasmic polyadenylation element-binding protein 4), an RNA binding protein encoded by the CPEB4 gene. To date, the role of CPEB4 molecules in the progression of human cancer is not fully understood. The inventors first discovered that the molecule and gene have the function of inhibiting breast cancer cell cycle progression and proliferation, and can inhibit the metastasis and growth of breast tumors in vivo. Specifically, the inventors found that CPEB4 can bind to and degrade mRNAs of genes such as cell cycle promoting genes CDK2, CDK4, CCNE1 and MCM2, and inhibit their expression. CPEB4 can prevent tumor cells from transitioning from the G1 / S phase. In addition, the inventors further found that the RRM1 functional region of CPEB4 is critical for specific binding to the mRNA of cell cycle promoting genes, and this functional region can control the progression of tumor cell cycle. Importantly, by analyzing the human breast cancer sample database, it was found that the expression of CPEB4 was significantly positively correlated with patient survival. In summary, the inventors first identified CPEB4 as a tumor suppressor gene in human breast cancer that exerts anti-cancer function. Summary of the invention

[0005] The purpose of the present invention is to provide a new use of CPEB4 protein and CPEB4 gene. More specifically, the present invention relates to a new use of CPEB4 protein and CPEB4 gene in the preparation of tumor treatment drugs.

[0006] The present invention firstly protects the application of CPEB4 protein in preparing tumor therapeutic drugs.

[0007] The present invention also protects the use of the CPEB4 gene or biological materials having the CPEB4 gene in preparing tumor therapeutic drugs.

[0008] The present invention also protects the use of a substance that can increase the abundance of CPEB4 protein in an organism in the preparation of a tumor treatment drug. The substance that can increase the abundance of CPEB4 protein in an organism can be the CPEB4 protein itself, or other proteins located upstream of the CPEB4 protein in the organism that can promote the production of the CPEB4 protein, or other proteins located downstream of the CPEB4 protein in the organism that can reduce the degradation of the CPEB4 protein, or compounds or other small molecules that can promote the increase of the CPEB4 protein level in the organism.

[0009] The present invention also protects the use of a substance that can increase the abundance of the CPEB4 gene in an organism in the preparation of a tumor treatment drug. The substance that can increase the abundance of the CPEB4 gene in an organism can be the CPEB4 gene itself, or other proteins or nucleic acid molecules located upstream of the CPEB4 gene in an organism that can promote the expression of the CPEB4 gene, or other proteins or nucleic acid molecules located downstream of the CPEB4 gene in an organism that can reduce the degradation of the CPEB4 gene, or compounds or other small molecules that can promote the expression of the CPEB4 gene in an organism.

[0010] The present invention also protects a tumor treatment drug, which degrades the mRNAs of cell cycle promoting genes in tumor cells by up-regulating the abundance of CPEB4 protein in an organism or up-regulating the abundance of CPEB4 gene in an organism, thereby preventing the progression of the tumor cell cycle. The substance that can up-regulate the CPEB4 protein in an organism can be the CPEB4 protein itself, or other proteins located upstream of the CPEB4 protein in an organism that can promote the production of the CPEB4 protein, or other proteins located downstream of the CPEB4 protein in an organism that can reduce the degradation of the CPEB4 protein, or compounds or other small molecules that can promote the increase of the CPEB4 protein level in an organism. The substance that can up-regulate the CPEB4 gene in an organism can be the CPEB4 gene itself, or other proteins or nucleic acid molecules located upstream of the CPEB4 gene in an organism that can promote the expression of the CPEB4 gene, or other proteins or nucleic acid molecules located downstream of the CPEB4 gene in an organism that can reduce the degradation of the CPEB4 gene, or compounds or other small molecules that can promote the expression of the CPEB4 gene in an organism.

[0011] The present invention also protects a truncated form of the CPEB4 protein, namely the RRM1 functional region of the CPEB4 protein.

[0012] The RRM1 functional region of CPEB4 protein is the amino acid residues 472-563 of CPEB4 protein.

[0013] The gene encoding the truncated CPEB4 protein also falls within the protection scope of the present invention.

[0014] The present invention also protects the use of CPEB4 protein truncations in the preparation of tumor therapeutic drugs.

[0015] The present invention also protects the use of a gene encoding the CPEB4 protein truncation or a biological material having a gene encoding the CPEB4 protein truncation in the preparation of tumor therapeutic drugs.

