Application of N4BP1 protein and coding gene thereof in preparation of tumor treatment medicine, and tumor treatment medicine

Abstract

The invention relates to a novel application of an N4BP1 protein and an N4BP1 gene in tumor treatment. Specifically, the invention relates to a new effect of N4BP1 protein and N4BP1 gene in preparation of a tumor treatment drug and the tumor treatment drug. The invention has important theoretical value for mechanism research on inhibition 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 N4BP1 protein and N4BP1 gene. More specifically, the present invention relates to a new use of N4BP1 protein and N4BP1 gene in preparing tumor therapeutic drugs and tumor therapeutic drugs. Background Art

[0002] One of the common characteristics of cancer is the uncontrolled cell cycle progression, which leads to unlimited proliferation of cancer cells (Xiong Y, et al. 2018), which is also one of the reasons why tumor cells resist traditional treatments. Although many studies have increased 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 regulation of molecules such as RNA binding proteins, and its mechanism are still 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 uncontrollability 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 (Orega SM et al., 2002; Yu D et al., 2006; Leonard JP et al., 2012; Sheppard KE et al., 2013; Sawai CM et al., 2012). Therefore, clinically, targeted drugs are mainly used to inhibit the expression of cell cycle-promoting genes or their molecular activities to prevent the uncontrolled cell cycle progression and proliferation of cancer cells.

[0004] N4BP1, an RNA-binding protein encoded by the N4BP1 gene, remains unclear as to the role of N4BP1 in the progression of human cancer. Summary of the invention

[0005] The purpose of the present invention is to provide a new use of N4BP1 protein and N4BP1 gene. More specifically, the present invention relates to a new use of N4BP1 protein and N4BP1 gene in the preparation of tumor therapeutic drugs and tumor therapeutic drugs.

[0006] The present invention firstly protects the application of N4BP1 protein in preparing tumor therapeutic drugs.

[0007] The present invention also protects the use of the N4BP1 gene or a biological material having the N4BP1 gene in preparing tumor therapeutic drugs.

[0008] The present invention also protects the use of a substance that can increase the abundance of N4BP1 protein in an organism in the preparation of a tumor treatment drug. The substance that can increase the abundance of N4BP1 protein in an organism can be the N4BP1 protein itself, or other proteins located upstream of the N4BP1 protein in the organism that can promote the production of the N4BP1 protein, or other proteins located downstream of the N4BP1 protein in the organism that can reduce the degradation of the N4BP1 protein, or compounds or other small molecules that can promote the increase of the level of the N4BP1 protein in the organism.

[0009] The present invention also protects the use of a substance that can increase the abundance of N4BP1 gene in an organism in the preparation of a tumor treatment drug. The substance that can increase the abundance of N4BP1 gene in an organism can be the N4BP1 gene itself, or other proteins or nucleic acid molecules located upstream of the N4BP1 gene in an organism that can promote the expression of the N4BP1 gene, or other proteins or nucleic acid molecules located downstream of the N4BP1 gene in an organism that can reduce the degradation of the N4BP1 gene, or compounds or other small molecules that can promote the expression of the N4BP1 gene in an organism.

[0010] The present invention also protects a tumor treatment drug, which degrades mRNAs of cell cycle promoting genes in tumor cells by upregulating the abundance of N4BP1 protein or upregulating the abundance of N4BP1 gene in the organism, thereby preventing the progression of tumor cell cycle.

[0011] Substances that can up-regulate the N4BP1 protein in an organism can be the N4BP1 protein itself, or other proteins located upstream of the N4BP1 protein in an organism that can promote the production of the N4BP1 protein, or other proteins located downstream of the N4BP1 protein in an organism that can reduce the degradation of the N4BP1 protein, or compounds or other small molecules that can promote the increase of the level of the N4BP1 protein in an organism. Substances that can up-regulate the N4BP1 gene in an organism can be the N4BP1 gene itself, or other proteins or nucleic acid molecules located upstream of the N4BP1 gene in an organism that can promote the expression of the N4BP1 gene, or other proteins or nucleic acid molecules located downstream of the N4BP1 gene in an organism that can reduce the degradation of the N4BP1 gene, or compounds or other small molecules that can promote the expression of the N4BP1 gene in an organism.

[0012] The present invention also protects a truncated N4BP1 protein, namely the RNase functional region of the N4BP1 protein.

