Application of ZCCHC24 protein and its encoding gene in the preparation of anti-tumor drugs and tumor therapeutic drugs

Abstract

The present invention relates to a new use of ZCCHC24 protein and ZCCHC24 gene in tumor treatment. Specifically, the present invention relates to a new role of ZCCHC24 protein and ZCCHC24 gene in preparing tumor treatment drugs. The present invention has important theoretical value for the study of the mechanism of tumor cell proliferation, tumor cell cycle progression, tumor growth and metastasis. The present 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 ZCCHC24 protein and ZCCHC24 gene. More specifically, the present invention relates to a new use of ZCCHC24 protein and ZCCHC24 gene in preparing tumor therapeutic drugs and a new use of ZCCHC24 protein and ZCCHC24 gene as a target molecule in the research and development of anti-tumor drugs. Background Art

[0002] One of the main characteristics of cancer is unlimited proliferation, and uncontrolled cell cycle progression is one of the main causes of its formation (Xiong Y, et al. 2018). Therefore, in-depth research on the mechanism of uncontrolled tumor cell cycle and the development of targeted therapeutic drugs still have important scientific significance and application value. Although existing studies have increased people's understanding of the regulation of cell cycle progression, the post-transcriptional regulation mechanism 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 regulation of cell cycle-related gene expression is a fundamental cause of uncontrolled cancer cell cycle progression (Fischer M 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 restricted 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 (Yu D et al., 2006; Leonard JP et al., 2012; Sheppard KE et al., 2013; Sawai CM et al., 2012). Therefore, drugs targeting cell cycle signaling pathways in clinical practice mainly prevent cancer cell cycle progression and proliferation by inhibiting cell cycle-promoting gene expression or its molecular activity.

[0004] ZCCHC24 (Zinc finger CCHC domain-containing protein 24), an RNA binding protein encoded by the ZCCHC24 gene. To date, the role of the ZCCHC24 molecule in the progression of human cancer is still unclear. The inventors first discovered that the molecule and gene have the function of inhibiting breast cancer cell proliferation and preventing cell cycle progression, and can inhibit breast tumor metastasis and growth in vivo. Specifically, the inventors found that ZCCHC24 can bind to and degrade mRNAs of genes such as cell cycle promoting genes CDK2, CDK4, CCNE1 and CDKN3, and inhibit their expression. ZCCHC24 can prevent tumor cells from G1 / S phase transition. In addition, the inventors further found that the zinc finger structure functional region of ZCCHC24 is critical for specific binding to the mRNA of cell cycle promoting genes, and this functional region can inhibit tumor cell cycle progression. Importantly, by analyzing the human breast cancer sample database, it was found that the expression of ZCCHC24 was significantly positively correlated with patient survival. In summary, the inventors first identified ZCCHC24 as a tumor suppressor gene of human breast cancer to play an anti-cancer function. Summary of the invention

[0005] The purpose of the present invention is to provide a new use of ZCCHC24 protein and ZCCHC24 gene. More specifically, the present invention relates to a new use of ZCCHC24 protein and ZCCHC24 gene in the preparation of tumor therapeutic drugs and tumor therapeutic drugs.

[0006] The present invention firstly protects the application of ZCCHC24 protein in preparing tumor therapeutic drugs.

[0007] The present invention also protects the use of the ZCCHC24 gene or a biological material having the ZCCHC24 gene in preparing tumor therapeutic drugs.

[0008] The present invention also protects the use of a substance that can increase the abundance of ZCCHC24 protein in an organism in the preparation of a tumor treatment drug. The substance that can increase the abundance of ZCCHC24 protein in an organism can be the ZCCHC24 protein itself, or other proteins located upstream of the ZCCHC24 protein in the organism that can promote the production of the ZCCHC24 protein, or other proteins located downstream of the ZCCHC24 protein in the organism that can reduce the degradation of the ZCCHC24 protein, or compounds or other small molecules that can promote the increase of the ZCCHC24 protein level in the organism.

[0009] The present invention also protects the use of a substance that can increase the abundance of the ZCCHC24 gene in an organism in the preparation of a tumor treatment drug. The substance that can increase the abundance of the ZCCHC24 gene in an organism can be the ZCCHC24 gene itself, or other proteins or nucleic acid molecules located upstream of the ZCCHC24 gene in an organism that can promote the expression of the ZCCHC24 gene, or other proteins or nucleic acid molecules located downstream of the ZCCHC24 gene in an organism that can reduce the degradation of the ZCCHC24 gene, or compounds or other small molecules that can promote the expression of the ZCCHC24 gene in an organism.