[0016] The present invention also protects a tumor treatment drug, which can upregulate the abundance of the CPEB4 protein truncate according to claim 6 in an organism or upregulate the abundance of the gene encoding the CPEB4 protein truncate according to claim 6 in an organism, and degrade the mRNAs of the cell cycle promoting gene in the tumor cells through the CPEB4 protein truncate, thereby preventing the progression of the tumor cell cycle. The substance that can upregulate the CPEB4 protein truncate in an organism can be the CPEB4 protein truncate itself, or other proteins, peptides, compounds or other small molecules that can promote the increase of the level of the CPEB4 protein truncate in an organism. The substance that can upregulate the gene encoding the CPEB4 protein truncate in an organism can be the gene encoding the CPEB4 protein truncate itself, or other proteins, polypeptides, nucleic acid molecules, compounds or other small molecules that promote the expression of the gene encoding the CPEB4 protein truncate.

[0017] The functions of any of the above drugs are as follows (a1) and / or (a2) and / or (a3) ​​and / or (a4): (a1) treating tumors; (a2) inhibiting tumor growth and / or metastasis; (a3) ​​inhibiting tumor cell proliferation; (a4) preventing tumor cell cycle progression.

[0018] Exemplarily, any of the above CPEB4 proteins may be human CPEB4 proteins.

[0019] Any of the above CPEB4 proteins may specifically be the following (b1), (b2), (b3) or (b4):

[0020] (b1) the protein shown in Sequence 1 in the sequence listing;

[0021] (b2) a fusion protein obtained by connecting a tag to the amino terminus or carboxyl terminus of (b1);

[0022] (b3) a protein having any of the functions of (a1) to (a4) obtained by substituting and / or deleting and / or adding one or more amino acid residues in (b1);

[0023] (b4) A protein derived from humans, having 98% or more identity with (b1) and having any one of the functions of (a1) to (a4).

[0024] The specific labels are shown in Table 1.

[0025] Table 1 Tag sequences

[0026]

[0027] The CPEB4 protein may also be a homologous protein having any of the functions (a1) to (a4) in other species, including but not limited to mice, rats, rabbits, dogs, monkeys, gorillas, apes, cattle, sheep, pigs, horses, sheep, goats, cats, etc.

[0028] The CPEB4 gene is a gene encoding the CPEB4 protein or a CPEB4 protein.

[0029] Any of the above CPEB4 genes may specifically be as follows (c1) or (c2) or (c3):

[0030] (c1) a DNA molecule whose coding region is shown in Sequence 2 in the sequence listing;

[0031] (c2) a DNA molecule derived from humans and having 95% or more identity with (c1) and encoding the protein;

[0032] (c3) A DNA molecule that hybridizes to the nucleotide sequence defined in (c1) under stringent conditions and encodes the protein.

[0033] The CPEB4 gene may also be a homologous gene in other species, including but not limited to mice, rats, rabbits, dogs, monkeys, gorillas, apes, cattle, sheep, pigs, horses, sheep, goats, cats, etc.

[0034] The CPEB4 protein truncation may specifically be as follows (d1), (d2), (d3) or (d4):

[0035] (d1) the protein represented by amino acid residues 472-563 and 580-662 in SEQ ID NO: 1 of the sequence listing;

[0036] (d2) a fusion protein obtained by connecting a tag to the amino terminus or carboxyl terminus of (d1);

[0037] (d3) a protein having any of the functions of (a1) to (a4) obtained by substituting and / or deleting and / or adding one or more amino acid residues in (d1);

[0038] (d4) A protein derived from humans, having 98% or more identity with (d1) and having any one of the functions of (a1) to (a4).

[0039] The specific labels are shown in Table 1.

[0040] The CPEB4 protein truncation may also be a homologous protein having any of the functions (a1) to (a4) in other species, including but not limited to mice, rats, rabbits, dogs, monkeys, gorillas, apes, cattle, sheep, pigs, horses, sheep, goats, cats, etc.

[0041] Any of the above genes encoding CPEB4 protein truncations may specifically be as follows (e1) or (e2) or (e3):

[0042] (e1) a DNA molecule encoding a truncated form of the CPEB4 protein in the DNA molecule shown in Sequence 2 in the sequence listing;

[0043] (e2) a DNA molecule derived from humans and having more than 95% identity with (e1) and encoding the truncated form of the CPEB4 protein;

[0044] (e3) A DNA molecule that hybridizes with the nucleotide sequence defined in (e1) under stringent conditions and encodes the CPEB4 protein truncation.