[0013] The RNase functional region of N4BP1 protein is the amino acid residues 617-769 of N4BP1 protein.

[0014] The gene encoding the N4BP1 protein truncation also falls within the protection scope of the present invention.

[0015] The present invention also protects the use of N4BP1 protein truncations in the preparation of tumor therapeutic drugs.

[0016] The present invention also protects the use of a gene encoding the N4BP1 protein truncation or a biological material having a gene encoding the N4BP1 protein truncation in the preparation of tumor therapeutic drugs.

[0017] The present invention also protects a tumor treatment drug, which degrades mRNAs of cell cycle promoting genes in tumor cells by upregulating the abundance of N4BP1 protein truncations in an organism or upregulating the abundance of genes encoding the N4BP1 protein truncations in an organism, thereby preventing tumor cell cycle progression.

[0018] The substance that can up-regulate the N4BP1 protein truncation in an organism can be the N4BP1 protein truncation itself, or other proteins, peptides, compounds or other small molecules that can promote the increase of the level of the N4BP1 protein truncation in an organism. The substance that can up-regulate the gene encoding the N4BP1 protein truncation in an organism can be the gene encoding the N4BP1 protein truncation itself, or other proteins, polypeptides, nucleic acid molecules, compounds or other small molecules that promote the expression of the gene encoding the N4BP1 protein truncation.

[0019] 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.

[0020] Exemplarily, any of the above-mentioned N4BP1 proteins may be human N4BP1 proteins.

[0021] Any of the above N4BP1 proteins may specifically be the following (b1), (b2), (b3) or (b4):

[0022] (b1) the protein shown in Sequence 1 in the sequence listing;

[0023] (b2) a fusion protein obtained by connecting a tag to the amino terminus or carboxyl terminus of (b1);

[0024] (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);

[0025] (b4) A protein derived from humans, having 98% or more identity with (b1) and having any one of the functions of (a1) to (a4).

[0026] The specific labels are shown in Table 1.

[0027] Table 1 Tag sequences

[0028]

[0029]

[0030] The N4BP1 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.

[0031] The N4BP1 gene is a gene encoding the N4BP1 protein or the N4BP1 protein.

[0032] Any of the above N4BP1 genes may specifically be as follows (c1) or (c2) or (c3):

[0033] (c1) a DNA molecule whose coding region is shown in Sequence 2 in the sequence listing;

[0034] (c2) a DNA molecule derived from humans and having 95% or more identity with (c1) and encoding the protein;

[0035] (c3) A DNA molecule that hybridizes to the nucleotide sequence defined in (c1) under stringent conditions and encodes the protein.

[0036] The N4BP1 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.

[0037] The N4BP1 protein truncation may specifically be as follows (d1), (d2), (d3) or (d4):

[0038] (d1) the protein represented by amino acid residues 205-257 in SEQ ID NO: 1 of the sequence listing;

[0039] (d2) a fusion protein obtained by connecting a tag to the amino terminus or carboxyl terminus of (d1);

[0040] (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);

[0041] (d4) A protein derived from humans, having 98% or more identity with (d1) and having any one of the functions of (a1) to (a4).

[0042] The specific labels are shown in Table 1.

[0043] The N4BP1 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.

[0044] Any of the above genes encoding N4BP1 protein truncations may specifically be as follows (e1) or (e2) or (e3):

[0045] (e1) a DNA molecule encoding a truncated N4BP1 protein in the DNA molecule shown in Sequence 2 in the sequence listing;

[0046] (e2) a DNA molecule derived from humans and having more than 95% identity with (e1) and encoding the N4BP1 protein truncation;

[0047] (e3) A DNA molecule that hybridizes with the nucleotide sequence defined in (e1) under stringent conditions and encodes the N4BP1 protein truncation.

[0048] The gene encoding the truncate N4BP1 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.

[0049] Any of the above-mentioned biological materials having the N4BP1 gene may be an expression vector having the N4BP1 gene. Any of the above-mentioned biological materials having a gene encoding a truncate of the N4BP1 protein may be an expression vector having a gene encoding a truncate of the N4BP1 protein. The expression vector is capable of carrying a nucleotide sequence, integrating this sequence into the cell genome, and being able to replicate in the cell. "Expression vectors" include plasmids, cosmids, viruses (bacteriophages, animal viruses, plant viruses, etc.) and artificial chromosomes (such as YACs). The viruses (also called viral vectors) that may currently 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.

