BRIX1 inhibitor and application thereof in tumor resistance

Abstract

The invention discloses a BRIX1 inhibitor and application of the BRIX1 inhibitor in tumor resistance, and particularly discloses a nucleoprotein BRIX1 which is a key regulatory factor of a steady state between ribosome biogenesis and p53 activation. Research results show that inhibition of BRIX1 can induce nucleolus stress to activate p53, so that growth of tumors is inhibited. Besides, the engineered exosome is constructed, the surface of the engineered exosome is decorated with iRGD, and the engineered exosome is loaded with siRNA with BRIX1 specificity and is used for treating cancers.

Description

Technical Field

[0001] The present invention belongs to the fields of molecular biology and biomedicine. Specifically, the present invention relates to a BRIX1 inhibitor and its use in anti-tumor treatment. Background Art

[0002] Ribosome biogenesis is a multi-step process, including three basic steps: the synthesis of ribosomal RNA (rRNA) and ribosomal proteins (RPs), the processing of precursor rRNA, and the assembly of 40S and 60S ribosomal subunits in the nucleolus. An abnormal increase in the size and number of nucleoli is considered an important hallmark of malignant cells and is associated with poor prognosis in cancer patients. This is because cancer cells need to produce ribosomes faster than normal cells during rapid growth and proliferation to meet the needs of protein synthesis. Currently, various oncogenic signals have been found to promote cancer growth by increasing ribosome production. For example, c-Myc promotes the synthesis of all three rRNAs and the basic factors involved in ribosome biogenesis. Therefore, targeting nucleolar function may be an effective method to inhibit tumor cells while minimizing cytotoxic effects on normal cells.

[0003] Studies have shown that any key step in ribosome biogenesis being disrupted may lead to nucleolar stress, also known as ribosomal stress. Under such stress conditions, a portion of ribosomal proteins, especially RPL5 and RPL11, translocate from the nucleolus to the nucleoplasm, interact with MDM2, and inhibit its E3 ligase-mediated ubiquitination and degradation of p53, thereby activating p53. p53 is located on the short arm of human chromosome 17 (17p13.1), consisting of 11 exons, 10 introns, and 393 amino acid residues. p53 is an important tumor suppressor gene and is also the most commonly mutated gene in human cancers.

[0004] In addition, nucleolar stress may lead to tumor suppression through p53-independent signaling pathways. Some ribosomal proteins, such as RPL5, RPL11, and RPS14, have been shown to activate the p53 homolog TAp73 and inactivate the oncoprotein c-Myc under nucleolar stress. Multiple strategies have been shown to trigger nucleolar stress. For example, various chemotherapeutic drugs can induce ribosomal DNA (rDNA) damage or hinder pre-rRNA processing, thereby impairing ribosome biosynthesis. Some compounds limit rRNA synthesis by inhibiting the activity of RNA polymerase (Pol) I, such as CX-5461 and BMH-21. However, it was later found that these drugs also cause DNA damage or chromatin damage, and some RPs or nucleolar proteins have been shown to be essential for pre-rRNA processing and can specifically induce nucleolar stress.

[0005] Therefore, those skilled in the art are committed to developing the potential of inducing nucleolar stress as a cancer treatment strategy in order to obtain more efficient anti-tumor drugs. Summary of the Invention

[0006] An object of the present invention is to provide a BRIX1 inhibitor and its use in anti-tumor.

[0007] In a first aspect of the present invention, there is provided a use of a BRIX1 gene or its inhibitor for:

[0008] (1) Preparing a drug for preventing or treating tumors;

[0009] (2) Preparing a reagent for activating p53;

[0010] (3) Preparing a drug for inhibiting tumors through a p53-dependent mechanism; or

[0011] (4) Preparing a drug for inhibiting tumors through a p53-independent mechanism.

[0012] In another preferred embodiment, the BRIX1 gene is selected from the group consisting of:

[0013] (A) A polynucleotide sequence encoding a polypeptide as shown in SEQ ID NO.2;

[0014] (B) A polynucleotide sequence as shown in SEQ ID NO.1;

[0015] (C) A polynucleotide sequence formed by substituting, deleting or adding one or several nucleotides to the polynucleotide sequence shown in SEQ ID NO.1;

[0016] (D) A polynucleotide sequence having a homology of ≥90%, preferably ≥95%, more preferably ≥98%, and most preferably ≥99% compared to the polynucleotide sequence shown in SEQ ID NO.:1;

[0017] (E) A polynucleotide sequence complementary to any one of the polynucleotide sequences of (A)-(D).

[0018] In another preferred embodiment, the BRIX1 gene is derived from mammals (including humans).

[0019] In another preferred embodiment, the inhibitor of the BRIX1 gene is: a BRIX1 gene-specific siRNA or its precursor, a BRIX1 gene-specific microRNA or its precursor, an inhibitor that inhibits the BRIX1 gene promoter, or a combination thereof.

[0020] In another preferred embodiment, the inhibitor of the BRIX1 gene is a BRIX1 gene-specific siRNA or its precursor, or a microRNA or its precursor.

[0021] In another preferred embodiment, the siRNA specifically targets the sequence shown in SEQ ID NO.4 or SEQ ID NO.8 in the BRIX1 gene or its complementary sequence.

[0022] In another preferred embodiment, the inhibitor of the BRIX1 gene is an exosome, and the exosome is loaded with the siRNA or its precursor, or a microRNA or its precursor.

[0023] In another preferred embodiment, the inhibitor of the protein encoded by the BRIX1 gene is selected from the group consisting of: an antibody against the protein encoded by the BRIX1 gene, a binding protein of the protein encoded by the BRIX1 gene.

[0024] In another preferred embodiment, the tumor is a p53 wild-type tumor.

[0025] In another preferred embodiment, the tumor is a p53 mutant tumor.

[0026] In a second aspect of the present invention, there is provided a pharmaceutical composition comprising a pharmaceutically acceptable carrier and an effective amount of an active ingredient, wherein the active ingredient comprises an inhibitor of the BRIX1 gene or its encoded protein.

[0027] In another preferred embodiment, the pharmaceutical composition is used for preventing or treating tumors.

[0028] In another preferred embodiment, the inhibitor of the protein encoded by the BRIX1 gene is selected from the group consisting of: an antibody specifically against the protein encoded by the BRIX1 gene, a binding protein specifically binding to the protein encoded by the BRIX1 gene, a compound inhibiting the activity of the protein encoded by the BRIX1 gene.

[0029] In another preferred embodiment, the inhibitor of the BRIX1 gene is: a BRIX1 gene-specific siRNA or its precursor, a BRIX1 gene-specific microRNA or its precursor, an inhibitor inhibiting the BRIX1 gene promoter, or a combination thereof.

[0030] In another preferred embodiment, the inhibitor of the BRIX1 gene is a BRIX1 gene-specific siRNA or its precursor, or a microRNA or its precursor.

[0031] In another preferred embodiment, the siRNA specifically targets the sequence shown in SEQ ID NO.4 or 8 in the BRIX1 gene or its complementary sequence.

[0032] In another preferred example, the inhibitor of the BRIX1 gene is an exosome, and the exosome is loaded with the siRNA or its precursor, or microRNA or its precursor.

[0033] In another preferred example, the active ingredient includes 5-FU.

[0034] In the third aspect of the present invention, a method for non-therapeutically inhibiting tumor cells in vitro is provided, including the steps of: culturing tumor cells in the presence of an inhibitor of the BRIX1 gene or its encoded protein, thereby inhibiting the tumor cells.

[0035] In another preferred example, the BRIX1 gene inhibitor inhibits the expression of the BRIX1 gene in the tumor cells; or, the inhibitor of the BRIX1 gene-encoded protein inhibits the activity of the BRIX1 gene-encoded protein in the tumor cells.

[0036] In another preferred example, the tumor is a p53 wild-type tumor.

[0037] In another preferred example, the tumor is a p53 mutant tumor.

[0038] In another preferred example, the tumor is breast cancer or colorectal cancer; preferably, the tumor cells are CAL-51 breast cancer cells, MCF-7 breast cancer cell line, or HCT116 colorectal cancer cell line.

[0039] In another preferred example, the inhibition of tumor cells is to inhibit the growth of tumor cells or inhibit tumorigenesis of tumor cells.

[0040] In another preferred example, the inhibitor of the BRIX1 gene is an exosome, and the exosome is loaded with the siRNA or its precursor, or microRNA or its precursor.

[0041] In another preferred example, compared with control tumor cells, the activity of the BRIX1 gene-encoded protein in the tumor cells is reduced by more than 10%, preferably more than 20%, more preferably more than 30%, more preferably more than 40%, more preferably more than 50%, more preferably more than 60%, more preferably more than 70%, more preferably more than 80%, more preferably more than 90%, and most preferably completely without the activity of the BRIX1 gene-encoded protein.

[0042] In another preferred embodiment, compared with control tumor cells, the expression of the BRIX1 gene in the tumor cells is reduced by more than 10%, preferably by more than 20%, more preferably by more than 30%, more preferably by more than 40%, more preferably by more than 50%, more preferably by more than 60%, more preferably by more than 70%, more preferably by more than 80%, more preferably by more than 90%, and most preferably there is no expression of the BRIX1 gene at all.