[0045] The gene encoding the truncate form of the CPEB4 protein may also be a homologous gene in other species, including but not limited to mice, rats, rabbits, dogs, monkeys, gorillas, apes, cattle, sheep, pigs, horses, sheep, goats, cats, etc.

[0046] Any of the above-mentioned biological materials having the CPEB4 gene may be an expression vector having the CPEB4 gene. Any of the above-mentioned biological materials having a gene encoding a truncate of the CPEB4 protein may be an expression vector having a gene encoding a truncate of the CPEB4 protein. The expression vector can carry a nucleotide sequence, integrate this sequence into the cell genome, and can replicate in the cell. "Expression vector" includes plasmids, cosmids, viruses (bacteriophages, animal viruses, plant viruses, etc.) and artificial chromosomes (such as YACs). The viruses (also called viral vectors) that may be suitable for clinical gene therapy as expression vectors are as follows: adenovirus vectors, retrovirus vectors, adeno-associated virus vectors, lentivirus vectors, herpes virus vectors, chimeric virus vectors and other viral vectors.

[0047] Any of the above mentioned tumors include but are not limited to breast cancer.

[0048] Any of the above mentioned tumor cells include but are not limited to breast cancer cells.

[0049] The inventors of the present invention have found that CPEB4 protein has significant tumor suppressor function and can induce tumor cell cycle arrest. Its mechanism is to inhibit its expression by targeting and degrading cell cycle promoting gene transcripts (including CDK2, CDK4, CCNE1, MCM2, etc.). The inventors of the present invention found that CPEB4 gene expression is inhibited in breast cancer tissues and cells, which may help tumor cells escape strict cell cycle regulation. Overexpression of CPEB4 gene can block G1 / S transition of breast cancer cell cycle. Consistent with these in vitro findings, overexpression of CPEB4 gene in tumor tissues of tumor-bearing animals can significantly inhibit tumor growth and metastasis. By analyzing the human tumor tissue sample database, the inventors found that low expression levels of CPEB4 are strongly associated with poor survival of breast cancer patients. In addition, the expression level of CPEB4 gene in cancer patient tissues is significantly negatively correlated with the expression levels of its target genes CDK2, CDK4, CDK6, CCNE1, MCM2 and CDKN3. These results indicate that CPEB4 is a potential tumor suppressor gene that participates in regulating cell cycle signaling pathways by inhibiting cell cycle promoting gene expression. Based on this, using the tumor suppressor protein CPEB4 to improve the clinical tumor treatment effect has very important application prospects.

[0050] The present invention identifies for the first time that the human CPEB4 gene is a tumor cell cycle inhibitory gene, and overexpression of the CPEB4 gene in tumor cells can significantly inhibit tumor cell proliferation and metastasis. The present invention has important theoretical value for the study of the mechanism of tumor cell cycle, tumor cell proliferation, tumor growth and metastasis. The present invention has very important theoretical and practical significance for clinical tumor treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 CPEB4 is expressed at low levels in human breast cancer tissues and can inhibit tumor cell proliferation and cell cycle progression. TCGA database analysis found that the mRNA expression level of CPEB4 in 1097 primary breast cancer tumors was significantly lower than that in normal breast tissues (114 cases) ( Figure 1 A); TCGA database analysis found that the expression level of CPEB4 in different subtypes of breast cancer tissues (Luminal, HER2+, Triple negative) was significantly lower than that in normal breast tissues ( Figure 1 B); Analysis of the breast cancer database showed that CPEB4 expression level was significantly positively correlated with patient survival ( Figure 1 C); Overexpression of CPEB4 gene significantly inhibits tumor cell proliferation ( Figure 1 D and Figure 1 E); Overexpression of CPEB4 gene prevents G1 / S phase transition of tumor cells ( Figure 1F); Western blotting results showed that overexpression of CPEB4 gene could effectively increase p21 expression and slightly increase Cleaved-PARP1 and -Caspase3 expression, indicating that ZCCHC24 could significantly inhibit cell cycle progression but did not significantly affect cell apoptosis.

[0052] Figure 2 CPEB4 specifically inhibits the expression of cell cycle-promoting genes by targeting 3'UTRs. After overexpression of the CPEB4 gene, the mRNAs and protein expressions of cell cycle-promoting genes were downregulated ( Figure 2 A. Figure 2 B. Figure 2 C. Figure 2 D and Figure 2 E); CPEB4 protein can target and bind to the 3'UTRs of cell cycle-promoting genes ( Figure 2 F and Figure 2 G).