[0050] Any of the above mentioned tumors include but are not limited to breast cancer.

[0051] Any of the above mentioned tumor cells include but are not limited to breast cancer cells.

[0052] The inventors of the present invention have found that the N4BP1 protein has a significant tumor suppressor function and can induce tumor cell cycle arrest. Its mechanism is to specifically inhibit the expression of cell cycle promoting gene transcripts (including CDK4, E2F1, CDKN3, MCM2, etc.). The inventors of the present invention have found that N4BP1 gene expression is inhibited in breast cancer tissues and cells, which may help tumor cells escape cell cycle regulation. Overexpression of the N4BP1 gene can block the G1 / S progression of breast cancer cell cycle. Consistent with these in vitro observations, overexpression of the N4BP1 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 N4BP1 are strongly associated with poor survival of breast cancer patients. In addition, the expression level of the N4BP1 gene in cancer patient tissues is significantly negatively correlated with the expression levels of its target genes CDK4, E2F1, CDKN3, MCM2 and CDC25A. These results indicate that N4BP1 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 N4BP1 to improve the clinical tumor treatment effect has very important application prospects.

[0053] The present invention identifies for the first time that the human N4BP1 gene is a tumor cell cycle inhibitory gene, and overexpression of the N4BP1 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

[0054] Figure 1 N4BP1 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 N4BP1 in 1097 primary breast cancer tumors was significantly lower than that in normal tissues (114 cases) ( Figure 1 A); TCGA database analysis found that the expression level of N4BP1 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 N4BP1 was significantly positively correlated with patient survival ( Figure 1 C); Overexpression of N4BP1 gene significantly inhibited tumor cell proliferation ( Figure 1 D and Figure 1 E); Overexpression of N4BP1 gene prevents G1 / S phase transition of tumor cells ( Figure 1 F); Western blot results showed that overexpression of N4BP1 gene could effectively increase p21 expression and slightly increase Cleaved-PARP1 and -Caspase3 expression, indicating that N4BP1 could significantly inhibit cell cycle progression but did not significantly affect cell apoptosis ( Figure 1 G).

[0055] Figure 2 N4BP1 specifically inhibits the expression of cell cycle-promoting genes by targeting 3'UTRs. After overexpression of the N4BP1 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); N4BP1 protein can target and bind to 3'UTRs of cell cycle-promoting genes ( Figure 2 F and Figure 2 G).

[0056] Figure 3 The RNase functional domain of N4BP1 degrades the mRNAs of cell cycle-promoting genes. Overexpression of the N4BP1 gene significantly reduces the half-life of the mRNAs of cell cycle-promoting genes ( Figure 3A); Schematic diagram of the N4BP1 functional region and the preparation strategies of different mutants as well as the crystal structure of the RNase functional region ( Figure 3 B); After overexpression of the T2 truncated gene (shown as aa 616-896 in the figure), the mRNAs of cell cycle-promoting genes were downregulated ( Figure 3 C); Overexpression of the T2 truncated gene prevents tumor cells from progressing through the G1 / S phase of the cycle ( Figure 3 D).

[0057] Figure 4 Overexpression of the N4BP1 gene in vivo inhibits tumor growth and metastasis. Overexpression of the N4BP1 gene significantly inhibits tumor growth in nude mice ( Figure 4 A). Recombinant adenovirus expressing N4BP1 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 N4BP1 in breast cancer tissues was significantly negatively correlated with the expression of cell cycle promoting genes ( Figure 4 E). DETAILED DESCRIPTION

[0058] 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.

[0059] 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 .

[0060] By analyzing the TCGA database, it was found that N4BP1 was expressed at low levels in human breast cancer tissue samples, and its low expression was significantly associated with poor prognosis of patients. Figure 1A, The mRNA expression level of N4BP1 in 1097 primary breast cancer tumors was significantly lower than that in normal tissues (114 cases); TCGA database analysis found that the expression level of N4BP1 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 N4BP1 was significantly positively correlated with patient survival ( Figure 1 C).

