Application of MBNL3 protein and coding gene thereof in preparation of tumor medicine and tumor treatment medicine

Abstract

The invention relates to oncology, cytobiology, genetics, molecular biology and the like. Specifically, the invention relates to application of the MBNL3 protein and the coding gene thereof in preparation of tumor drugs and the tumor treatment drugs. The amino acid sequence of the protein is as shown in SEQ ID NO. 1, and the gene sequence is as shown in SEQ ID NO. 2. The MBNL3 protein and the coding gene of the MBNL3 protein have remarkable theoretical and clinical application potentials for controlling tumor-induced angiogenesis and inhibiting tumor metastasis and growth processes.

Description

Technical Field

[0001] The present invention relates to the fields of oncology, genetics and molecular biology, and in particular, to a new use of MBNL3 protein and MBNL3 gene, and more particularly, to a new use of MBNL3 protein and MBNL3 gene in preparing tumor therapeutic drugs and tumor therapeutic drugs. Background Art

[0002] Tumor-induced angiogenesis is a key factor in the further growth of solid tumors and metastasis to distant organs (Teleanu RL, et al. 2019). However, the molecular mechanisms that regulate tumor angiogenesis are still unclear. It is known that tumor cells can activate vascular endothelial cells and pericytes by secreting a series of angiogenesis-related factors, and then form a rich vascular network in the tumor tissue (Lugano R, et al. 2020). These newly formed blood vessels help provide nutrition and promote further growth and distant metastasis of solid tumors (Singh P, et al. 2018). Therefore, a series of angiogenic factors secreted by tumor cells become a link for mutual communication between tumor cells and endothelial cells (Kumar M, et al. 2016), which control the activation of tumor angiogenesis. Under normal circumstances, the activation of tumor angiogenesis is either through increasing the expression of pro-angiogenic factor genes or reducing the expression of anti-angiogenic factor genes. The balance of angiogenesis-related factors in local tissues will determine whether angiogenesis is induced or inhibited (Gacche RN, et al. 2013). Although extensive research progress has expanded our understanding of tumor-induced angiogenesis, the post-transcriptional regulation of angiogenesis-related gene expression in tumor cells, especially the role and mechanism of RNA-binding proteins in controlling tumor angiogenesis, remains unclear.

[0003] MBNL3 (Muscleblind-like protein 3) is an RNA-binding protein encoded by the MBNL3 gene and belongs to the MBNL family (composed of MBNL1-3). To date, the role of MBNL3 molecules in the progression of human cancer is still not fully understood. Summary of the invention

[0004] The purpose of the present invention is to provide a new application of MBNL3 protein and MBNL3 gene. More specifically, the present invention relates to a new application of MBNL3 protein and MBNL3 gene in the preparation of tumor treatment drugs.

[0005] The present invention firstly protects the application of MBNL3 protein in preparing tumor therapeutic drugs.

[0006] The present invention also protects the use of the MBNL3 gene or the biological material having the MBNL3 gene in the preparation of tumor therapeutic drugs.

[0007] The present invention also protects the use of a substance that can increase the abundance of MBNL3 protein in an organism in the preparation of a tumor treatment drug. The substance that can increase the abundance of MBNL3 protein in an organism can be the MBNL3 protein itself, or other proteins located upstream of the MBNL3 protein in the organism that can promote the expression of the MBNL3 protein, or other proteins located downstream of the MBNL3 protein in the organism that can reduce the degradation of the MBNL3 protein, or compounds or other small molecules that promote the increase of the MBNL3 protein level in the organism.

[0008] The present invention also protects the use of a substance that can increase the abundance of the MBNL3 gene in an organism in the preparation of a tumor treatment drug. The substance that can increase the abundance of the MBNL3 gene in an organism can be the MBNL3 gene itself, or other proteins or nucleic acid molecules located upstream of the MBNL3 gene in an organism that can promote the expression of the MBNL3 gene; or other proteins or nucleic acid molecules located downstream of the MBNL3 gene in an organism that can reduce the degradation of the MBNL3 gene; or a compound or other small molecule that promotes the expression of the MBNL3 gene in an organism.