[0053] Figure 3 The RRM1 functional region of CPEB4 is responsible for degrading the mRNAs of cell cycle-promoting genes. Overexpression of the CPEB4 gene significantly reduces the half-life of the mRNAs of cell cycle-promoting genes ( Figure 3 A); Schematic diagram of CPEB4 functional region and preparation strategies of different mutants and crystal structure of RRM1 functional region ( Figure 3 B); After overexpression of the T1 truncated gene (shown as aa 1-564 in the figure), the mRNAs of cell cycle-promoting genes were downregulated ( Figure 3 C); Overexpression of the T1 truncated gene prevents tumor cells from progressing through the G1 / S phase of the cycle ( Figure 3 D).

[0054] Figure 4 Overexpression of CPEB4 gene in vivo inhibits tumor growth and metastasis. Overexpression of CPEB4 gene significantly inhibits tumor growth in nude mice ( Figure 4 A). Recombinant adenovirus expressing CPEB4 gene can inhibit tumor growth and metastasis in tumor-bearing nude mice ( Figure 4 B. Figure 4 C. and Figure 4 D). Analysis of the clinical breast cancer database showed that the expression of CPEB4 in breast cancer tissues was significantly negatively correlated with the expression of cell cycle promoting genes ( Figure 4 E). DETAILED DESCRIPTION

[0055] The principles and features of the present invention are described below in conjunction with the accompanying drawings. The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. Unless otherwise specified, the quantitative tests in the following examples are repeated more than 3 times, and the results are averaged.

[0056] pEGFP-N1 vector: Clontech; pEGFP-N1 vector expresses EGFP protein, which is also called GFP protein. MDA-MB-468 cells (human breast cancer cells): HTB-132 TM MCF7 cells (human breast cancer cells): HTB-22 TM MDA-MB-231 cells (human breast cancer cells): HTB-26 TM .

[0057] Example 1: Analysis of the expression of CPEB4 in human breast cancer tissues and its correlation with patient prognosis, and identification of a new function of CPEB4 in preventing tumor cell cycle progression.

[0058] 1. By analyzing the TCGA database, it was found that CPEB4 was expressed at a low level in human breast cancer tissue samples, and its low expression was significantly associated with the poor prognosis of patients. The CPEB4 gene (CPEB4 gene is shown in sequence 2 of the sequence table, and the insertion sequence removes the stop codon) was inserted between the Hind III and BamH I restriction sites of the pEGFP-N1 vector to obtain the recombinant plasmid pEGFP-N1-CPEB4-GFP. The recombinant plasmid pEGFP-N1-CPEB4-GFP has been sequenced and verified. In the recombinant plasmid pEGFP-N1-CPEB4-GFP, the inserted DNA molecule forms a fusion gene with the EGFP gene in the vector to express the CPEB4-EGFP fusion protein (referred to as T10-GFP fusion protein).

[0059] 2. Introduce the recombinant plasmid pEGFP-N1-CPEB4-GFP into MDA-MB-468 cells to obtain recombinant cells, named MDA-MB-468 / T10-GFP cells.

[0060] 3. Introduce the pEGFP-N1 vector into MDA-MB-468 cells to obtain recombinant cells, named MDA-MB-468 / GFP cells.

[0061] 4. Introduce the recombinant plasmid pEGFP-N1-CPEB4-GFP into MCF7 cells to obtain recombinant cells, named MCF7 / T10-GFP cells.

[0062] 5. Introduce the pEGFP-N1 vector into MCF7 cells to obtain recombinant cells, named MCF7 / GFP cells.

[0063] 6. Take MDA-MB-468 / T10-GFP cells and MDA-MB-468 / GFP cells and perform the following steps respectively: culture them under parallel conditions, lyse the cells after 36 hours, and then collect the lysate for immunoblotting. The results of immunoblotting using GFP antibody are shown in Figure 1 G. Figure 1 Above the lanes in G, Con represents MDA-MB-468 / GFP cells.

[0064] 7. Take MDA-MB-468 cells (marked as MDA468 / Parental in the figure), MDA-MB-468 / GFP cells (marked as Empty control in the figure) and MDA-MB-468 / CPEB4-GFP cells (marked as CPEB4 / GFP in the figure) and perform the following steps respectively: culture them under parallel conditions, count the cells at different culture times (0h, 24h, 48h, 72h) to detect the effect of overexpression of CPEB4 gene on cell proliferation. The results are shown in Figure 1 D on.