[0010] The present invention also protects the use of ZCCHC24 protein or ZCCHC24 gene as a target molecule in the development of anti-tumor drugs. ZCCHC24 protein as a target molecule can specifically be ZCCHC24 protein as a target molecule that is upregulated. Substances that can upregulate ZCCHC24 protein in an organism can be ZCCHC24 protein itself, or other proteins located upstream of ZCCHC24 protein in an organism that can promote the production of ZCCHC24 protein, or other proteins located downstream of ZCCHC24 protein in an organism that can reduce the degradation of ZCCHC24 protein, or compounds or other small molecules that can promote the increase of ZCCHC24 protein levels in an organism. ZCCHC24 gene as a target molecule can specifically be ZCCHC24 gene as a target molecule that is upregulated. The substance that can up-regulate the ZCCHC24 gene in an organism can be the ZCCHC24 gene itself, or it can be other proteins or nucleic acid molecules located upstream of the ZCCHC24 gene in the organism that can promote the expression of the ZCCHC24 gene, or it can be other proteins or nucleic acid molecules located downstream of the ZCCHC24 gene in the organism that can reduce the degradation of the ZCCHC24 gene, or it can be a compound or other small molecule that can promote the expression of the ZCCHC24 gene in an organism.

[0011] The present invention also protects a ZCCHC24 protein truncation, namely the ZF (ZnicFinger, zinc finger structure) functional region of the ZCCHC24 protein.

[0012] The ZF functional region of ZCCHC24 protein is the amino acid residues 132-149 of ZCCHC24 protein.

[0013] The gene encoding the ZCCHC24 protein truncation also falls within the protection scope of the present invention.

[0014] The present invention also protects the use of ZCCHC24 protein truncations in the preparation of tumor therapeutic drugs.

[0015] The present invention also protects the use of a gene encoding the ZCCHC24 protein truncation or a biological material having a gene encoding the ZCCHC24 protein truncation in the preparation of tumor therapeutic drugs.

[0016] The present invention also protects the use of ZCCHC24 protein truncation or the gene encoding the ZCCHC24 protein truncation as a target in the development of anti-tumor drugs. ZCCHC24 protein truncation as a target molecule can be specifically upregulated as a target molecule. The substance that can upregulate the ZCCHC24 protein truncation in an organism can be the ZCCHC24 protein truncation itself, or other proteins, peptides, compounds or other small molecules that can promote the increase of the level of ZCCHC24 protein truncation in an organism. The gene encoding the ZCCHC24 protein truncation as a target molecule can be specifically upregulated as a target molecule. The substance that can upregulate the gene encoding the ZCCHC24 protein truncation in an organism can be the gene encoding the ZCCHC24 protein truncation itself, or other proteins, polypeptides, nucleic acid molecules, compounds or other small molecules that promote the expression of the gene encoding the ZCCHC24 protein truncation.

[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 (G1 / S phase progression).

[0018] Exemplarily, any of the above-mentioned ZCCHC24 proteins may be human ZCCHC24 proteins.

[0019] Any of the above ZCCHC24 proteins may specifically be as follows (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 ZCCHC24 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 ZCCHC24 gene is a gene encoding the ZCCHC24 protein or the ZCCHC24 protein.

[0029] Any of the above ZCCHC24 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 ZCCHC24 gene may also be a homologous gene in other species. The other species include but are not limited to mice, rats, rabbits, dogs, monkeys, gorillas, apes, cattle, sheep, pigs, horses, sheep, goats, cats, etc.

[0034] The truncated form of the ZCCHC24 protein may specifically be as follows (d1), (d2), (d3) or (d4):

[0035] (d1) the protein represented by amino acid residues 205-257 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 ZCCHC24 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 the ZCCHC24 protein truncation may specifically be as follows (e1) or (e2) or (e3):

[0042] (e1) a DNA molecule encoding a truncated ZCCHC24 protein in the DNA molecule shown in Sequence 2 in the sequence list;

[0043] (e2) a DNA molecule derived from humans and having more than 95% identity with (e1) and encoding the ZCCHC24 protein truncation;

[0044] (e3) A DNA molecule that hybridizes with the nucleotide sequence defined in (e1) under stringent conditions and encodes the ZCCHC24 protein truncation.