[0009] The present invention also protects a tumor treatment drug, which can upregulate the abundance of MBNL3 protein or MBNL3 gene in an organism. The substance that can upregulate MBNL3 protein in an organism can be the MBNL3 protein itself, or other proteins located upstream of the MBNL3 protein in the organism that can promote the production of MBNL3 protein, or other proteins located downstream of the MBNL3 protein in the organism that can reduce the degradation of MBNL3 protein, or compounds or other small molecules that promote the increase of MBNL3 protein levels in an organism. The substance that can upregulate MBNL3 gene in an organism can be the MBNL3 gene itself, or other proteins or nucleic acid molecules located upstream of the MBNL3 gene in the organism that can promote the expression of MBNL3 gene, or other proteins or nucleic acid molecules located downstream of the MBNL3 gene in the organism that can reduce the degradation of MBNL3 gene, or compounds or other small molecules that promote the expression of MBNL3 gene in an organism.

[0010] The present invention also protects a truncated form of MBNL3 protein, namely, the ZF1 and ZF2 functional regions of MBNL3 protein, and / or, the ZF3 and ZF4 functional regions of MBNL3 protein.

[0011] The ZF1 functional region of MBNL3 protein is the 14th to 42nd amino acid residues of MBNL3 protein, the ZF2 functional region is the 48th to 74th amino acid residues of MBNL3 protein, the ZF4 functional region is the 174th to 202nd amino acid residues of MBNL3 protein, and the ZF5 functional region is the 210th to 236th amino acid residues of MBNL3 protein.

[0012] The gene encoding the MBNL3 protein truncation also falls within the protection scope of the present invention.

[0013] The present invention also protects the use of MBNL3 protein truncations in the preparation of tumor therapeutic drugs.

[0014] The present invention also protects the use of a gene encoding the MBNL3 protein truncation or a biological material having a gene encoding the MBNL3 protein truncation in the preparation of tumor therapeutic drugs.

[0015] The present invention also protects a tumor treatment drug, which can upregulate the abundance of MBNL3 protein truncations or the abundance of genes encoding MBNL3 protein truncations in an organism. The substance that can upregulate MBNL3 protein truncations in an organism can be the MBNL3 protein truncations themselves, or other proteins, peptides, compounds or other small molecules that promote the increase in the level of MBNL3 protein truncations in an organism. The substance that can upregulate the gene encoding MBNL3 protein truncations in an organism can be the gene encoding MBNL3 protein truncations themselves, or other proteins, polypeptides, nucleic acid molecules, compounds or other small molecules that promote the expression of genes encoding MBNL3 protein truncations.

[0016] 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 angiogenesis; (a4) inhibiting the growth of tumor cells.

[0017] Exemplarily, any of the above-mentioned MBNL3 proteins may be human MBNL3 proteins.

[0018] Any of the above MBNL3 proteins may specifically be the following (b1), (b2), (b3) or (b4):

[0019] (b1) the protein shown in Sequence 1 in the sequence listing;

[0020] (b2) a fusion protein obtained by connecting a tag to the amino terminus or carboxyl terminus of (b1);

[0021] (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);

[0022] (b4) A protein derived from humans, having 98% or more identity with (b1) and having any one of the functions of (a1) to (a4).

[0023] The specific labels are shown in Table 1.

[0024] Table 1 Tag sequences

[0025]

[0026]

[0027] The MBNL3 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] MBNL3 gene is a gene encoding MBNL3 protein.

[0029] Any of the above MBNL3 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 MBNL3 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 MBNL3 protein truncations may specifically be as follows (d1), (d2), (d3) or (d4):

[0035] (d1) a protein comprising amino acid residues 14-42 and 48-74 of sequence 1 in the sequence listing, and / or a protein comprising amino acid residues 174-202 and 210-236 of sequence 1 in 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 MBNL3 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 MBNL3 protein truncations may specifically be as follows (e1) or (e2) or (e3):

[0042] (e1) a DNA molecule encoding a truncated form of the MBNL3 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 MBNL3 protein truncation;

[0044] (e3) A DNA molecule that hybridizes with the nucleotide sequence defined in (e1) under stringent conditions and encodes the MBNL3 protein truncated form.

[0045] The gene encoding the MBNL3 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 MBNL3 gene may be an expression vector having MBNL3 gene. Any of the above-mentioned biological materials having genes encoding MBNL3 protein truncations may be an expression vector having genes encoding MBNL3 protein truncations. 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 liver cancer.