[0065] 8. Take MCF7 cells (labeled as MCF7 / Parental in the figure), MCF7 / GFP cells (labeled as Empty control in the figure) and MCF7 / CPEB4-GFP cells (labeled as CPEB4 / GFP in the figure) and perform the following steps respectively: culture them under parallel conditions and count the cells at different culture times (0h, 24h, 48h, 72h) to detect the effect of overexpression of CPEB4 gene on cell proliferation. The results are shown in Figure 1 D below.

[0066] 9. Take MDA-MB-468 cells (marked as MDA468 / Parental in the figure), MDA-MB-468 / GFP cells (marked as Empty control in the figure) and MDA-MB-468 / CPEB4-GFP cells (marked as CPEB4 / GFP in the figure) and perform the following steps respectively: culture them under parallel conditions and perform MTT staining at different culture times (0h, 24h, 48h, 72h) to detect the effect of overexpression of CPEB4 gene on cell proliferation. The results are shown in Figure 1 On E.

[0067] 10. Take MCF7 cells (labeled as MCF7 / Parental in the figure), MCF7 / GFP cells (labeled as Empty control in the figure) and MCF7 / CPEB4-GFP cells (labeled as CPEB4 / GFP in the figure) and perform the following steps respectively: culture them under parallel conditions and perform MTT staining at different culture times (0h, 24h, 48h, 72h) to detect the effect of overexpression of CPEB4 gene on cell proliferation. The results are shown in Figure 1 E below.

[0068] 11. Take MDA-MB-468 / CPEB4-GFP cells (labeled as CPEB4-GFP in the figure) and MDA-MB-468 / GFP cells (labeled as Empty control in the figure) and perform the following steps respectively: culture them under parallel conditions. After 36 hours, collect the cells for flow cytometry analysis to obtain the ratio of cells in each cell cycle. The results are shown in Figure 1 F on.

[0069] 12. Take MCF7 / CPEB4-GFP cells (labeled as CPEB4-GFP in the figure) and MCF7 / GFP cells (labeled as Empty control in the figure) and perform the following steps respectively: culture them under parallel conditions. After 36 hours, collect the cells for flow cytometry analysis to obtain the ratio of cells in each cell cycle. The results are shown in Figure 1 F below.

[0070] 13. Take MDA-MB-468 / CPEB4-GFP cells (marked as MDA-MB-468 in the figure) and MDA-MB-468 / GFP cells (marked as Con in the figure) and perform the following steps respectively: culture under parallel conditions, collect cell protein lysate after 36 hours and perform immunoblotting, and use different antibodies to detect the expression of cell cycle inhibitor p21 and apoptosis indicator proteins Cleaved-PARP1 and -Caspase3. The results are shown in Figure 1 G.

[0071] Figure 1 The results showed that the expression of CPEB4 in human breast tumor tissue was significantly lower than that in normal breast tissue and was significantly positively correlated with the survival of breast cancer patients ( Figure 1 A. Figure 1 B and Figure 1 C); Overexpression of CPEB4 gene significantly inhibits tumor cell proliferation ( Figure 1 D. Figure 1 E); Overexpression of CPEB4 gene prevents tumor cells from progressing through the G1 / S phase of the cell cycle ( Figure 1 F and Figure 1 G).

[0072] The above results show that overexpression of the CPEB4 gene and thus increase in CPEB4 protein levels can inhibit tumor cell proliferation and prevent tumor cell cycle progression, that is, overexpression of the CPEB4 gene and thus increase in CPEB4 protein levels have the effect of treating tumors. The above functions of the CPEB4 protein / CPEB4 gene were discovered for the first time by the inventors of the present invention.

[0073] Example 2: CPEB4 can degrade cell cycle promoting gene mRNAs

[0074] 1. CPEB4 can degrade cell cycle-promoting gene mRNAs

[0075] In order to further examine the mechanism by which CPEB4 protein inhibits tumor cell proliferation and blocks cell cycle progression, we examined how it regulates the expression of tumor cell cycle-related genes.

[0076] 1. Take the MDA-MB-468 / CPEB4-GFP cells in Example 1, culture them for 36 hours, extract total RNA and reverse transcribe it into cDNA, and then perform RNA-seq. The results are shown in Figure 2 A.

[0077] 2. Take the MCF7 / CPEB4-GFP cells in Example 1, culture them for 36 hours, extract total RNA and reverse transcribe it into cDNA, and then perform RNA-seq. The results are shown in Figure 2 B.