[0043] In a fourth aspect of the present invention, there is provided an siRNA targeting the BRIX1 gene, and the siRNA is selected from the following group:

[0044] siBRIX1-1: 5'-CGTGTTTACTTTCACCATT-3' (SEQ ID NO.4), and

[0045] siBRIX1-2: 5'-GCATCGGCGTGTCATAAGA-3' (SEQ ID NO.8).

[0046] In a fifth aspect of the present invention, there is provided an siRNA precursor (shRNA), and the siRNA precursor is a precursor of the siRNA described in the fourth aspect of the present invention.

[0047] In another preferred embodiment, the 5' to 3' end of the siRNA precursor sequentially includes: a first sequence unit, a stem-loop sequence unit, and a second sequence unit. The first sequence unit and the second sequence unit are reverse complementary so that the siRNA precursor forms a hairpin structure, and the first sequence unit is selected from the following group:

[0048] 5'-CGTGTTTACTTTCACCATT-3' (SEQ ID NO.4), and

[0049] 5'-GCATCGGCGTGTCATAAGA-3' (SEQ ID NO.8).

[0050] In a sixth aspect of the present invention, there is provided an exosome, and the exosome is loaded with the siRNA or its precursor described in the fourth aspect of the present invention.

[0051] In another preferred embodiment, the exosome is further loaded with the surface protein LAMP2B, and the N-terminus of the surface protein LAMP2B is fused with the iRGD peptide.

[0052] In a seventh aspect of the present invention, there is provided a method for knocking down the expression level of the BRIX1 gene in cells, including the step: culturing cells in the presence of the siRNA described in the fourth aspect of the present invention, thereby achieving knocking down the expression level of the BRIX1 gene in cells.

[0053] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described hereinafter (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be elaborated one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] The following drawings are used to illustrate the specific implementation manners of the present invention, and are not used to limit the scope of the present invention defined by the claims.

[0055] Figure 1 : Knockdown of BRIX1 activates p53. (A) Venn diagram depicting the screening of potential regulators of ribosome biogenesis. (B) Heatmap showing that BRIX1 depletion significantly inhibits cell growth in four cancer cell lines as detected by cell viability assays. (C) Heatmap analysis results show that the expression of many p53 target genes is elevated in CAL-51 cells with BRIX1 depletion. (D) KEGG pathway enrichment analysis results show that target genes in the p53 pathway are enriched in cells with BRIX1 depletion. (E-G) Knockdown of BRIX1 can increase the mRNA levels of p53 target genes. CAL-51 (E), MCF-7 (F), and HCT116 p53 + / + cells were transfected with control or BRIX1 siRNA for 48 h, and then RT-qPCR analysis was performed. (H) Knockdown of BRIX1 has no effect on the mRNA levels of p53 target genes in HCT116 p53- / - cells. (I-K) Knockdown of BRIX1 increases the protein levels of p53 and its target genes. CAL-51 (I), MCF-7 (J), and HCT116 p53+ / + cells were transfected with control or BRIX1 siRNA for 48 h, and then IB analysis was performed. (L) Knockdown of BRIX1 has no effect on the protein levels of p53 target genes in HCT116 p53- / - cells. (M) Knockdown of BRIX1 does not increase the phosphorylation level of γ-H2AX. Cells were treated with drugs for 24 h or transfected with siRNA for 48 h, and then IB analysis was performed. ***p < 0.001.

[0056] Figure 2 : BRIX1 promotes pre-rRNA processing through the PeBoW complex. (A,B) Knockdown of BRIX1 reduces the level of 28S rRNA. CAL-51 (A) and HCT116 were transfected with control or BRIX1 siRNA p53+ / +(B) The cells were subjected to agarose gel electrophoresis experiment and ImageJ quantitative analysis at 48 h. (C, D) Knockdown of BRIX1 decreased the level of 28S rRNA detected by RT-qPCR. (E-H) BRIX1 interacted with BOP1 and PES1. Cells were transfected with plasmids for 48 h and then co-IP-IB analysis was performed using the indicated antibodies. (I) Endogenous interaction between BRIX1 and BOP1. HCT116 cells were treated with MG132 for 6 h and then co-IP-IB analysis was performed. p53+ / + Cells were transfected with the indicated siRNA and plasmid, and then co-IP-IB analysis was performed using the indicated antibodies. (L) Knockdown of BRIX1 impaired pre-rRNA processing. Cells were transfected with control or BRIX1 siRNA and then Northern blot analysis was performed. The schematic diagram of the ITS-2 probe (orange) in the experiment is shown as follows. ***p < 0.001.

[0057] Figure 3 : Knockdown of BRIX1 activated the nucleolar stress-p53 pathway and inhibited tumor growth. (A-D) Knockdown of RPL5 or RPL11 inhibited the activation of p53 after BRIX1 deletion. CAL-51 (A, B) and HCT116 cells were transfected with siRNA for 48 h and then IB analysis was performed. p53+ / + (E, F) Knockdown of BRIX1 increased the interaction between MDM2 and RPL5 and RPL11, respectively. Co-IP-IB analysis was performed using cells stably expressing shNC or shBRIX1 and antibodies. Cells were treated with the proteasome inhibitor MG132 for 5 h before harvesting. (G) Knockdown of BRIX1 decreased MDM2-induced p53 ubiquitination. HCT116 cells stably expressing control or BRIX1 shRNA were transfected with plasmids. p53- / -Cells were treated for 48 hours and then with MG132 for 5 hours before in vivo ubiquitination assay and IB analysis. (H) Knockdown of BRIX1 prolonged the half-life of p53 protein. Cells were transfected with control or BRIX1 siRNA. Before harvesting cells for IB analysis, cells were treated with cycloheximide (CHX) (100 μg / ml) at the indicated time. (I, J) Knockdown of BRIX1 inhibited the proliferation of wild-type p53 breast cancer cells. CAL-51 (I) and MCF-7 (J) cells were transfected with control or BRIX1 siRNA for 6 - 12 hours and then seeded in 96-well plates for cell viability assay. (K, L) Knockdown of BRIX1 impaired the colony formation ability of wild-type p53 cancer cells. CAL-51 (K) and MCF-7 (L) cells were transfected with control or BRIX1 siRNA for 6 - 12 hours and then seeded in 6-well plates for about 14 days. Colonies were fixed with methanol and stained with crystal violet. (M, N) In breast cancer cells carrying wild-type p53, knockdown of BRIX1 induced G1 cell cycle arrest. CAL-51 (M) and MCF-7 (N) cells were transfected with control or BRIX1 siRNA for 48 hours and then subjected to flow cytometry analysis. (O, P) Knockdown of BRIX1 inhibited the migration of wild-type p53-containing cancer cells. CAL-51 (O) and MCF-7 (P) cells were transfected with control or BRIX1 siRNA for 6 - 12 hours and then subjected to cell migration assay. ***p < 0.001.

[0058] Figure 4 : Overexpression of BRIX1 impaired the activation of p53 under nucleolar stress. (A - D) Overexpression of BRIX1 inhibited the expression of p53 and its target genes under nucleolar stress. CAL-51 (A, C) and HCT116 p53+ / + (B, D) cells were transfected with plasmids as indicated, treated with 10 nM Act D for 24 h, and then subjected to IB or RT-qPCR analysis. (E) Under actinomycin D (Act D) (10 nM), BRIX1 translocated from the nucleolus to the nucleoplasm, while under Nutlin-3 (20 μM), BRIX1 did not translocate. Cells were transfected with plasmids and treated with drugs as indicated, and then subjected to immunofluorescence staining. (F) BRIX1 interacted with RPL5 and RPL11. HCT116 p53+ / + cells were treated with Act D (10 nM) and MG132 (20 μM) for 24 h and 6 h respectively, and then subjected to co-IP-IB analysis. (G) BRIX1 inhibited the interaction between MDM2 and RPL5 / RPL11. HCT116 p53+ / +Cells were treated with Act D (10 nM) for 24 h and MG132 (20 μM) for 6 h, followed by co-IP-IB analysis. (H) Overexpression of BRIX1 increased the ubiquitination of p53. HCT116 cells stably overexpressing control or BRIX1 were transfected with the indicated plasmids for 48 h, then treated with Act D (10 nM) for 24 h and MG132 (20 μM) for 6 h, followed by in vivo ubiquitination assay. (I, J) Plasmids were transfected into HCT116 p53- / - and CAL-51 cells, which were treated with Act D (10 nM) for 24 h and MG132 (20 μM) for 6 h, followed by IB analysis. (K) Overexpression of BRIX1 shortened the half-life of p53 protein. HCT116 cells were transfected with plasmids and treated with Act D (10 nM) and CHX (100 mg / ml) at different time points for 24 h, followed by IB analysis. (L, M) Overexpression of BRIX1 increased the proliferation of CAL51 (L) and HCT116 p53+ / + (M) cells. Cells were transfected with plasmids and treated with Act D (10 nM), followed by cell viability assay. (N, O) Overexpression of BRIX1 enhanced the colony formation ability of CAL51 (N) and HCT116 p53+ / + (O) cells. Cells stably overexpressing control or BRIX1 were treated with 10 nM Act D, followed by colony formation assay. (P, Q) Overexpression of BRIX1 increased the migration of CAL51 (P) and HCT116 p53+ / + (Q) cells. Cells stably overexpressing control or BRIX1 were treated with 10 nM Act D, followed by cell migration assay. *p < 0.05, **p < 0.01, ***p < 0.001. (R-U) Overexpression of BRIX1 promoted the growth rate (R), weight (S), and size (T) of HCT116 p53+ / + cell-derived xenograft tumors, but did not affect the body weight of mice (U). On the designated first day after inoculation, mice were intraperitoneally injected with 30 μg / kg Act D. Data are presented as mean ± SD, n = 5. **p < 0.01, **p < 0.001. p53+ / + (O) The knockdown of BRIX1 had a significant inhibitory effect on tumors, which was largely dependent on p53. (A, B) Compared with HCT116 p53+ / + cells (B), the growth inhibitory effect of BRIX1 knockdown on HCT116