[0045] The gene encoding the ZCCHC24 protein truncation 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 ZCCHC24 gene may be an expression vector having the ZCCHC24 gene. Any of the above-mentioned biological materials having a gene encoding a truncate of the ZCCHC24 protein may be an expression vector having a gene encoding a truncate of the ZCCHC24 protein. The expression vector can carry a nucleotide sequence, integrate this sequence into the cell genome, and 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) currently applicable to 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 the ZCCHC24 protein has a significant tumor suppressor function and can induce tumor cell cycle arrest. Its mechanism is to specifically target and bind to cell cycle promoting gene transcripts (including CDK2, CDK4, CCNE1, CDKN3, etc.) and inhibit their expression. The inventors of the present invention have found that the expression of the ZCCHC24 gene is suppressed in breast cancer tissues and cells, which may be conducive to tumor cells escaping cell cycle regulation. Overexpression of the ZCCHC24 gene can block the G1 / S progression of the breast cancer cell cycle. Consistent with these in vitro findings, overexpression of the ZCCHC24 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 ZCCHC24 are strongly associated with poor survival of breast cancer patients. In addition, the expression level of the ZCCHC24 gene in cancer patient tissues is significantly negatively correlated with the expression levels of its target genes CDK2, CDK4, CCNE1, CDKN3, and MCM2 and other cell cycle-related genes. These results indicate that ZCCHC24 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 ZCCHC24 to improve the clinical tumor treatment effect has very important application prospects.

[0050] The present invention identifies for the first time that the human ZCCHC24 gene is a tumor cell cycle inhibitory gene, and overexpression of the ZCCHC24 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 ZCCHC24 is expressed at a low level 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 ZCCHC24 in 1085 breast cancer tumor tissues was significantly lower than that in normal tissues (291 cases) ( Figure 1 A); TCGA database analysis found that the expression level of ZCCHC24 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 the expression level of ZCCHC24 was significantly positively correlated with patient survival ( Figure 1 C); Overexpression of ZCCHC24 gene significantly inhibited tumor cell proliferation ( Figure 1 D and Figure 1 E); Overexpression of ZCCHC24 gene prevents G1 / S phase transition of tumor cells ( Figure 1 F); Western blotting results showed that overexpression of the ZCCHC24 gene could significantly increase the expression of p21 and slightly increase the expression of Cleaved-PARP1 and -Caspase3, indicating that ZCCHC24 could significantly inhibit cell cycle progression but did not significantly affect cell apoptosis.

[0052] Figure 2 ZCCHC24 inhibits the expression of cell cycle-promoting genes by targeting 3'UTRs. After overexpression of the ZCCHC24 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); ZCCHC24 protein can target and bind to 3'UTRs of cell cycle promoting genes ( Figure 2 F and Figure 2 G).

[0053] Figure 3 The ZF functional region of ZCCHC24 is responsible for degrading the mRNAs of cell cycle-promoting genes. Overexpression of the ZCCHC24 gene significantly reduces the half-life of the mRNAs of cell cycle-promoting genes ( Figure 3 A); Schematic diagram of the ZCCHC24 functional region and the preparation strategies of different mutants as well as the crystal structure of the ZF functional region ( Figure 3 B); Immunoblotting method to verify the expression of different truncations ( Figure 3 C); After overexpression of the T2 truncated gene (shown as aa 132-241 in the figure), the mRNAs of cell cycle-promoting genes were downregulated ( Figure 3 D); Overexpression of the T2 truncated gene prevents tumor cells from progressing through the G1 / S phase of the cycle ( Figure 3 E).

[0054] Figure 4 Overexpression of the ZCCHC24 gene in vivo inhibits tumor growth and metastasis. Overexpression of the ZCCHC24 gene significantly inhibits tumor growth in nude mice ( Figure 4 A). Recombinant adenovirus overexpressing the ZCCHC24 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 ZCCHC24 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: The expression of ZCCHC24 in human breast cancer tissues and its correlation with patient prognosis were detected, and the new function of ZCCHC24 in preventing tumor cell cycle progression was identified.

[0058] 1. By analyzing the TCGA database, it was found that ZCCHC24 was expressed at a low level in human breast cancer tissue samples, and its low expression was significantly associated with poor prognosis of patients. The ZCCHC24 gene (ZCCHC24 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-ZCCHC24-GFP. The recombinant plasmid pEGFP-N1-ZCCHC24-GFP has been sequenced and verified. In the recombinant plasmid pEGFP-N1-ZCCHC24-GFP, the inserted DNA molecule forms a fusion gene with the EGFP gene in the vector to express the ZCCHC24-EGFP fusion protein (referred to as T10-GFP fusion protein).