[0061] Example 1. Expression of N4BP1 in human breast cancer tissue and identification of its association with prognosis, and identification of a new function of N4BP1 in preventing tumor cell cycle progression

[0062] 1. Insert the N4BP1 gene (the N4BP1 gene is shown in sequence 2 of the sequence table, and the terminator is removed from the inserted sequence) between the Hind III and BamH I restriction sites of the pEGFP-N1 vector to obtain the recombinant plasmid pEGFP-N1-N4BP1-GFP. The recombinant plasmid pEGFP-N1-N4BP1-GFP has been sequenced and verified. In the recombinant plasmid pEGFP-N1-N4BP1-GFP, the inserted DNA molecule forms a fusion gene with the EGFP gene in the vector to express the N4BP1-EGFP fusion protein (referred to as T10-GFP fusion protein).

[0063] 2. Introduce the recombinant plasmid pEGFP-N1-N4BP1-GFP into MDA-MB-468 cells to obtain recombinant cells, named MDA-MB-468 / T10-GFP cells.

[0064] 3. Introduce the pEGFP-N1 vector into MDA-MB-468 cells to obtain recombinant cells, named MDA-MB-468 / GFP cells.

[0065] 4. Introduce the recombinant plasmid pEGFP-N1-N4BP1-GFP into MCF7 cells to obtain recombinant cells, named MCF7 / T10-GFP cells.

[0066] 5. Introduce the pEGFP-N1 vector into MCF7 cells to obtain recombinant cells, named MCF7 / GFP cells.

[0067] 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 1Above the lanes in G, Con represents MDA-MB-468 / GFP cells.

[0068] 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 / N4BP1-GFP cells (marked as N4BP1 / 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 N4BP1 gene on cell proliferation. The results are shown in Figure 1 D on.

[0069] 8. Take MCF7 cells (marked as MCF7 / Parental in the figure), MCF7 / GFP cells (marked as Empty control in the figure) and MCF7 / N4BP1-GFP cells (marked as N4BP1 / 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 N4BP1 gene on cell proliferation. The results are shown in Figure 1 D below.

[0070] 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 / N4BP1-GFP cells (marked as N4BP1 / 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 N4BP1 gene on cell proliferation. The results are shown in Figure 1 On E.

[0071] 10. Take MCF7 cells (marked as MCF7 / Parental in the figure), MCF7 / GFP cells (marked as Empty control in the figure) and MCF7 / N4BP1-GFP cells (marked as N4BP1 / 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 N4BP1 gene on cell proliferation. The results are shown in Figure 1 E below.

[0072] 11. Take MDA-MB-468 / N4BP1-GFP cells (labeled as N4BP1-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.

[0073] 12. Take MCF7 / N4BP1-GFP cells (labeled as N4BP1-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.

[0074] 13. Take MDA-MB-468 / N4BP1-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.

[0075] Figure 1 The results showed that the expression of N4BP1 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 N4BP1 gene significantly inhibited tumor cell proliferation ( Figure 1 D and Figure 1 E); Overexpression of N4BP1 gene prevents tumor cells from progressing through the G1 / S phase of the cycle ( Figure 1 F and Figure 1 G).

[0076] The above results show that overexpression of the N4BP1 gene to increase the level of N4BP1 protein can prevent tumor cell cycle progression and inhibit tumor cell proliferation, that is, overexpression of the N4BP1 gene to increase the level of N4BP1 protein has the effect of treating tumors. The above functions of N4BP1 protein / N4BP1 gene were discovered for the first time by the inventors of the present invention.

[0077] Example 2: N4BP1 can degrade cell cycle promoting gene mRNAs

[0078] 1. N4BP1 can degrade cell cycle-promoting gene mRNAs

[0079] In order to further examine the mechanism by which N4BP1 protein inhibits tumor cell proliferation and blocks cell cycle progression, genes regulating tumor cell cycle were detected.

[0080] 1. Take the MDA-MB-468 / N4BP1-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.

[0081] 2. Take the MCF7 / N4BP1-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.

[0082] 3. Take the MDA-MB-468 / N4BP1-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.

[0083] 3. Take the MCF7 / N4BP1-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.

[0084] 4. Take MDA-MB-468 / N4BP1-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 2 The lane corresponding to Con in E.

[0085] 5. The 3'UTRs sequences of CDK4, CDKN3, E2F1 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.

[0086] 6. Luciferase reporter assay

[0087] 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-N4BP1-GFP (labeled as N4BP1 in the figure) or pEGFP-N1 vector (labeled as Control in the figure). The reporter vectors were: reporter vector with β-actin gene 3'UTR, reporter vector with CDK4 gene 3'UTR, reporter vector with CDKN3 gene 3'UTR, reporter vector with E2F1 gene 3'UTR or reporter vector with MCM2 gene 3'UTR.