[0059] Figure 5 cells (A) was more significant. (C, D) Knockdown of BRIX1 reduced HCT116 p53- / - cells (C) more than HCT116 p53+ / + cells (A). p53+ / + cells (C) compared with HCT116 p53- / -Cells (D) more significantly reduced the colony formation ability. Cells were transfected with siRNA and subjected to colony formation assay. (E,F) Compared with HCT116 p53- / - cells (F), knockdown of BRIX1 had a greater impact on the migration of HCT116 p53 + / + cells (E). Cells were transfected with siRNA as indicated and subjected to cell migration assay. (G,H) In HCT116 p53+ / + (G) cells, knockdown of BRIX1 induced G1 phase arrest, while there was no such effect in HCT116 p53- / - cells (H). Cells were transfected with siRNA as shown and then analyzed by flow cytometry. (I-P) BRIX1 deficiency significantly inhibited the growth rate (I), weight (J) and size (K) of HCT116 p53 + / + cell-derived tumors, but had little effect on HCT116 p53- / - cell-derived tumors (M-O). The body weight of mice was not affected (L,P). Data are presented as mean±SD, n = 6. *p<0.05, **p<0.01, ***p<0.001.

[0060] Figure 6 : BRIX1 was highly expressed in breast cancer and colorectal cancer tissues and was associated with poor prognosis. (A,B) Compared with adjacent normal tissues, the protein and mRNA levels of BRIX1 were elevated in breast cancer samples. Five pairs of fresh tissues were analyzed by IB and RT-qPCR, respectively. (C,D) Elevated BRIX1 expression level was a factor for poor prognosis of breast cancer.

[0061] Figure 7 : Therapeutic BRIX1 siRNA delivered by iRGD-modified exosomes inhibited colorectal cancer in vitro and in vivo. (A,C) iRGD-modified exosomes transported fam-labeled siBRIX1 into cancer cells. CAL-51 and HCT116 p53+ / + cells were incubated with FAM-siBRIX1 or iRGD-Exo-FAM-siBRIX1 for 8 hours and then observed under a fluorescence microscope. FAM-siBRIX1, green; DAPI, blue for nuclei; scale bar, 10 μm. (B,D) iRGD-Exo-siBRIX1 activated p53. CAL-51 (B) and HCT116 p53+ / + (D) cells were incubated with the indicated exosomes for 48 hours and then analyzed by IB. (E-J) iRGD-Exo-siBRIX1 inhibited the growth (E,F), colony formation (G,H) and migration (I,J) of CAL-51 and HCT116 p53+ / + cells. CAL-51 and HCT116p53+ / + Cells were incubated with the designated exosomes for 24 hours, and then cell viability assay, colony formation assay or cell migration assay was performed. (K,L) iRGD increased the enrichment of exosomes in the tumor area. DIR-labeled exosomes were intravenously injected into BALB / c nude mice bearing transplanted tumors derived from HCT116 p53+ / + cells and analyzed by in vivo fluorescence imaging. (M-P) iRGD-Exo-siBRIX1 inhibited the growth of transplanted tumors derived from HCT116 p53+ / + cells. (Q) siBRIX1 combined with 5-FU synergistically inhibited the growth of cancer cells. HCT116 p53+ / + cells were treated with the combined drugs as indicated for 48 hours, and then cell viability assay and Chou-Talalay analysis were performed. (R-U) iRGD-Exo-siBRIX1 and 5-FU combined to inhibit the growth of transplanted tumors derived from HCT116 p53+ / + cells. Data are presented as mean ± SD, n = 6. **p < 0.01, **p < 0.001.

[0062] Figure 8 : Preparation of engineered exosomes expressing iRGD. (A) Preparation route for generating engineered exosomes with surface expression of iRGD. (B) Transmission electron microscopy verified the morphology of purified exosomes. (C) Nanoparticle tracking analysis verified the morphology of purified exosomes. (D) Detection results of exosome markers. Detailed implementation mode

[0063] The present inventors have conducted extensive and in-depth research and found that BRIX1 mainly promotes the processing of 12S and 32S rRNA precursors by binding to the PES1-BOP1-WDR12 (PeBoW) complex. Deletion of BRIX1 triggers nucleolar stress and activates p53, while overexpression of BRIX1 alleviates nucleolar stress-induced p53 activation by blocking the interaction between RPL5 / RPL11 and MDM2. The research results of the present invention also show that BRIX1 is highly expressed in breast cancer and colorectal cancer, and the higher the BRIX1 level, the worse the prognosis. These findings indicate that BRIX1 is a viable target for cancer treatment. The present invention has developed a delivery system (Exo-siBRIX1) that uses engineered exosomes to load siRNA against BRIX1. The Exo-siBRIX1 has been proven to effectively inhibit cancer growth in vivo. In summary, the research of the present invention reveals the key role of BRIX1 in regulating the nucleolar stress-p53 pathway and provides a potential BRIX1-targeted strategy for cancer treatment.

[0064] Terms

[0065] BRIX1 gene and its encoded protein

[0066] BRIX (Biogenesis of ribosomes in Xenopus laevis) was first discovered in frogs and later found to be involved in rRNA processing in yeast. The expression of human BRIX1 has recently been shown to be associated with rRNA synthesis and to promote the translation of GLUT1 in colorectal cancer. However, the role and mechanism of BRIX1 in rRNA synthesis and cancer development remain largely unknown.

[0067] The human BRIX1 gene (NCBI accession number Gene ID: 55299) is located on human chromosome 5.

[0068]

[0069] In a preferred embodiment of the present invention, the sequence of the protein encoded by the BRIX1 gene is as shown in SEQ ID NO.2:

[0070] MAATKRKRRGGFAVQAKKPKRNEIDAEPPAKRHATAEEVEEEERDRIPGPVC

[0071] KGKWKNKERILIFSSRGINFRTRHLMQDLRMLMPHSKADTKMDRKDKLFVINE

[0072] VCEMKNCNKCIYFEAKKKQDLYMWLSNSPHGPSAKFLVQNIHTLAELKMTGN

[0073] CLKGSRPLLSFDPAFDELPHYALLKELLIQIFSTPRYHPKSQPFVDHVFTFTILDN

[0074] RIWFRNFQIIEEDAALVEIGPRFVLNLIKIFQGSFGGPTLYENPHYQSPNMHRRVI

[0075] RSITAAKYREKQQVKDVQKLRKKEPKTLLPHDPTADVFVTPAEEKPIEIQWVKPEPKVDLKARKKRIYKRQRKMKQRMDSGKTK(SEQ ID NO.2)

[0076] The protein encoded by the BRIX1 gene of the present invention can be a recombinant polypeptide, a natural polypeptide or a synthetic polypeptide.

[0077] Inhibitor of the BRIX1 gene

[0078] As used herein, the term "substance that inhibits gene expression" means any substance that inhibits the mRNA transcription of a target gene, decomposes the transcribed mRNA, or inhibits the translation of mRNA into protein, without particular limitation. As such substances, nucleic acids can be cited, for example: siRNA, miRNA, etc. Among them, siRNA is preferred. As the "substance that inhibits gene expression", in addition to the above, it also includes proteins or peptides, or other small molecules. It should be noted that in the present invention, the target gene is the BRIX1 gene.

[0079] As used herein, the term "inhibitor of the BRIX1 gene" refers to a substance that inhibits the replication or transcription of the BRIX1 gene, or a substance that reduces the expression of the BRIX1 gene. Inhibitors of the BRIX1 gene include (but are not limited to): siRNA, microRNA, compounds, or combinations thereof. Preferred inhibitors of the BRIX1 gene are siRNA or microRNA.

[0080] As used herein, the term "RNAi" (RNA interference) refers to a phenomenon that is highly conserved in the process of evolution, induced by double-stranded RNA (dsRNA), and highly efficiently and specifically degrades RNA with complementary pairing sequences. Since the RNAi technology can specifically turn off the expression of specific genes, this technology has been widely used in fields such as exploring gene functions and gene therapy for infectious diseases and tumors.

[0081] As used herein, the term "siRNA" (Small interfering RNA) refers to a small RNA molecule (about 21-25 nucleotides), which can be processed from its precursors (such as dsRNA, shRNA, etc.) by Dicer (an enzyme specific for double-stranded RNA in the RNase III family), or can be chemically synthesized or produced by other proteins. siRNA is the main member of siRISC, which stimulates the target RNA complementary to its sequence to be rapidly cleaved and degraded, resulting in the silencing of the target gene, and thus becomes a key functional molecule in RNAi.