[0059] 2. Introduce the recombinant plasmid pEGFP-N1-ZCCHC24-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-ZCCHC24-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 / ZCCHC24-GFP cells (marked as ZCCHC24 / 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 ZCCHC24 gene on cell proliferation. The results are shown in Figure 1 D on.

[0065] 8. Take MCF7 cells (marked as MCF7 / Parental in the figure), MCF7 / GFP cells (marked as Empty control in the figure) and MCF7 / ZCCHC24-GFP cells (marked as ZCCHC24 / 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 ZCCHC24 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 / ZCCHC24-GFP cells (marked as ZCCHC24 / GFP in the figure) and perform the following steps respectively: culture under parallel conditions, perform MTT staining at different culture times (0h, 24h, 48h, 72h) to detect the effect of overexpression of ZCCHC24 gene on cell proliferation. The results are shown in Figure 1 On E.

[0067] 10. Take MCF7 cells (marked as MCF7 / Parental in the figure), MCF7 / GFP cells (marked as Empty control in the figure) and MCF7 / ZCCHC24-GFP cells (marked as ZCCHC24 / GFP in the figure) and perform the following steps respectively: culture under parallel conditions, perform MTT staining at different culture times (0h, 24h, 48h, 72h) to detect the effect of overexpression of ZCCHC24 gene on cell proliferation. The results are shown in Figure 1 E below.

[0068] 11. Take MDA-MB-468 / ZCCHC24-GFP cells (marked as ZCCHC24-GFP in the figure) and MDA-MB-468 / GFP cells (marked 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 / ZCCHC24-GFP cells (marked as ZCCHC24-GFP in the figure) and MCF7 / GFP cells (marked 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 / ZCCHC24-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 detect the expression of cell cycle inhibitor p21 and apoptosis indicator proteins Cleaved-PARP1 and -Caspase3 by different antibodies. The results are shown in Figure 1 G.

[0071] Figure 1 The results showed that the expression of ZCCHC24 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 ZCCHC24 gene significantly inhibited tumor cell proliferation ( Figure 1 D. Figure 1E); Overexpression of ZCCHC24 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 ZCCHC24 gene to increase the level of ZCCHC24 protein can inhibit tumor cell proliferation and prevent tumor cell cycle progression, that is, overexpression of the ZCCHC24 gene to increase the level of ZCCHC24 protein has the effect of treating tumors. The above functions of the ZCCHC24 protein / ZCCHC24 gene were discovered for the first time by the inventors of the present invention.

[0073] Example 2: ZCCHC24 can degrade cell cycle promoting gene mRNAs

[0074] 1. ZCCHC24 can degrade cell cycle-promoting gene mRNAs

[0075] In order to further detect the mechanism by which ZCCHC24 protein inhibits tumor cell proliferation and prevents cell cycle progression, genes related to regulating tumor cell cycle were detected.

[0076] 1. Take the MDA-MB-468 / ZCCHC24-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 / ZCCHC24-GFP cells in Example 1, culture 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 / ZCCHC24-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 C.

[0079] 3. Take the MCF7 / ZCCHC24-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 / ZCCHC24-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 2The 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 2 The lane corresponding to Con in E.

[0081] 5. After cloning the 3'UTRs sequences of CDK2, CDK4, CCNE1 and CDKN3 genes into the luciferase gene of the luciferase reporter vector pGL3 by PCR, they formed fusion genes with the luciferase gene and prepared luciferase reporters containing different 3'UTR sequences. See the schematic diagram of the prepared reporter vector for details. 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-ZCCHC24-GFP (labeled as ZCCHC24 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 CDKN3 gene 3'UTR.

[0084] 8. Take MDA-MB-468 / ZCCHC24-GFP cells (marked as ZCCHC24-GFP in the figure) and MDA-MB-468 / GFP cells (marked 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 ZCCHC24 gene, the half-life of cell cycle promoting gene mRNAs was reduced ( Figure 2 A). This indicates that ZCCHC24 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] Through the above detection, the inventors discovered for the first time a new function of the ZCCHC24 protein, namely, it can degrade the mRNAs of cell cycle-promoting genes in tumor cells to downregulate their expression, thereby preventing the progression of the tumor cell cycle. This new function explains the essential reason why overexpression of the ZCCHC24 gene can prevent tumor cell proliferation.