[0088] 8. Take MDA-MB-468 / N4BP1-GFP cells (labeled as N4BP1-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.

[0089] These results showed that after overexpression of the N4BP1 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 2 E); N4BP1 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).

[0090] The above results indicate that the inventors have discovered for the first time a new function of the N4BP1 protein, namely, 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 N4BP1 gene can prevent tumor cell proliferation.

[0091] 2. The RNase domain of N4BP1 protein is responsible for preventing tumor cell cycle progression

[0092] The schematic diagram of the domain structure of N4BP1 protein is shown in Figure 3 B. The amino acid residues 59-143 are the KH domain; the amino acid residues 617-769 are the RNase domain.

[0093] 1. Prepare several recombinant plasmids as follows (each recombinant plasmid has been sequenced and verified):

[0094] 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 1-616 in sequence 1 of the sequence table.

[0095] 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 by the amino acid residues at positions 616-896 in sequence 1 of the sequence table. Due to the shearing site that can be recognized by gene shearing, the truncation 2 obtained by shearing is shown by the amino acid residues at positions 616-896 in sequence 1 of the sequence table, but the redundant amino acid residues of truncation 2 relative to the RNase functional region cannot form an independent functional region, and the function realized by truncation 2 is the same as that of the RNase functional region.

[0096] 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.

[0097] 3. Take the MDA-MB-468 / N4BP1-GFP cells (labeled as N4BP1-GFP in the figure) in Example 1, take the MDA-MB-468 / T1-GFP cells (labeled as aa1-616 in the figure) and MDA-MB-468 / T2-GFP cells (labeled as aa 616-896 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 3C.

[0098] 4. Take the MDA-MB-468 / N4BP1-GFP cells (labeled as N4BP1-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 1-616 in the figure) and MDA-MB-468 / T2-GFP cells (labeled as aa 616-896 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.

[0099] The results of step 2 showed that after overexpression of the T2 truncated gene, the mRNAs of cell cycle-promoting genes were downregulated ( Figure 3 C); 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 D).

[0100] The above results indicate that the RNase functional domain of N4BP1 protein plays an important role in degrading cell cycle promoting genes.

[0101] Example 3: Overexpression of the N4BP1 gene (increasing the level of N4BP1 protein) inhibits tumor growth and metastasis

[0102] 1. Take the MDA-MB-468 / N4BP1-GFP cells (labeled as N4BP1-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).

[0103] 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). 10pfu), and injected 5 times in total. Two treatment groups were set up, and the recombinant adenovirus expressing the N4BP1 gene (the N4BP1 gene is shown in Sequence 2 of the sequence table, and the recombinant adenovirus expressing the N4BP1 gene is represented by Ad-N4BP1) or the control adenovirus (represented by Ad-Control, the difference between the control adenovirus and the recombinant adenovirus expressing the N4BP1 gene is that the control adenovirus does not have the N4BP1 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 showed a significant negative correlation between the expression level of N4BP1 and the expression of cell cycle promoting genes. The results showed Figure 4 E.

[0104] The results showed that N4BP1 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 N4BP1 gene (increasing the level of N4BP1 protein) in vivo can significantly inhibit tumor growth and tumor metastasis.

[0105] 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 N4BP1 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 N4BP1 gene or a biomaterial having the N4BP1 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 N4BP1 protein in an organism 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.

4. Use of a substance that can upregulate the abundance of the N4BP1 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 N4BP1 protein or N4BP1 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 N4BP1 protein, namely the RNase functional region of the N4BP1 protein.

7. A gene encoding the N4BP1 protein truncation according to claim 6.

8. Use of N4BP1 protein truncation 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 N4BP1 protein truncation according to claim 7 or a biological material having a gene encoding the N4BP1 protein truncation according to claim 7 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 achieves the following functions (a1) and / or (a2) and / or (a3) ​​and / or (a4) by upregulating the abundance of N4BP1 protein truncation in an organism or upregulating the abundance of a gene encoding the N4BP1 protein truncation in an organism, degrading the mRNAs of cell cycle promoting genes in tumor cells, thereby preventing tumor cell cycle progression: (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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