[0082] In a preferred embodiment of the present invention, the present invention provides an siRNA precursor having the siRNA sequence of the present invention. As used herein, the term "siRNA precursor" refers to an RNA molecule that can be processed in mammalian cells to produce siRNA. Specifically, it is selectively processed by Dicer or other similar proteins to produce mature siRNA, thereby implementing RNAi.

[0083] In a preferred embodiment of the present invention, the present invention provides a nucleic acid construct. As used herein, the term "construct" refers to a nucleic acid construct containing the siRNA precursor of the present invention.

[0084] In a preferred embodiment of the present invention, the present invention provides an expression cassette. As used herein, the term "expression cassette" refers to an expression cassette containing the coding sequence of the siRNA precursor of the present invention and a promoter and a termination signal operably linked to the coding sequence, and the expression cassette produces the siRNA precursor of the present invention after transcription.

[0085] One way to generate "small interfering RNA" (siRNA) in vivo is to clone the siRNA sequence as part of a "short hairpin" into a plasmid vector. When introduced into an animal, the hairpin sequence is expressed to form a "double-stranded RNA" (shRNA) with a stem-loop structure, which is recognized and processed by the Dicer protein in the cell to produce functional siRNA.

[0086] As used herein, the term "shRNA" refers to a specific shRNA constructed using the precursor of human miR-26b as a backbone. The shRNA, from the 5' end to the 3' end, consists of: (a) a 5' flanking sequence region; (b) a 5' paired siRNA region; (c) a stem-loop region; (d) a 3' paired siRNA region, and the 5' paired siRNA region and the 3' paired siRNA region form a double-stranded region; (e) a 3' flanking sequence region; the shRNA generates siRNA, and the nucleotide sequence of the siRNA corresponds to the 3' paired siRNA region or the 5' paired siRNA region.

[0087] Broadly speaking, shRNA is an abbreviation of short hairpin RNA, that is, "short hairpin RNA". shRNA includes two short inverted complementary sequences separated by a stem-loop sequence, forming a hairpin structure, which is usually transcribed under the control of an endogenous RNA polymerase III (RNA polymerase III) promoter in the cell. 5-6 Ts are linked to the ends of the shRNA sequence as a transcription terminator for RNA polymerase III. shRNA can also be transcribed from promoters of other RNA polymerases.

[0088] As used herein, the term "miRNA" (microRNA) refers to a class of non-coding single-stranded RNA molecules encoded by endogenous genes, with a length of approximately 20 - 24 nucleotides, which participate in the regulation of the expression of a large number of genes in animals and plants. So far, more than four thousand miRNA molecules have been discovered in animals, plants and viruses. Most miRNA genes exist in the genome in the form of single copies, multiple copies or gene clusters. Each miRNA can regulate multiple target genes, and several miRNAs can also jointly participate in the regulation of the same gene, forming a complex regulatory network. It is speculated that miRNAs regulate the expression of more than half of the genes in humans. miRNAs exist in multiple forms, and the most primitive one is pri-miRNA; after being processed by Drosha, pri-miRNA becomes pre-miRNA, that is, the miRNA precursor, with a length of approximately 50 - 90 nucleotides; after further digestion by Dicer enzyme, pre-miRNA becomes a mature miRNA with a length of about 20 - 24 nucleotides. miRNAs mainly inhibit target gene expression by inhibiting translation and accelerating the deadenylation of mRNA, and its mechanism is different from that of siRNA-mediated mRNA degradation.

[0089] Exosome

[0090] As used herein, exosomes are tiny vesicles secreted by cells, with a diameter of about 30 - 200 nm, a density of 1.13 - 1.21 g / ml, having a cup-shaped morphology and a double-membrane structure, and are naturally present in biological fluids such as blood, urine, saliva, breast milk and cell culture medium. Almost all types of cells (immune cells, nerve cells, stem cells), including tumor cells, can produce and release exosomes.

[0091] In the present invention, the exosomes include substances (such as siRNA, miRNA, etc.) that inhibit the expression of the BRIX1 gene and / or its protein activity.

[0092] The present invention constructs engineered exosomes, which are decorated with iRGD (a tumor-targeting peptide) on the surface and loaded with BRIX1-specific siRNA for the treatment of cancer. It is worth noting that these engineered exosomes iRGD-Exo-siBRIX1 significantly inhibit the growth of colorectal cancer in vivo and improve the efficacy of 5-FU chemotherapy.

[0093] Drug composition

[0094] The present invention provides a drug composition, comprising a pharmaceutically acceptable carrier and an effective amount of the following active ingredient: an inhibitor of the BRIX1 gene or its encoded protein.

[0095] As used herein, the term "effective amount" or "effective dose" refers to an amount that can produce a function or activity in humans and / or animals and is acceptable to humans and / or animals.

[0096] In a preferred embodiment, the active ingredient of the pharmaceutical composition of the present invention is an exosome preparation.

[0097] The exosome preparation of the present invention contains a safe and effective amount of exosomes and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, powder, and combinations thereof. The pharmaceutical preparation should be matched with the administration method.

[0098] In a preferred embodiment, the pharmaceutical composition of the present invention may also contain a safe and effective amount of other drugs for preventing and / or treating tumors.

[0099] The pharmaceutical composition of the present invention can be made into a liquid preparation, and its preparation can be carried out by conventional methods. The liquid preparation should be manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, such as about 1 μg / kg body weight - about 50 mg / kg body weight per day, about 5 μg / kg body weight - about 10 mg / kg body weight, about 10 μg / kg body weight - about 5 mg / kg body weight. In addition, the preparation of the present invention can also be used together with other therapeutic agents.

[0100] When using the preparation of the present invention, a safe and effective amount of the drug is administered to a mammal, and the safe and effective amount is usually at least about 10 μg / kg body weight, and in most cases does not exceed about 50 mg / kg body weight. Preferably, the dose is about 10 μg / kg body weight - about 20 mg / kg body weight. Of course, the specific dose should also consider factors such as the administration route and the health status of the patient, which are within the scope of the skills of a skilled physician.

[0101] As used herein, a "pharmaceutically acceptable" component is a substance that is suitable for humans and / or mammals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), that is, a substance having a reasonable benefit / risk ratio. The term "pharmaceutically acceptable carrier" refers to a carrier used for administering a therapeutic agent, including various excipients and diluents.

[0102] The pharmaceutical composition of the present invention contains a safe and effective amount of the active ingredient of the present invention and a pharmaceutically acceptable carrier. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. Usually, the pharmaceutical preparation should be matched with the administration method. The dosage form of the pharmaceutical composition of the present invention is an injection, an oral preparation (tablet, capsule, oral liquid), a transdermal agent, a sustained-release agent. For example, it is prepared by conventional methods with physiological saline or an aqueous solution containing glucose and other adjuvants. The said pharmaceutical composition should be manufactured under sterile conditions.

[0103] The effective amount of the active ingredient described in the present invention may vary depending on the mode of administration, the severity of the disease to be treated, etc. The selection of the preferred effective amount can be determined by those of ordinary skill in the art based on various factors (e.g., through clinical trials). Such factors include, but are not limited to: the pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated in the patient, the patient's body weight, the patient's immune status, the route of administration, etc. Generally, when the active ingredient of the present invention is administered at a dose of about 0.00001 mg - 50 mg / kg of animal body weight per day (preferably 0.0001 mg - 10 mg / kg of animal body weight), satisfactory effects can be obtained. For example, depending on the urgency of the treatment situation, several separate doses may be administered per day, or the dose may be proportionally reduced.

[0104] The pharmaceutically acceptable carriers described in the present invention include (but are not limited to): water, saline, liposomes, lipids, proteins, protein - antibody conjugates, peptides, cellulose, nanogels, or combinations thereof. The selection of the carrier should match the mode of administration, which is well known to those of ordinary skill in the art.

[0105] Application

[0106] The present invention provides a method for treating tumors, comprising the step of administering a therapeutically effective amount of an inhibitor of the BRIX1 gene or its encoded protein to a tumor patient.

[0107] The present invention also provides a method for non - therapeutically inhibiting tumor cells in vitro, comprising the step of culturing tumor cells in the presence of an inhibitor of the BRIX1 gene or its encoded protein, thereby inhibiting the tumor cells.

[0108] In another preferred embodiment, the tumor is a p53 wild - type tumor, or the tumor cells are p53 wild - type tumor cells.

[0109] In a preferred embodiment of the present invention, the inhibitor of the BRIX1 gene - encoded protein inhibits the activity of the BRIX1 gene - encoded protein in the tumor cells; or the inhibitor of the BRIX1 gene inhibits the expression of the BRIX1 gene in the tumor cells.

[0110] In a preferred embodiment of the present invention, the inhibition of tumor cells is to inhibit the growth, metastasis or tumorigenesis of tumor cells.