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

[0049] The inventors of the present invention have found that MBNL3 protein has significant anti-tumor angiogenesis function and can inhibit tumor cell-induced angiogenesis. Its mechanism is to target and bind to the transcripts of pro-angiogenic genes (including but not limited to PTGS1, ID1, FGF1 and ANG, etc.) and inhibit their expression, specifically by degrading the mRNAs of pro-angiogenic factor genes in cancer cells through MBNL3 protein, thereby specifically inhibiting the expression of pro-angiogenic genes in tumor cells and inhibiting tumor cell-induced angiogenesis. The inventors of the present invention have found that MBNL3 gene expression is inhibited in liver cancer tissue, which may be beneficial to promote liver cancer angiogenesis. Overexpression of MBNL3 gene can significantly inhibit liver cancer-induced angiogenesis. Consistent with this, overexpression of MBNL3 gene in vivo can significantly inhibit angiogenesis and tumor growth of liver transplant tumors. By analyzing the human liver cancer sample database, the inventors found that low expression levels of MBNL3 were significantly associated with poor survival of liver cancer patients. Moreover, the expression level of MBNL3 gene in liver cancer patient tissues was significantly negatively correlated with the expression levels of a series of pro-angiogenic genes PTGS1, ID1, TYMP, FGF1, ENG, MMP2, IL6, etc. These results indicate that MBNL3 is a potential liver cancer angiogenesis inhibitor gene, which participates in regulating the tumor angiogenesis signaling pathway by inhibiting the expression of pro-angiogenic genes. Based on this, the use of the tumor suppressor protein MBNL3 to improve the clinical targeted angiogenesis treatment of tumors has a very important application prospect.

[0050] The present invention identifies for the first time that the human MBNL3 gene is a liver cancer suppressor gene. Overexpression of the MBNL3 gene in liver cancer cells can significantly inhibit angiogenesis in tumor tissues and inhibit tumor growth. The present invention provides theoretical support for a deeper understanding of the molecular mechanism of regulating tumor-induced angiogenesis, and at the same time provides a new molecular target for clinical anti-angiogenesis treatment of tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 MBNL3 is expressed at low levels in human liver cancer tissues and is associated with the prognosis of liver cancer patients. Analysis of the TCGA database found that the mRNA expression level of MBNL3 in 371 primary liver cancer tumors was significantly lower than that in normal tissues (50 cases) ( Figure 1 A); qPCR detection found that the expression level of MBNL3 in 24 liver cancer tissues was significantly lower than that in paired normal adjacent cancer tissues ( Figure 1 B); The results of immunohistochemical analysis of 22 liver cancer tissues and their paired adjacent adjacent tissues showed that the expression of MBNL3 protein in liver cancer tissues was significantly lower than that in normal liver tissues ( Figure 1 C); Analysis of the TCGA liver cancer database showed that the expression level of MBNL3 was significantly positively correlated with the survival of liver cancer patients ( Figure 1 D)

[0052] Figure 2 MBNL3 inhibits the expression of pro-angiogenic genes in liver cancer cells and inhibits liver cancer cell-induced angiogenesis. After overexpression of MBNL3 gene in liver cancer cells HepG2 and SMMC7721, the mRNAs of pro-angiogenic genes were downregulated, and some anti-angiogenic genes were upregulated ( Figure 2 A. Figure 2 B); Western blot and ELISA results showed that MBNL3 could downregulate the expression of angiogenic factors (including ID1, TYMP, FGF1; COX1, ANG) ( Figure 2 C. Figure 2 MBNL3 protein targets and binds to the mRNA of angiogenic factor genes ( Figure 2 D); Supernatants of different liver cancer cells overexpressing MBNL3 gene significantly inhibited endothelial cell migration ( Figure 2 E) and pipe ( Figure 2 F).

[0053] Figure 3 MBNL3 inhibits the expression of angiogenic gene mRNAs by targeting the 3'UTRs of angiogenic genes through its ZF functional region. Overexpression of MBNL3 gene significantly reduces the half-life of angiogenic gene mRNAs ( Figure 3 MBNL3 protein can target and bind to the mRNAs of angiogenic factor genes ( Figure 3B), but could not target the mRNAs of anti-angiogenic genes (THBS1, COL4A3, SERPINF1, CXCL10) ( Figure 3 B); MBNL3 targets and binds to the 3'UTR of angiogenic gene mRNA ( Figure 3 C. Figure 3 D); Schematic diagram of MBNL3 functional region and preparation strategies of different mutants and crystal structure of ZF functional region ( Figure 3 E); After overexpression of T1 truncated gene or T2 truncated gene, the mRNAs of angiogenic factor genes were downregulated, indicating that T1 truncated gene or T2 truncated gene can target and bind to the 3'UTR of angiogenic genes and degrade their mRNAs ( Figure 3 F).