[0087] 2. The ZF domain of the ZCCHC24 protein is responsible for preventing tumor cell cycle progression

[0088] Schematic diagram of the domain structure of ZCCHC24 protein is shown in Figure 3 B. Amino acid residues 132-149 are the ZF 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 in the amino acid residues 1-131 in sequence 1 of the sequence table.

[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 132-241 amino acid residues in sequence 1 of the sequence table. It can be understood by those skilled in the art that the sequence of truncation 2 is not completely consistent with the ZF functional region of the ZCCHC24 protein, but only the ZF functional region exists in truncation 2, and other regions cannot constitute other functional regions.

[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 / ZCCHC24-GFP cells in Example 1 (marked as WT in the figure), take the MDA-MB-468 / T1-GFP cells (marked as 1-131 in the figure) and MDA-MB-468 / T2-GFP cells (marked as 132-241 in the figure) prepared in step 2, and perform the following steps: culture for 72 hours under parallel conditions, perform western blot, and detect the expression of each target protein. The results are shown in Figure 3 The corresponding lane in C.

[0094] 4. Take the MDA-MB-468 / ZCCHC24-GFP cells in Example 1 (labeled as ZCCHC24-GFP in the figure), take the MDA-MB-468 / T1-GFP cells (labeled as aa 1-131 in the figure) and MDA-MB-468 / T2-GFP cells (labeled as aa 132-241 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 D.

[0095] 4. Take the MDA-MB-468 / GFP cells in Example 1 (marked as EmptyControl in the figure), take the MDA-MB-468 / T1-GFP cells (marked as aa1-131 in the figure) and MDA-MB-468 / T2-GFP cells (marked as aa 132-241 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 cell ratio in each cell cycle. The results are shown in Figure 3 E.

[0096] The results showed that after overexpression of the T2 truncated gene, the mRNAs of cell cycle-promoting genes were downregulated ( Figure 3 D); Cell function detection found that overexpression of the T2 truncated gene prevented the progression of tumor cells into the G1 / S phase of the cycle ( Figure 3 E).

[0097] The above results indicate that the ZF functional region of ZCCHC24 protein plays an important role in degrading cell cycle promoting genes.

[0098] Example 3: Overexpression of the ZCCHC24 gene (increasing the level of ZCCHC24 protein) inhibits tumor growth and metastasis

[0099] 1. Take the MDA-MB-468 / ZCCHC24-GFP cells (labeled as ZCCHC24-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 (average value of 6 mice).

[0100] 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 ZCCHC24 gene (the ZCCHC24 gene is shown in Sequence 2 of the sequence table, and the recombinant adenovirus expressing the ZCCHC24 gene is represented by Ad-ZCCHC24) or the control adenovirus (represented by Ad-Control, the difference between the control adenovirus and the recombinant adenovirus expressing the ZCCHC24 gene is that the control adenovirus does not have the ZCCHC24 gene). The in situ tumor volume was measured every day from the 24th day to the 60th day, and the changes in tumor volume over time are 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 breast cancer database found that the expression level of ZCCHC24 in human breast cancer tissue was significantly negatively correlated with the expression of cell cycle promoting genes. The results showed Figure 4 E.

[0101] The results showed that ZCCHC24 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 above results show that overexpression of the ZCCHC24 gene (increasing the level of ZCCHC24 protein) in vivo can significantly inhibit tumor growth and tumor metastasis.

[0102] 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. Application of ZCCHC24 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 ZCCHC24 gene or a biomaterial having the ZCCHC24 gene 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.

3. Use of a substance that can upregulate the abundance of ZCCHC24 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 ZCCHC24 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 can upregulate the abundance of ZCCHC24 protein in an organism or upregulate the abundance of ZCCHC24 gene in an organism, degrade the mRNAs of cell cycle promoting genes in tumor cells through ZCCHC24 protein, and then prevent the progression of 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 tumor cell cycle.

6. A ZCCHC24 protein truncation, namely the ZF functional region of the ZCCHC24 protein.

7. A gene encoding the ZCCHC24 protein truncate according to claim 6.

8. Use of ZCCHC24 protein truncations 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.

9. Use of a gene encoding the ZCCHC24 protein truncation according to claim 6 or a biomaterial having the gene encoding the ZCCHC24 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 ZCCHC24 protein truncations in an organism or upregulate the abundance of a gene encoding the ZCCHC24 protein truncations according to claim 6 in an organism, and degrade mRNAs of cell cycle-promoting genes in tumor cells through ZCCHC24 protein truncations, thereby preventing tumor cell cycle progression, 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 tumor cell cycle progression.

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