[0111] Preferably, compared with control tumor cells, the activity of the protein encoded by the BRIX1 gene in the tumor cells is reduced by more than 10%, preferably more than 20%, more preferably more than 30%, more preferably more than 40%, more preferably more than 50%, more preferably more than 60%, more preferably more than 70%, more preferably more than 80%, more preferably more than 90%, and most preferably there is no activity of the protein encoded by the BRIX1 gene at all.

[0112] Or preferably, compared with control tumor cells, the expression level of the BRIX1 gene in the tumor cells is reduced by more than 10%, preferably more than 20%, more preferably more than 30%, more preferably more than 40%, more preferably more than 50%, more preferably more than 60%, more preferably more than 70%, more preferably more than 80%, more preferably more than 90%, and most preferably there is no expression of the BRIX1 gene at all.

[0113] The main advantages of the present invention are as follows:

[0114] (1) It is verified that the BRIX1 gene and its encoded protein can be used as novel tumor treatment targets.

[0115] (2) siRNAs that highly inhibit the BRIX1 gene are obtained.

[0116] (3) Exosomes that target the BRIX1 gene and have excellent anti-tumor effects are obtained.

[0117] The present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are usually carried out under conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under the conditions recommended by the manufacturer. Unless otherwise stated, parts and percentages are by weight.

[0118] Examples

[0119] (I) Materials and Methods

[0120] Cell culture and transient transfection

[0121] The human colorectal cancer cell line HCT116 p53+ / + 、HCT116 p53- / -HeLa, RKO, breast cancer cell lines CAL-51 and MCF-7, and embryonic kidney cell line 293T were cultured in DMEM (Dulbecco's modified Eagle’s medium) supplemented with 10% fetal bovine serum (Yeasen, Shanghai, China), 100 units / mL penicillin, and 100 μg / mL streptomycin (BasalMedia, Shanghai, China). All cell lines were obtained from ATCC and were mycoplasma-free as determined by PCR. Cells were seeded onto plates at the optimal density 12 - 24 h prior to transfection. Transfection was performed using Hieff Trans Liposomal Transfection Reagent (Yeasen) according to the manufacturer's protocol.

[0122] Plasmids and Antibodies

[0123] Design primers to amplify BRIX1, BOP1, PES1, and WDR12 cDNAs, and then clone the amplified sequences into Flag-pCDNA3.1, Myc-pCDNA3.1, and HA-pCMV vectors. The BRIX1 cDNA sequence was ligated into a lentiviral vector to generate pCDH-Flag-BRIX1. Anti-Flag (F1804, Sigma-Aldrich, St. Louis, MO, USA), anti-Myc (Cat; No. 60003-2-Ig, Proteintech), anti-HA (Cat; No. 2367, Cell Signaling Technology, Danvers, MA, USA), anti-BRIX1 (Cat; No. 17295-1-AP, Proteintech), anti-p53 (Cat; No. sc-126, DO-1, Santa Cruz Biotechnology), anti-mdm2 (Cat; No. ab16895, 2A10, Abcam), anti-phospho-histone H2A (Ser139) (Cat; No. 9718, Cell Signaling Technology), anti-GAPDH (Cat; No. 60004-1-Ig, Proteintech), anti-RPL5 (Cat; No. ab86863, Abcam), anti-RPL11 (Cat; No. ab79352, Abcam), anti-p21 (Cat; No. 2947, Cell Signaling Technology), anti-NPM1 (Cell Signaling Technology; No. sc-271737, Santa Cruz Biotechnology), anti-BOP1 (Cat; No. 28366-1-AP, Proteintech), anti-TSG10 (Cat; No. 28283-1-AP, Proteintech), anti-CD81 (Cat; No. sc-166029, Santa Cruz Biotechnology), anti-CD9 (Cat; No. sc-13118, Santa Cruz Biotechnology), anti-coxⅣ (Cat; No. 11242-1-AP, Proteintech) were commercially purchased.The secondary antibodies were enzyme-labeled affinity-purified goat anti-rabbit IgG (Cat. No. SA00001-2, Proteintech) and anti-mouse IgG (Cat. No. SA00001-1, Proteintech). ECL chemiluminescent reagent (Yeasen) was used for development.

[0124] Reverse transcription and real-time quantitative PCR

[0125] Total RNA was isolated from cells using RNAiso Plus (Takara, Japan) according to the manufacturer's protocol, and cDNA was synthesized using Hiscript III qRT SuperMix (Vazyme). Quantitative PCR (qPCR) was performed using SYBR qPCR Master Mix according to the manufacturer's protocol (Vazyme). The relative expression levels of mRNA were calculated using the comparative Ct method with normalized GAPDH.

[0126] Transcriptome sequencing

[0127] After siNC or siBRIX1 transfected CAL-51 cells for 48 h, total RNA was isolated using RNAiso Plus (Takara, Japan), and RNA sequencing was provided by OE Biotech Co, Ltd (shanghai, China).

[0128] Immunoblotting

[0129] Proteins were extracted using RIPA buffer [50 mM Tris / HCl (pH 8.0), 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 0.2 mM phenylmethylsulfonyl fluoride, and 10% protease inhibitor mixture] and quantified using BCA reagent (Yeasen). Equal amounts of clear cell lysates (20 - 80 μg) were used for immunoblotting (IB) analysis.

[0130] Northern blotting

[0131] Northern blotting assay was performed using the NorthernMax-Gly kit (Thermo Fisher Scientific, USA). RNA (30 - 60 μg) denatured with glyoxal loading dye was separated by electrophoresis on a 1% agarose gel and transferred to BrightStar TM-Plus nylon membrane (Thermo Fisher Scientific, USA). Then, the RNA was cross-linked to the membrane under ultraviolet irradiation at 1.5 J cm2, pre-hybridized at 65 °C for 30 minutes, and hybridized with the probe overnight at 42 °C. The next day, it was washed with Low Stringency Wash Solution at room temperature for 10 minutes, washed at 42 °C for 2 minutes, and blocked with blocking buffer for 30 minutes. Subsequently, it was incubated with anti-DIG at room temperature for 1 hour (Universal Biotech Co., shanghai), washed twice with washing buffer for 15 minutes each, and washed with detection buffer for 5 minutes. Finally, the membrane was developed with CDP-Star (Roche, USA). The digoxigenin (DIG)-labeled probe was synthesized by GENEWIZ (Suzhou, China).

[0132] Immunofluorescence staining

[0133] Cells transfected with siRNA were fixed overnight at -20 °C with methanol, washed three times with PBS, and incubated with blocking buffer (8% BSA and 0.3% Triton X-100) at room temperature for 1 hour. Then, it was incubated with primary antibodies (anti-BRIX1, 1:100; anti-NPM1, diluted 1:50) overnight at 4 °C. The next day, the cells were washed with PBS and incubated with fluorescent secondary antibodies (Yeasen) and DAPI (Sigma-Aldrich). Images were obtained using an inverted fluorescence microscope (Leica, Wetzlar, Germany).

[0134] Protein immunoprecipitation

[0135] Proteins were extracted with lysis buffer [50 mM Tris / HCl (pH 7.5), 0.5% Nonidet P-40 (NP-40), 1 mM EDTA, 150 mM NaCl, 1 mM dithiothreitol (DTT), 0.2 mM phenylmethylsulfonyl fluoride, and 10% protease inhibitor mixture], and immunoprecipitated (IP) with antibodies as Figure 4 shown. Briefly, the protein (0.5 - 1 mg) was incubated with the specified antibody at 4 °C for 5 hours, and then incubated with protein A / G magnetic beads (Santa Cruz Biotechnology) for 2 hours. The magnetic beads containing the immunoprecipitated protein were washed 6 - 8 times with lysis buffer and analyzed by IB.

[0136] In vivo ubiquitination assay

[0137] As shown in the figure, plasmids of p53, HA-MDM2, His-Ub, and BRIX1 siRNA were transfected into HCT116 p53- / -Cells were treated with MG132 for 4 - 6 hours and then harvested for in vivo ubiquitination assays. Briefly, 48 hours after transfection, cells were collected and divided into two parts, one for IB and the other for ubiquitination assays. Cell pellets were lysed in buffer I [8 M urea, 0.1 M Na2HPO4 / NaH2PO4 (pH 8.0)), 10 mM Tris - HCl (pH 8.0), 10 mM β - mercaptoethanol, and 5 mM imidazole] and incubated with Ni - NTA beads (Takara) at room temperature for 4 hours. The Ni - NTA beads (Takara) captured his - tagged proteins / complexes. The beads were washed twice with buffer I and then twice with buffer II [8 M urea, 0.1 M Na2HPO4 / NaH2PO4 (pH 6.3), 10 mM Tris - HCl (pH 6.3), and 10 mM β - mercaptoethanol] to elute the captured proteins, and IB experiments were performed with the designated antibodies.

[0138] RNA Interference and Generation of Stable Cell Lines

[0139] Small interfering RNAs (siRNAs) targeting BRIX1, RPL5, and RPL11 were synthesized and purified by GenePharma (Shanghai, China).

[0140] siRNAs were designed against the BRIX1 gene and the knockdown efficiency was detected.

[0141] siRNA Knockdown Efficiency Screening:

[0142] After the designed siRNAs were synthesized, cell RNA was extracted after transfection of cells, reverse transcription reaction was carried out, and the knockdown efficiency of the BRIX1 gene was detected by qPCR.