[0054] Figure 4 Overexpression of MBNL3 gene in vivo inhibits angiogenesis and tumor growth in liver transplanted tumors. Recombinant adenovirus expressing MBNL3 gene can significantly inhibit tumor growth in nude mice bearing tumors ( Figure 4 A. Figure 4 B); Using recombinant adenovirus expressing MBNL3 gene can significantly inhibit angiogenesis in tumor tissue ( Figure 4 C); Compared with liver cancer tissues with low MBNL3 expression (MBNL3-negative), liver cancer tissues with high MBNL3 expression (MBNL3-Positive) had lower microvessel density ( Figure 4 D); Analysis of the clinical liver cancer database showed that the expression of MBNL3 in liver cancer tissues was significantly negatively correlated with the expression of pro-angiogenic 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. HepG2 cells (human hepatoma cells): HB-8065 TM SMMC-7721 cells (human hepatoma cells): from Shanghai Institute of Biological Sciences; human umbilical vein endothelial cells: HUVEC (Human Umbilical Vein Endothelial Cells).

[0057] Example 1: The expression of MBNL3 in human liver cancer tissues and its correlation with patient prognosis were identified, and a new function of MBNL3 in preventing angiogenesis in liver cancer was identified.

[0058] By analyzing the TCGA database, it was found that MBNL3 was expressed at a low level in human liver cancer tissue samples, and its low expression was significantly associated with poor prognosis of patients. The expression of MBNL3 in human liver cancer tissue was significantly reduced by qPCR. Immunohistochemical analysis found that the expression level of MBNL3 protein in human liver cancer tissue was significantly lower than that in normal liver tissue.

[0059] Figure 1 The results showed that the expression of MBNL3 in human hepatocellular carcinoma tissue was significantly lower than that in normal liver tissue ( Figure 1 A. Figure 1 B and Figure 1 C), and its expression level was significantly positively correlated with the survival of liver cancer patients ( Figure 1 D).

[0060] Example 2: MBNL3 can inhibit the expression of pro-angiogenic genes and suppress angiogenesis induced by liver cancer cells.

[0061] In order to detect the role and mechanism of MBNL3 protein-regulated angiogenesis in liver cancer, the expression of angiogenesis-related genes in liver cancer cells was detected.

[0062] 1. Insert the MBNL3 gene (MBNL3 gene is shown in sequence 2 of the sequence table, and the stop codon is removed from the inserted sequence) between the EcoR I and AgeI restriction sites of the pEGFP-N1 vector to obtain the recombinant plasmid pEGFP-N1-MBNL3-GFP. The recombinant plasmid pEGFP-N1-MBNL3-GFP has been sequenced and verified. In the recombinant plasmid pEGFP-N1-MBNL3-GFP, the inserted DNA molecule forms a fusion gene with the EGFP gene in the vector to express the MBNL3-EGFP fusion protein.

[0063] 2. Introduce the recombinant plasmid pEGFP-N1-MBNL3-GFP into HepG2 cells to obtain recombinant cells, named HepG2 / MBNL3-GFP cells.

[0064] 3. Introduce the pEGFP-N1 vector into HepG2 cells to obtain recombinant cells, named HepG2 / GFP cells.

[0065] 4. Introduce the recombinant plasmid pEGFP-N1-MBNL3-GFP into SMMC-7721 cells to obtain recombinant cells, named SMMC-7721 / MBNL3-GFP cells.

[0066] 5. Introduce the pEGFP-N1 vector into SMMC-7721 cells to obtain recombinant cells, named SMMC-7721 / GFP cells.

[0067] 6. After culturing HepG2 / MBNL3-GFP cells for 36 hours, total RNA was extracted and reverse transcribed into cDNA, and then RNA-seq was performed to analyze the changes in the expression of pro-angiogenic genes and anti-angiogenic genes. The results are shown in Figure 2 A on.

[0068] 7. After 36 hours of culturing SMMC-7721 / MBNL3-GFP cells, total RNA was extracted and reverse transcribed into cDNA. RNA-seq was then performed to analyze the changes in the expression of pro-angiogenic genes and anti-angiogenic genes. The results are shown in Figure 2 A down.