[0078] 3. Take the MDA-MB-468 / CPEB4-GFP cells in Example 1, culture them for 36 hours, extract total RNA and reverse transcribe it into cDNA, and then perform qPCR to detect the expression of the target gene. Figure 2 C.

[0079] 3. Take the MCF7 / CPEB4-GFP cells in Example 1, culture for 36 hours, extract total RNA and reverse transcribe it into cDNA, and then perform qPCR to detect the expression of the target gene. Figure 2 D.

[0080] 4. Take MDA-MB-468 / CPEB4-GFP cells and perform the following steps: culture them under parallel conditions, perform western blot at different culture times (24h, 72h) to detect the abundance of each target protein. The results are shown in Figure 2 The lanes corresponding to each time point in E. MDA-MB-468 / GFP cells were taken and the following steps were performed: cultured for 72 hours under parallel conditions, and western blot was performed to detect the abundance of each target protein. The results are shown in Figure 2The lane corresponding to Con in E.

[0081] 5. The 3'UTRs sequences of CDK2, CDK4, CDKN3, CCNE1 and MCM2 genes were cloned into the luciferase gene of the luciferase reporter vector pGL3 by PCR, and then formed fusion genes with the luciferase gene to prepare luciferase reporters containing different 3'UTR sequences. The schematic diagram of the prepared reporter vector is shown in Figure 2 F. Figure 2 In G, a reporter containing the 3'UTR of human β-actin was used as a negative control.

[0082] 6. Luciferase reporter assay

[0083] The test plasmid and reporter vector were co-transfected into HEK293 cells. The cells were lysed 36 hours after transfection, and the lysate was collected. The luciferase activity was detected using the Dual-LuciferaseReporter Assay System (Promega). The results are shown in Figure 2 G. The test plasmids were: recombinant plasmid pEGFP-N1-CPEB4-GFP (labeled as CPEB4 in the figure) or pEGFP-N1 vector (labeled as Control in the figure). The reporter vectors were: a reporter vector with β-actin gene 3'UTR, a reporter vector with CDK2 gene 3'UTR, a reporter vector with CDK4 gene 3'UTR, a reporter vector with CCNE1 gene 3'UTR or a reporter vector with MCM2 gene 3'UTR.

[0084] 8. Take MDA-MB-468 / CPEB4-GFP cells (labeled as CPEB4-GFP in the figure) and MDA-MB-468 / GFP cells (labeled as Empty control in the figure), and perform the following steps to detect the half-life of cell cycle promoting gene mRNAs: Treat cells with ActD and DRB (the working concentration of ActD is 5μg / mL, and the working concentration of DRB is 5μg / mL), take cells after 0min, 60min, 120min and 240min, extract total RNA, and then perform qRT-PCR to detect the target gene. The results are shown in Figure 3 A.

[0085] The results showed that after overexpression of the CPEB4 gene, the mRNAs and proteins of cell cycle-promoting genes were downregulated ( Figure 2 A. Figure 2 B. Figure 2 C. Figure 2 D. Figure 2E); CPEB4 protein can target and bind to the 3'UTR of mRNAs of cell cycle-promoting genes and degrade their mRNAs ( Figure 2 F. Figure 2 G and Figure 3 A).

[0086] The above results indicate that the inventors have discovered for the first time a new function of the CPEB4 protein, which is that it can downregulate the expression of cell cycle-promoting genes in tumor cells by degrading their mRNAs, thereby preventing the progression of the tumor cell cycle. This new function explains the essential reason why overexpression of the CPEB4 gene can prevent tumor cell proliferation.

[0087] 2. The RRM1 domain of CPEB4 protein is responsible for preventing tumor cell cycle progression

[0088] The schematic diagram of the domain structure of CPEB4 protein is shown in Figure 3 B. Amino acid residues 472-563 are the RRM1 domain; amino acid residues 580-662 are the RRM2 domain.

[0089] 1. Prepare several recombinant plasmids as follows (each recombinant plasmid has been sequenced and verified):

[0090] The DNA molecule encoding truncation 1 in sequence 2 of the sequence table is inserted into the pEGFP-N1 vector to obtain the recombinant plasmid pEGFP-N1-T1-GFP; the recombinant plasmid pEGFP-N1-T1-GFP expresses the T1-EGFP fusion protein. Truncation 1 is represented by T1, as shown by the amino acid residues 1-564 in sequence 1 of the sequence table. It can be understood by those skilled in the art that there is only one functional region in truncation 1, namely the RRM1 functional region.