[0143] Some representative siRNAs designed are as follows:

[0144] Number Sequence (DNA) SEQ ID NO. BRIX1 gene knockdown efficiency 01# CGAGGTTTGTGAAATGAAG 3 7.6％ 02# CGTGTTTACTTTCACCATT 4 88.2％ 03# GGACCAACTTTATATGAAA 5 12.4％ 04# TCGCTGAACTGAAGATGAC 6 6.1％ 05# GGAACGGATTCTCATCTTT 7 3.8％ 06# GCATCGGCGTGTCATAAGA 8 92.5％ 07# AAACATGCATCGGCGTGTC 9 6.8％ 08# GGCTTTGCAGTTCAGGCGA 10 12.9％ 09# GTGTTTACTTTCACCATTT 11 28.6％ 10# GAACAAGACATTTAATGCA 12 21.2％ 11# GGATAATAGGATATGGTTT 13 3.0％ 12# GATATGGTTTCGGAACTTT 14 9.7％ 13# AAAGTTGATTTGAAAGCAA 15 6.4％ 14# AACAGGATCTCTATATGTG 16 53.2％ 15# TTGACCCTGCTTTTGATGA 17 16.3％ 16# AATGTTGATGCCTCATTCT 18 2.7％ Control siNC / 0

[0145] According to the primary screening results, 10 siRNAs showing knockdown effects on the BRIX1 gene were re - screened. According to the re - screening results, the siRNAs with the best knockdown effects, 02# and 06#, were named si BRIX1 - 1 (SEQ ID NO.4) and si BRIX1 - 2 (SEQ ID NO.8) respectively for subsequent experiments.

[0146] Transfection of siRNA was performed using Hieff reverse liposome transfection reagent according to the manufacturer's protocol (Yeasen). All transfections were verified by qPCR or western blot. The shRNA specific sequence targeting BRIX1 was obtained from Sigma-Aldrich and cloned into the PLKO.1 vector. The plasmid containing shRNA, together with the packaging plasmids psPAX2 and pMD2G, was transfected into HEK293T cells. Lentiviral particles were collected 48 hours after transfection for cell infection. Stably infected cells were selected with 1 μg / ml puromycin.

[0147] Cell viability assay

[0148] To evaluate cell viability, 2 - 4×10 3 cells were seeded per well in 96-well culture plates 6 - 12 h after transfection. Cell Counting Kit-8 (CCK-8) reagent (Yeasen) was added to each well at a final concentration of 10%. After incubation at 37 °C for 2 h, the absorbance at 450 nm was measured using a microplate reader. For combination drug analysis, CompuSyn software was used, and the Chou-Talalay method was adopted to calculate the relationship between the combination index (CI) and the fraction affected (FA). When the CI value is less than, equal to, or greater than 1, it indicates synergistic, additive, or antagonistic effects.

[0149] Colony formation assay

[0150] 6 - 18 h after transfection of cells with siRNA, 1×10 3 cells were seeded on 6-cm plates and cultured for 14 days. The culture medium was changed every 3 days until colonies were visible, and then stained with methanol solution containing 0.2% crystal violet at room temperature for 30 minutes. The number of cell colonies was determined by ImageJ.

[0151] Cell cycle assay

[0152] 48 h after transfection of cells with siRNA, the cells were fixed in 70% ethanol containing PBS overnight, treated with 50 μg / ml RNase A (Sangon) and 0.1% Triton X-100 (Sangon) in PBS at 37 °C for 30 minutes, and stained with 50 μg / ml propidium iodide (PI) (Vazyme) in the dark for 30 minutes. Finally, flow cytometry was used to analyze the cell cycle (CytoFLEX S, Beckman Coulter, Indianapolis, IN, USA).

[0153] Transwell cell migration assay

[0154] In the cell migration assay, 200 μl of serum-free medium was inoculated into the upper chamber, and 800 μl of 20% FBS medium was inoculated into the lower well plate, and 5 - 10×10 4 cells were inoculated. After culturing at 37 °C for 36 - 48 hours, the cells on the lower surface of the chamber were fixed with methanol and stained with 0.2% crystal violet (BBI Life Sciences) for 30 min. The migrated cells were counted under a microscope and quantified using ImageJ.

[0155] Cell apoptosis detection

[0156] The cell apoptosis assay was performed according to the manufacturer's protocol of the cell apoptosis detection kit (Vazyme). Briefly, the cells were washed twice with cold PBS and then resuspended in 1× Binding Buffer at a concentration of 1×10 6 cells / ml. The cells were incubated with Annexin V-PE and 7-aminoactinomycin D (7AAD) at room temperature for 15 min. Finally, the cell apoptosis level was detected by flow cytometry (CytoFLEX S, Beckman Coulter, Indianapolis, In, USA).

[0157] In vivo transplantation assay

[0158] Five-week-old female BALB / c nude mice were obtained from the Experimental Animal Science Center of Fudan University Shanghai Cancer Center. To study the function of BRIX1, 5×10 6 HCT116 p53+ / + and HCT116 p53- / - cells, as well as PCDH or PCDH-BRIX1 resuspended in 100 μL of serum-free medium, were injected into the flanks of the mice. The tumor volume and weight of the mice were measured as shown. The formula for calculating the tumor volume was: Tumor volume (mm 3 ) = (length × width 2) × 0.52. To study the anti-tumor effect of iRGD-Exo-siBRIX1, in this invention, exosomes were injected into tumor-bearing mice via the tail vein three times a week. To explore the combined effect of iRGD-Exo-siBRIX1 and chemotherapy, the tumor-bearing mice were randomly divided into 4 groups and were respectively given normal saline, 10 mg / kg 5-FU, 10 mg / kg 5-FU combined with 150 μg exosomes containing 3.75 μg siNC (iRGD-Exo-siNC), or 150 μg exosomes containing 3.75 μg siBRIX1 (iRGD-Exo-siBRIX1). Normal saline and 5-FU were injected intraperitoneally, and the engineered exosomes were injected intravenously. When the tumors reached an appropriate volume, the nude mice were sacrificed, the tumors were dissected, weighed, and photographed. All studies were approved by the Animal Welfare Committee of Shanghai Cancer Center, Fudan University, and all animals were handled in accordance with institutional guidelines.

[0159] Human breast cancer and colorectal cancer specimens

[0160] Five pairs of fresh breast cancer and adjacent normal tissues were selected from the First Affiliated Hospital of China Medical University to detect the BRIX1 protein and mRNA levels. Ninety-one paraffin sections of breast cancer were obtained from the First Affiliated Hospital of China Medical University for immunohistochemical analysis. In addition, a total of 10 pairs of colorectal cancer and adjacent normal tissues and 62 paraffin-embedded sections of colorectal cancer were obtained from the First Affiliated Hospital of Nanchang University for immunohistochemical analysis. This study was approved by the Human Research Ethics Committees of the First Affiliated Hospital of China Medical University and the First Affiliated Hospital of Nanchang University.

[0161] Immunohistochemical staining

[0162] Paraffin-embedded breast cancer or colorectal cancer tissue samples were dewaxed, rehydrated, and treated with sodium citrate-edta antigen retrieval solution for antigen extraction. Subsequently, the tissues were incubated with the primary antibody at room temperature for 1 - 2 hours. After extensive washing with PBS, the tissues were treated with the secondary antibody (Cat; No. GK500705, GeneTech) at room temperature for 1 hour. Subsequently, the sections were stained with 3'-diaminobenzidine (Cat; No. GK500705, GeneTech) for 5 minutes to show the staining signal, and then counterstained with hematoxylin. Photographs were taken using a Leica DMIRE2 microscope (Leica Microsystems Imaging Solutions Ltd.).

[0163] Isolation and purification of exosomes

[0164] Exosomes expressing the plasmid of the iRGD-LAMP2B fusion protein were collected from HEK293T cell culture medium (for the preparation method, refer to the reference PMID: 24345736). The sample was centrifuged at 4500 rpm for 20 minutes to collect the supernatant, filtered through a 0.45 μm membrane, and then through a 0.22 μm membrane. The exosome sample was concentrated using a hollow fiber column. The concentrated sample was chromatographically purified and then passed through the hollow fiber column again. Ultrafiltration concentration: The sample was added to an ultrafiltration tube and centrifuged at 3000 rpm for 10 - 15 minutes at 4°C. The retentate was filtered and sterilized through a 0.22 μm filter and stored at -80°C. Shanghai Youmi Biotechnology Co., Ltd. provided transmission electron microscopy and nanoparticle tracking analysis (NTA) services.

[0165] Loading siRNA into exosomes

[0166] The 2'-methoxy-modified siRNA was commissioned from GenePharma (Shanghai, China). The siRNA solution was combined with the exosome suspension (the ratio of siRNA to exosomes was 1 μg / 4 μg) in an electroporation cuvette. To effectively uptake siRNA without damaging exosomes, the voltage, pulse length, and number of pulses were optimized. After electroporation, it was incubated at room temperature for 60 minutes, and then centrifuged at 1×10 4 g for 60 minutes to remove free siRNA and isolate the exosomes.