[0069] 8. Take HepG2 / MBNL3-GFP cells, 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 B on.

[0070] 9. Take SMMC-7721 / MBNL3-GFP cells, 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 B down.

[0071] 10. Take HepG2 / MBNL3-GFP cells (marked as HepG2 / MBNL3 in the figure) and HepG2 / GFP cells (marked as Con in the figure) and perform the following steps respectively: culture under parallel conditions, collect cell protein lysates and perform immunoblotting at different time points (24h, 72h), and detect the expression abundance of angiogenic factors ID1, TYMP, and FGF1 using different antibodies. The results are shown in Figure 2 The lanes in C correspond to the various time points.

[0072] 11. HepG2 / MBNL3-GFP cells were cultured at 0h and 72h under parallel conditions, and their culture supernatants (CM, Conditioned Medium) were collected, filtered at 1500rpm, and labeled as 0h CM and 72h CM. The protein content of COX1 and ANG in CM was detected by ELISA. The results are shown in Figure 2 D.

[0073] 12. Take the CM of HepG2 / GFP cells and HepG2 / MBNL3-GFP cells, as well as the CM of SMMC-7721 / GFP cells and SMMC-7721 / MBNL3-GFP cells, and perform the following steps respectively: treat HUVEC under parallel conditions, take photos and record the migration of endothelial cells after 24 hours to detect the effect of overexpression of MBNL3 gene on endothelial cell migration. The results are shown in Figure 2 E.

[0074] 13. Take the CM of HepG2 / GFP cells and HepG2 / MBNL3-GFP cells, as well as the CM of SMMC-7721 / GFP cells and SMMC-7721 / MBNL3-GFP cells, and perform the following steps respectively: treat HUVEC under parallel conditions, take photos and record the tube formation ability of endothelial cells after 12 hours to detect the effect of overexpression of MBNL3 gene on endothelial cell tube formation. The results are shown in Figure 2 F.

[0075] The above results show that after overexpression of MBNL3 gene, the expression of angiogenic factor genes in liver cancer cells was downregulated ( Figure 2 A. Figure 2 B. Figure 2 C. Figure 2 D), some anti-angiogenic genes were upregulated ( Figure 2 A); Overexpression of the MBNL3 gene to increase the level of MBNL3 protein can inhibit the expression of pro-angiogenic genes in liver cancer cells and inhibit liver cancer cell-induced angiogenesis, indicating that overexpression of the MBNL3 gene to increase the level of MBNL3 protein has the function of inhibiting tumor angiogenesis. The above functions of MBNL3 protein / MBNL3 gene are first discovered by the inventors of the present invention.

[0076] Example 3: MBNL3 specifically degrades pro-angiogenic gene mRNAs through its ZF functional region.

[0077] In order to further examine the mechanism by which MBNL3 protein inhibits tumor angiogenesis, the angiogenesis-related genes regulated by it were detected.

[0078] 1. Take HepG2 / MBNL3-GFP cells (marked as MBNL3-GFP in the figure) and HepG2 / 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.

[0079] 2. Take HepG2 / MBNL3-GFP cells and perform the following steps: after 36 hours of culture, cell lysis was performed and lysate was collected; GFP antibody (or isotype IgG) was incubated with Protein A / G Beads (Santa Cruz) for 2 hours, beads were washed by centrifugation, and then the above lysis solution was added and incubated for another 2 hours; then centrifuged at 4°C and 2700rpm to fully wash beads; finally, total RNA was collected using Trizol, and qRT-PCR was performed after reverse transcription to detect the target gene. Results are shown in Figure 3 B.

[0080] 3. After cloning the 3'UTRs sequences of PTGS1, ID1, FGF1 and ANG 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. The schematic diagram of the prepared reporter vector is shown in Figure 3 C. Figure 3 In D, a reporter containing the 3'UTR of human β-actin served as a negative control.

[0081] 4. Luciferase reporter assay

[0082] 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 3D. The test plasmids were: recombinant plasmid pEGFP-N1-MBNL3-GFP (labeled as MBNL3 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 PTGS1 gene 3'UTR, a reporter vector with ID1 gene 3'UTR, a reporter vector with FGF1 gene 3'UTR or a reporter vector with ANG gene 3'UTR.