[0091] The DNA molecule encoding truncation 2 in sequence 2 of the sequence table is inserted into the pEGFP-N1 vector to obtain the recombinant plasmid pEGFP-N1-T2-GFP; the recombinant plasmid pEGFP-N1-T2-GFP expresses the T2-EGFP fusion protein. Truncation 2 is represented by T2, as shown in the amino acid residues 564-729 in sequence 1 of the sequence table.

[0092] 2. Introduce the recombinant plasmid pEGFP-N1-T1-GFP into MDA-MB-468 cells to obtain recombinant cells named MDA-MB-468 / T1-GFP cells. Introduce the recombinant plasmid pEGFP-N1-T2-GFP into MDA-MB-468 cells to obtain recombinant cells named MDA-MB-468 / T2-GFP cells.

[0093] 3. Take the MDA-MB-468 / CPEB4-GFP cells in Example 1 (labeled as CPEB4-GFP in the figure), take the MDA-MB-468 / T1-GFP cells (labeled as aa1-564 in the figure) and MDA-MB-468 / T2-GFP cells (labeled as aa 564-729 in the figure) prepared in step 2, and perform the following steps respectively: culture under parallel conditions, take cells after 36 hours, extract total RNA and reverse transcribe it into cDNA, and then perform qPCR to detect the expression of cell cycle promoting genes. Results are shown in Figure 3 C.

[0094] 4. Take the MDA-MB-468 / CPEB4-GFP cells (labeled as CPEB4-GFP in the figure) and MDA-MB-468 / GFP cells (labeled as Empty Control in the figure) in Example 1, take the MDA-MB-468 / T1-GFP cells (labeled as aa 1-564 in the figure) and MDA-MB-468 / T2-GFP cells (labeled as aa 564-729 in the figure) prepared in step 2, and perform the following steps respectively: culture under parallel conditions, perform flow cytometry analysis after 36 hours, and obtain the ratio of cells in each cell cycle. The results are shown in Figure 3 D.

[0095] The results of step 2 showed that after overexpression of the T1 truncated gene, the mRNAs of cell cycle-promoting genes were downregulated ( Figure 3 C); Cell function detection found that overexpression of the T1 truncated gene prevented the progression of tumor cells into the G1 / S phase of the cycle ( Figure 3 D).

[0096] The above results indicate that the RRM1 functional region of CPEB4 protein plays an important role in degrading cell cycle promoting genes.

[0097] Example 3: Overexpression of the CPEB4 gene (increasing CPEB4 protein levels) inhibits tumor growth and metastasis

[0098] 1. Take the MDA-MB-468 / CPEB4-GFP cells (labeled as CPEB4-GFP in the figure) and MDA-MB-468 / GFP cells (labeled as Empty Vector in the figure) in Example 1 and perform the following steps: subcutaneously inject BALB / c nude mice (each mouse is injected with: 3×10 6 The number of days was counted from the injection. The volume of the orthotopic tumor was measured every day from the 18th day to the 56th day. The changes of the volume of the orthotopic tumor over time are shown in Figure 4 A (mean value of 6 mice).

[0099] 2. Take MDA-MB-231 cells and inject them subcutaneously into the back of BALB / c nude mice (3×10 per mouse) 6 cells / 100 μL PBS buffer). Days were counted from the injection of MDA-MB-231 cells. On day 40, the diameter of the orthotopic tumors of each mouse was >3 mm. Starting from day 40, the recombinant adenovirus was injected every other day (1×10 per mouse each time). 10 pfu), and injected 5 times in total. Two treatment groups were set up, and the recombinant adenovirus expressing the CPEB4 gene (the CPEB4 gene is shown in Sequence 2 of the sequence table, and the recombinant adenovirus expressing the CPEB4 gene is represented by Ad-CPEB4) or the control adenovirus (represented by Ad-Control, the difference between the control adenovirus and the recombinant adenovirus expressing the CPEB4 gene is that the control adenovirus does not have the CPEB4 gene). The in situ tumor volume was measured every day from the 20th day to the 60th day, and the change of the in situ tumor volume over time is shown in Figure 4 B (average of 6 mice). Photos of mouse transplanted tumors after 60 days are shown in Figure 4 C. On the 60th day, the mice were killed and dissected, and the whole lung tissue was taken for HE staining to detect lung metastasis. Figure 4 D. The left picture shows typical tumor metastatic nodules in lung tissue. The right picture shows the number of white nodules in lung tissue (average value of 6 mice). Analysis of the TCGA human breast cancer database found that the expression level of CPEB4 was significantly negatively correlated with the expression of cell cycle promoting genes. The results showed Figure 4 E.