[0167] Statistical analysis

[0168] All in vitro experiments were performed with three biological replicates. The in vivo experimental animals were randomly divided into different groups. The differences between two or more groups were analyzed by Student's t-test or one-way ANOVA. Statistical analysis was performed using GraphPad Prism 8.0 and expressed as mean ± standard deviation (SD). The Kaplan-Meier plot method and log-rank test were used to analyze the significant differences in patient survival rates. The multivariate Cox proportional hazards model generated the hazard ratio with a 95% confidence interval. Asterisks indicate statistically significant differences: *p < 0.05; **p < 0.01; ***p < 0.001.

[0169] (II) Results

[0170] Identifying the nucleolar protein BRIX1 as a regulator of p53

[0171] Due to the important role of ribosome homeostasis in controlling p53 activity and promoting cancer development, the present invention studied nucleolar proteins that may be crucial for ribosome biogenesis. Currently, 286 nucleolar proteins involved in yeast pre-rRNA processing have been identified.

[0172] These nucleolar proteins were selected according to the following criteria:

[0173] (i) Proteins that directly control pre-rRNA processing in yeast;

[0174] (ii) Proteins that are homologous between human and yeast;

[0175] (iii) Proteins that may have similar functions in ribosome biogenesis in humans and yeast;

[0176] (iv) Proteins whose role in cancer is not yet clear.

[0177] Therefore, six nucleolar proteins were obtained in the present invention: BRIX1, DHX35, EXOSC6, EXOSC7, LSM6, and PPAN for further analysis( Figure 1 A).

[0178] To test whether these nucleolar proteins are essential for the growth of cancer cells, the present invention knocked out six nucleolar proteins in four different cancer cell lines (including CAL-51, MCF-7, HCT116, and RKO), and performed cell viability assays. The research results of the present invention showed that the deletion of BRIX1 had a more significant inhibitory effect on the growth of all cell lines compared to the knockdown of other nucleolar proteins( Figure 1 B).

[0179] To explore the potential signaling pathways regulated by BRIX1, the present invention performed RNA sequencing (RNA-seq) analysis by knocking down BRIX1 in CAL-51 breast cancer cells. Heatmap and KEGG pathway enrichment analysis showed that knocking down BRIX1 activated the p53 pathway( Figure 1 C and 1D).

[0180] To verify this result, the present invention knocked down the expression of BRIX1 by two independent siRNAs and found that the lack of BRIX1 significantly increased the mRNA expression of p53 target genes CDKN1A (also known as p21), BTG2, and MDM2 in CAL-51, MCF-7, and HCT116 cells( Figure 1 E-1G).

[0181] Consistent with this, the knockdown of BRIX1 also induced the expression of p53 and p21 at the protein level( Figure 1 I-1K).

[0182] The upregulation of p21, BTG2, and MDM2 depends on p53, because the knockdown of BRIX1 had no effect on the expression in HCT116 p53- / - cells( Figure 1 H and 1L).

[0183] Interestingly, compared with the effects of treatment with cisplatin (DDP), 5-fluorouracil (5-FU), or actinomycin D (Act D), knockdown of BRIX1 did not result in an increase in γ-H2AX phosphorylation ( Figure 1 M), indicating that DNA damage stress was not induced.

[0184] Collectively, these results suggest that the nucleolar protein BRIX1 may act as a regulator of ribosome biogenesis and the p53 pathway.

[0185] BRIX1 promotes pre-rRNA processing through the PeBoW complex

[0186] It has been reported that Brix is involved in pre-rRNA processing and the synthesis of the large ribosomal subunit in yeast. However, it is not clear whether BRIX1 has a conserved function in human cells. To clarify this, the present invention performed gel electrophoresis analysis of total RNA in CAL-51 and HCT116 p53+ / + cells, and the results showed that when BRIX1 was knocked down, the level of 28S rRNA was significantly reduced ( Figure 2 A and 2B). These results were also confirmed in RT-qPCR analysis of the two cell lines ( Figure 2 C and 2D).

[0187] To investigate its potential mechanism, the present invention used the STRING database to predict the potential interacting proteins of BRIX1 and found that BOP1, PES1, and WDR12 had potential interaction relationships with BRIX1 (Supplementary Figure 1 A). These three proteins form the PeBoW complex, which is crucial for pre-rRNA processing and 60S ribosomal subunit maturation in mammalian cells. Therefore, the present invention speculated that BRIX1 may promote rRNA synthesis by interacting with the PeBoW complex.

[0188] To verify this hypothesis, the present invention performed a set of protein immunoprecipitation (co-IP) assays and found that exogenous BRIX1 interacted with exogenous BOP1 and PES1 ( Figure 2 E-2H), but did not interact with WDR12 (Supplementary Figure 1 B and 1C). Since the level of endogenous BOP1 is crucial for the formation of the PeBoW complex, the present invention further tested whether BRIX1 binds to endogenous BOP1 and regulates its level.

[0189] Although an endogenous interaction between BRIX1 and BOP1 was observed in cancer cells ( Figure 2I), but knocking out BRIX1 does not affect the protein level of BOP1. Notably, the research results of the present invention show that knocking down BRIX1 greatly reduces the interaction between BOP1 and PES1 ( Figure 2 J and 2K), which indicates that BRIX1 is necessary for the formation of the PeBoW complex. Since the PeBoW complex contributes to the synthesis and processing of 32S pre-rRNA, the present invention performed Northern blot analysis using a digoxigenin-labeled ITS2 probe, which can specifically detect 47S, 32S, and 12S pre-rRNA.

[0190] The research results of the present invention show that BRIX1 deletion mainly impairs the processing of 32S pre-rRNA, resulting in a significant reduction in 12S pre-rRNA, and the processing of 47S pre-rRNA may also be slightly affected ( Figure 2 L). In summary, these results indicate that BRIX1 is necessary for pre-rRNA processing by regulating the stability of the PeBoW complex.

[0191] Overexpression of BRIX1 inhibits nucleolar stress-induced p53 activation

[0192] Next, the present invention continued to explore the potential effect of overexpressing BRIX1 on p53 activity. To the surprise of the present invention, under normal growth conditions, overexpressing BRIX1 in cancer cells does not affect the expression level of p53. Therefore, the present invention attempted to detect whether overexpressing BRIX1 regulates p53 under cellular stress conditions.

[0193] Interestingly, the research results of the present invention show that overexpressing BRIX1 significantly reduces the p53 protein ( Figure 4 A and 4B) and its target genes ( Figure 4 C and 4D) in cancer cells during treatment with low-dose Act D and DDP or 5-FU. However, when overexpressing BRIX1 is treated with Nutlin-3 (an inducer that induces p53 by antagonizing MDM2), it cannot regulate the p53 level in cancer cells. It is well known that low concentrations of Act D preferentially inhibit RNA Pol I activity, and both DDP and 5-FU may impair ribosome biogenesis by inducing rDNA damage or inhibiting pre-rRNA processing. Therefore, these results indicate that BRIX1 inhibits the activation of p53 caused by nucleolar stress.

[0194] To clarify how BRIX1 inhibits the expression of p53, the present invention first detected the subcellular localization of BRIX1. The research results of the present invention show that BRIX1 is mainly distributed in the nucleolus under non-stress conditions or under Nultin-3 treatment, but under nucleolar stress induced by Act D, BRIX1 can migrate from the nucleolus to the nucleoplasm ( Figure 4 E).

[0195] Then, the present invention wanted to know whether overexpression of BRIX1 blocked the interaction between RPs and MDM2 in the nucleoplasm under nucleolar stress. Co-IP analysis showed that when cancer cells were treated with Act D, BRIX1 bound to RPL5 and RPL11 ( Figure 4 F).

[0196] In addition, overexpression of BRIX1 reduced the interaction between MDM2 and RPL5 and RPL11 ( Figure 4 G), probably because BRIX1 and these two nucleolar proteins competitively bind to MDM2. In addition, the present invention found that overexpression of BRIX1 enhanced MDM2-induced p53 ubiquitination ( Figure 4 H), and the p53 degradation caused by BRIX1 overexpression could be blocked by the proteasome inhibitor MG132 ( Figure 4 I and 4J).

[0197] Finally, cycloheximide chase analysis showed that overexpression of BRIX1 shortened the half-life of p53 protein ( Figure 4 K). These results indicate that BRIX1 translocates to the nucleoplasm to prevent the interaction between MDM2 and RPL5 / RPL11, thereby inhibiting the stabilization and activation of p53 induced by nucleolar stress.

[0198] Next, the present invention detected whether overexpression of BRIX1 promoted the survival and growth of cancer cells under nucleolar stress. The research results of the present invention showed that under low-dose Act d, overexpression of BRIX1 significantly increased the proliferation of cancer cells ( Figure 4 L and 4M) and colony formation ( Figure 4 N and 4O), and reduced apoptosis of cancer cells.

[0199] In addition, overexpression of BRIX1 promoted the migration of cancer cells ( Figure 4 P and 4Q).

[0200] In addition, the present invention determined the role of BRIX1 in vivo by establishing a transplanted tumor model. Consistent with the cell-based results, in HCT116 p53+ / + cells, stable overexpression of BRIX1 significantly promoted the growth rate, weight and size of the transplanted tumors derived from these cells ( Figure 4R-4T), without affecting the average body weight of mice ( Figure 4 U).