[0083] 8. Prepare several recombinant plasmids as follows (each recombinant plasmid has been sequenced and verified):

[0084] 8.1. Insert the DNA molecule encoding truncation 1 in sequence 2 of the sequence list 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 1-173 in sequence 1 of the sequence list. Due to the shearing site that can be recognized by gene shearing, the truncation 1 obtained by shearing is shown by the amino acid residues 1-173 in sequence 1 of the sequence list, but the redundant amino acid residues of truncation 1 relative to the ZF1 and ZF2 functional regions cannot form an independent functional region, and the function realized by truncation 1 is the same as that of the ZF1 and ZF2 functional regions.

[0085] 8.2. Insert the DNA molecule encoding truncation 2 in sequence 2 of the sequence list 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 173rd to 354th amino acid residues in sequence 1 of the sequence list. Due to the shearing site that can be recognized by gene shearing, the truncation 2 obtained by shearing is shown by the 173rd to 354th amino acid residues in sequence 1 of the sequence list, but the redundant amino acid residues of truncation 2 relative to the ZF3 and ZF4 functional regions cannot form an independent functional region, and the function realized by truncation 2 is the same as that of the ZF3 and ZF4 functional regions.

[0086] 8.3. Introduce the recombinant plasmid pEGFP-N1-T1-GFP into HepG2 cells to obtain recombinant cells named HepG2 / T1-GFP cells. Introduce the recombinant plasmid pEGFP-N1-T2-GFP into HepG2 cells to obtain recombinant cells named HepG2 / T2-GFP cells.

[0087] 8.4. Take the HepG2 / MBNL3-GFP cells in Example 2 (marked as MBNL3-GFP in the figure), take the HepG2 / T1-GFP cells (marked as aa 1-173 in the figure) and HepG2 / T2-GFP cells (marked as aa173-354 in the figure) prepared in step 8.2, and perform the following steps respectively: culture under parallel conditions, take the 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. The results are shown in Figure 3 F.

[0088] The results showed that MBNL3 could significantly reduce the half-life of angiogenic gene mRNAs ( Figure 3 A). MBNL3 can target and bind to the mRNAs of pro-angiogenic factor genes, but not the mRNAs of anti-angiogenic genes ( Figure 3 B); MBNL3 protein can target and bind to the 3'UTR of pro-angiogenic genes ( Figure 3 C and Figure 3 D); After overexpression of T1 truncated gene or T2 truncated gene, the mRNAs of angiogenic factor genes were downregulated ( Figure 3 F). These results indicate that the four ZF domains of MBNL3 protein play an important role in controlling the expression of pro-angiogenic genes.

[0089] The above results show that the inventors of the present invention have discovered for the first time a new function of the MBNL3 protein, namely, it can specifically degrade mRNAs of pro-angiogenic factor genes. This new function explains the essential reason why overexpression of the MBNL3 gene can prevent angiogenesis in liver cancer.

[0090] Example 4: Overexpression of the MBNL3 gene (increasing the level of MBNL3 protein) inhibits angiogenesis and growth of liver cancer.

[0091] 1. Take HepG2 / GFP cells (marked as Empty Vector in the figure) and HepG2 / MBNL3-GFP cells (marked as MBNL3-GFP in the figure) and perform the following steps: inject subcutaneously into the back of BALB / c nude mice (each mouse is injected with: 3×10 8 The number of days was counted from the injection. The tumor volume was measured every day from the 24th day to the 58th day. The changes in tumor volume over time are shown in Figure 1 A (average of 6 mice). Mice were killed on day 58 and tumors were removed by autopsy ( Figure 4 B). Neovascularization in tumor tissue was confirmed by immunohistochemical staining of CD31 ( Figure 4 C), CD31 + Quantitative analysis of the number of blood vessels is shown in Figure 4 C right.

[0092] 2. Immunohistochemical staining was used to identify 12 liver cancer tissues with low MBNL3 expression (labeled as MBNL3-Negative in the figure) and 12 liver cancer tissues with high MBNL3 expression (labeled as MBNL3-Positive in the figure). CD31 immunohistochemical analysis showed that the number of microvessels in the liver cancer tissues with high MBNL3 expression was significantly reduced ( Figure 4 D) In ​​addition to orthotopic tumors, all mice developed lung metastases.

[0093] 3. Analysis of the TCGA liver cancer database revealed that the expression level of MBNL3 in human liver cancer tissues was significantly negatively correlated with the expression of pro-angiogenic genes. Figure 4 E.