[0100] The results showed that CPEB4 can significantly inhibit tumor growth and inhibit lung metastasis of tumor cells in vivo, and clinical studies have found that its expression level in breast cancer tissue is significantly negatively correlated with cell cycle target genes. In summary, the results show that overexpression of the CPEB4 gene (increasing CPEB4 protein levels) in vivo can significantly inhibit tumor growth and tumor metastasis.

[0101] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. Use of CPEB4 protein in the preparation of tumor therapeutic drugs; the functions of the drugs are as follows (a1) and / or (a2) and / or (a3) ​​and / or (a4): (a1) treating tumors; (a2) inhibiting tumor growth and / or metastasis; (a3) ​​inhibiting tumor cell proliferation; (a4) preventing tumor cell cycle progression.

2. Use of the CPEB4 gene or a biomaterial having the CPEB4 gene in the preparation of a drug for treating tumors; the functions of the drug are as follows (a1) and / or (a2) and / or (a3) ​​and / or (a4): (a1) treating tumors; (a2) inhibiting tumor growth and / or metastasis; (a3) ​​inhibiting tumor cell proliferation; (a4) preventing tumor cell cycle progression.

3. Use of a substance that can upregulate the abundance of CPEB4 protein in an organism in the preparation of a tumor therapeutic drug; the functions of the drug are as follows (a1) and / or (a2) and / or (a3) ​​and / or (a4): (a1) treating tumors; (a2) inhibiting tumor growth and / or metastasis; (a3) ​​inhibiting tumor cell proliferation; (a4) preventing tumor cell cycle progression.

4. Use of a substance that can upregulate the abundance of the CPEB4 gene in an organism in the preparation of a tumor therapeutic drug; the functions of the drug are as follows (a1) and / or (a2) and / or (a3) ​​and / or (a4): (a1) treating tumors; (a2) inhibiting tumor growth and / or metastasis; (a3) ​​inhibiting tumor cell proliferation; (a4) preventing tumor cell cycle progression.

5. A tumor therapeutic drug, which achieves the following functions (a1) and / or (a2) and / or (a3) ​​and / or (a4) by upregulating the abundance of CPEB4 protein or CPEB4 gene in an organism, degrading the mRNAs of cell cycle promoting genes in tumor cells, and thereby preventing the progression of the tumor cell cycle: (a1) treating tumors; (a2) inhibiting tumor growth and / or metastasis; (a3) ​​inhibiting tumor cell proliferation; (a4) preventing the progression of the tumor cell cycle.

6. A truncated form of CPEB4 protein, namely the RRM1 functional region of CPEB4 protein.

7. A gene encoding the truncated form of the CPEB4 protein according to claim 6.

8. Use of the CPEB4 protein truncate according to claim 6 in the preparation of a tumor therapeutic drug; the functions of the drug are as follows (a1) and / or (a2) and / or (a3) ​​and / or (a4): (a1) treating tumors; (a2) inhibiting tumor growth and / or metastasis; (a3) ​​inhibiting tumor cell proliferation; (a4) preventing tumor cell cycle progression.

9. Use of a gene encoding the CPEB4 protein truncation according to claim 6 or a biological material having the gene encoding the CPEB4 protein truncation according to claim 6 in the preparation of a tumor therapeutic drug; the functions of the drug are as follows (a1) and / or (a2) and / or (a3) ​​and / or (a4): (a1) treating tumors; (a2) inhibiting tumor growth and / or metastasis; (a3) ​​inhibiting tumor cell proliferation; (a4) preventing tumor cell cycle progression.

10. A tumor therapeutic drug, which can upregulate the abundance of the CPEB4 protein truncation according to claim 6 in an organism or upregulate the abundance of the gene encoding the CPEB4 protein truncation according to claim 6 in an organism, degrade the mRNAs of cell cycle promoting genes in tumor cells through the CPEB4 protein truncation, thereby preventing the progression of the tumor cell cycle, thereby achieving the following functions (a1) and / or (a2) and / or (a3) ​​and / or (a4): (a1) treating tumors; (a2) inhibiting tumor growth and / or metastasis; (a3) ​​inhibiting tumor cell proliferation; (a4) preventing the progression of the tumor cell cycle.

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