[0094] The above results show that overexpression of MBNL3 gene (increasing MBNL3 protein level) can significantly inhibit liver cancer growth and its induced angiogenesis. In addition, clinical data analysis also confirmed that high expression of MBNL3 in human liver cancer tissue can inhibit liver cancer angiogenesis, which further confirms the research findings of the present invention.

[0095] 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 MBNL3 protein in the preparation of tumor drugs; the drug degrades the mRNAs of pro-angiogenic factor genes in cancer cells through MBNL3 protein, thereby specifically inhibiting the expression of pro-angiogenic genes in tumor cells and inhibiting tumor cell-induced angiogenesis. 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 angiogenesis; (a4) inhibiting the growth of tumor cells.

2. Use of the MBNL3 gene or a biomaterial having the MBNL3 gene in the preparation of an anti-tumor drug; the drug overexpresses the MBNL3 gene in vivo, and the MBNL3 protein degrades the mRNAs of pro-angiogenic factor genes in cancer cells, thereby specifically inhibiting the expression of pro-angiogenic genes in tumor cells and inhibiting tumor cell-induced angiogenesis. 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 angiogenesis; (a4) inhibiting tumor cell growth.

3. Use of a substance that can upregulate the abundance of MBNL3 protein in an organism in the preparation of an anti-tumor drug; the drug degrades the mRNAs of pro-angiogenic factor genes in cancer cells through MBNL3 protein, thereby specifically inhibiting the expression of pro-angiogenic genes in tumor cells and inhibiting tumor cell-induced angiogenesis, and 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 angiogenesis; (a4) inhibiting the growth of tumor cells.

4. Use of a substance that can upregulate the abundance of the MBNL3 gene in an organism in the preparation of an anti-tumor drug; the drug can overexpress the MBNL3 gene in an organism, degrade the mRNAs of pro-angiogenic factor genes in cancer cells through the MBNL3 protein, thereby specifically inhibiting the expression of pro-angiogenic genes in tumor cells and inhibiting tumor cell-induced angiogenesis, and the drug functions 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 angiogenesis; (a4) inhibiting the growth of tumor cells.

5. A tumor treatment drug, which can upregulate the abundance of MBNL3 protein or MBNL3 gene in an organism, degrade the mRNAs of pro-angiogenic factor genes in cancer cells through MBNL3 protein, thereby specifically inhibiting the expression of pro-angiogenic genes in tumor cells and inhibiting tumor cell-induced angiogenesis. The drug functions 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 angiogenesis; (a4) inhibiting the growth of tumor cells.

6. A truncate of MBNL3 protein, namely, ZF1 and ZF2 functional regions of MBNL3 protein, and / or, ZF3 and ZF4 functional regions of MBNL3 protein.

7. A gene encoding the MBNL3 protein truncation according to claim 6.

8. Use of the MBNL3 protein truncate according to claim 6 in the preparation of a tumor therapeutic drug, wherein the drug degrades the mRNAs of pro-angiogenic factor genes in cancer cells through the MBNL3 protein truncate, thereby specifically inhibiting the expression of pro-angiogenic genes in tumor cells and inhibiting tumor cell-induced angiogenesis, and the drug functions 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 angiogenesis; (a4) inhibiting tumor cell growth.

9. Use of a gene encoding the MBNL3 protein truncate according to claim 6 or a biological material having a gene encoding the MBNL3 protein truncate according to claim 6 in the preparation of a tumor therapeutic drug, wherein the drug is capable of overexpressing the gene of the MBNL3 protein truncate in vivo, degrading the mRNAs of pro-angiogenic factor genes in cancer cells through the MBNL3 protein truncate, thereby specifically inhibiting the expression of pro-angiogenic genes in tumor cells and inhibiting tumor cell-induced angiogenesis, wherein the drug has 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 angiogenesis; (a4) inhibiting tumor cell growth.

10. A tumor therapeutic drug, which can upregulate the abundance of the MBNL3 protein truncate according to claim 6 or the abundance of the gene encoding the MBNL3 protein truncate according to claim 6 in an organism, and degrade the angiogenic factor gene mRNAs in cancer cells through the MBNL3 protein truncate, thereby specifically inhibiting the expression of angiogenic genes in tumor cells and inhibiting tumor cell-induced angiogenesis, wherein the drug has 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 angiogenesis; (a4) inhibiting tumor cell growth.

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