[0201] In summary, these results indicate that excessive BRIX1 counteracts the anti-tumor effect mediated by nucleolar stress by inactivating p53, suggesting that BRIX1 is involved in the process of promoting chemoresistance.

[0202] The tumor-suppressive effect of knocking down BRIX1 largely depends on p53

[0203] Since BRIX1 plays an important role in regulating p53 activity, the present invention wanted to know whether the deletion of BRIX1 inhibits colorectal cancer in a p53-dependent manner. For this purpose, the isogenic colorectal cancer cell lines HCT116 p53+ / + and HCT116 p53- / - .

[0204] The research results of the present invention show that knockdown of BRIX1 significantly inhibits the proliferation of HCT116 p53+ / + cells, while only slightly inhibiting the proliferation of HCT116 p53- / - cells ( Figure 5 A and 5B). Similarly, compared with HCT116 p53- / - cells, the effect of knocking down BRIX1 on the colony formation ability of HCT116 p53+ / + cells is more significant ( Figure 5 C and 5D).

[0205] In addition, the inhibitory effect of knocking down BRIX1 on the migration of colorectal cancer cells also partially depends on the status of p53 ( Figure 5 E and 5F).

[0206] These results indicate that in the absence of p53, BRIX1 deletion can moderately inhibit the growth and migration of cancer cells by interfering with ribosome biogenesis. However, the induction of G1 cell cycle arrest by knocking down BRIX1 is completely dependent on the presence of p53 ( Figure 5 G and 5H), because this effect is specifically mediated by the p53 target gene p21 ( Figure 1 E-1L).

[0207] Finally, the present invention tested in vivo whether knocking down BRIX1 inhibits tumor growth through p53. The research results of the present invention show that BRIX1 deletion significantly reduces the growth rate, weight, and size of xenograft tumors derived from HCT116 p53+ / + cells ( Figure 5 I-5L), while having little effect on the growth of xenograft tumors derived from HCT116 p53- / - cells ( Figure 5M-5P). These results indicate that the impact of BRIX1 deficiency on colorectal cancer cells with wild-type p53 is more significant compared to that on colorectal cancer cells lacking p53.

[0208] High expression of BRIX1 is associated with poor prognosis in breast and colorectal cancers

[0209] Considering that BRIX1 is essential for the survival and proliferation of breast and colorectal cancer cells, the present invention investigated the clinical significance of BRIX1 in these two types of cancers.

[0210] First, the present invention evaluated the expression of BRIX1 in breast cancer and matched normal tissues by IB and RT-qPCR analysis. The research results of the present invention showed that the levels of BRIX1 protein and mRNA in breast cancer tissues were increased compared to normal tissues ( Figure 6 A and 6B). In addition, immunohistochemical (IHC) analysis of 91 breast cancer tissues showed that high expression of BRIX1 was significantly associated with higher tumor / node / metastasis (TNM) stage and poorer overall patient survival rate ( Figure 6 C).

[0211] Univariate and multivariate Cox regression analyses further confirmed that high expression of BRIX1 was a factor for poor prognosis in breast cancer ( Figure 6 D). The present invention used colorectal cancer samples with low p53 expression to determine the clinical relevance of BRIX1 levels because samples with low p53 expression may contain wild-type p53. The immunohistochemical analysis of the present invention showed that the expression level of BRIX1 in colorectal cancer was higher than that in normal tissues.

[0212] Furthermore, both univariate and multivariate analyses indicated that BRIX1 was a prognostic factor for colorectal cancer. Finally, the present invention verified the clinical relevance of BRIX1 through the TCGA database. Compared to normal tissues, the mRNA and protein levels of BRIX1 were upregulated in various cancer tissues, and in different types of cancers, higher levels of BRIX1 were associated with poorer prognosis. In summary, the above research results indicate that high levels of BRIX1 promote ribosome biogenesis and limit p53 activity, which is crucial for cancer progression, while reducing BRIX1 activates p53 through nucleolar stress, thereby inhibiting cancer development.

[0213] Inhibiting colorectal cancer development by delivering therapeutic BRIX1 siRNA via iRGD-modified exosomes

[0214] Based on the findings of the present invention, targeting BRIX1 may be a potential strategy for treating cancer by triggering nucleolar stress. Therapeutic RNAi has become a powerful method for treating various diseases, including cancer, by effectively inhibiting the expression of any specific gene.

[0215] However, the use of RNAi faces many challenges, such as the instability of siRNA, insufficient penetration of cells or tissues, and induction of immune responses. Exosomes are natural nanoparticles with diameters between 30 and 150 nanometers and have shown considerable potential as drug delivery carriers. For example, exosomes exhibit excellent biocompatibility because they do not carry unnecessary exogenous factors that stimulate the immune system. In addition, the surface expression of CD47 prevents the clearance of exosomes by monocytes and macrophages. Recently, some therapeutic exosomes delivering RNAi drugs have been reported to inhibit cancer growth. Therefore, the present invention attempts to establish an exosome-based delivery system loaded with siRNA against BRIX1 for cancer treatment.

[0216] First, the present invention fuses the tissue-penetrating iRGD peptide to the N-terminus of the exosome surface protein LAMP2B. The iRGD peptide may guide exosomes into tumors by binding to αv integrin and neuropilin-1. The plasmid encoding the fused iRGD-LAMP2B protein is stably transfected into HEK293T cells, and engineered exosomes with surface-expressed iRGD are produced as donor cells ( Figure 8 A).

[0217] The morphology of the purified exosomes is verified by transmission electron microscopy (TEM) ( Figure 8 B) and nanoparticle tracking analysis (NTA) ( Figure 8 C).

[0218] In addition, only exosome markers TSG101, CD81, and CD9 are detected in exosomes, with mitochondrial protein COXⅣ as a control ( Figure 8 D). Next, fam-labeled siRNA is loaded into iRGD-modified exosomes by electroporation.

[0219] CAL-51 and HCT116 p53+ / + cells are treated with these exosomes for 8 - 12 hours and observed under a fluorescence microscope. The research results of the present invention show that the siRNA loaded in exosomes (irgd-exo-fam-siRNA), rather than free fam-siRNA, can be taken up by cancer cells ( Figure 7 A and 7C).

[0220] In addition, iRGD-Exo-siBRIX1 significantly increased the levels of p53 and p21 by reducing the expression of BRIX1 in both cell lines ( Figure 7 B and 7D).

[0221] In addition, the present invention tested whether iRGD-Exo-siBRIX1 inhibits the growth and migration of cancer cells. As expected, treatment of CAL-51 and HCT116 p53+ / + cells with iRGD-Exo-siBRIX1 significantly reduced their proliferation ( Figure 7 E and 7F), colony formation ( Figure 7 G and 7H), and migration ( Figure 7 I and 7J). These results indicate that engineered exosomes can serve as carriers for BRIX1 siRNA to enter cancer cells.

[0222] To test whether iRGD-modified exosomes target tumors, the present invention intravenously injected exosomes labeled with DiR (a lipophilic fluorescent dye) into transplanted mice. Although exosomes were mainly located in the liver and spleen, iRGD could significantly increase the accumulation of exosomes in the grafted tumors ( Figure 7 K and 7L).

[0223] Next, the present invention studied in vivo whether iRGD-Exo-siBRIX1 inhibits tumor growth. The siRNAs used in the following experiments were 2'-O-methyl modified to enhance their stability and binding affinity and reduce immune activation. The method of 2'-O-methyl modification of siRNA is known in the art. BALB / c nude mice were subcutaneously inoculated with HCT116 p53+ / + cells, and then iRGD-Exo-siBRIX1 or iRGD-Exo-siNC was intravenously administered three times a week.

[0224] The research results of the present invention showed that iRGD-Exo-siBRIX1 significantly inhibited the growth rate ( Figure 7 M), weight ( Figure 7 N), and size (Figure 7O) of the transplanted tumors, while no obvious weight loss was observed ( Figure 7 P).

[0225] In addition, the present invention attempted to study whether the combination of iRGD-Exo-siBRIX1 and chemotherapy can synergistically inhibit colorectal cancer. To verify this hypothesis, the present invention first determined whether knockdown of BRIX1 enhanced the sensitivity of HCT116 p53+ / + cells to 5-FU.

[0226] The results showed that deletion of BRIX1 reduced the half-maximal inhibitory concentration (IC50) of 5-FU and significantly inhibited the combined growth of 5-FU and cells.

[0227] In addition, the combination of siBRIX1 and 5-FU had a strong synergistic effect on cell viability, and the combination index (CI) was around 0.5 or less than 0.5 ( Figure 7 Q).

[0228] Then, the present invention evaluated the combined effect of iRGD-Exo-siBRIX1 and 5-FU in vivo. Nude mice bearing transplanted tumors were randomly divided into four groups: normal saline, 5-FU, 5-FU combined with iRGD-Exo-siNC, and 5-FU combined with iRGD-Exo-siBRIX1 for different treatments.

[0229] The results showed that 5-FU inhibited the growth of the grafted tumors ( Figure 7 R-7T). Notably, the combination of 5-FU and iRGD-Exo-siBRIX1 produced a more significant anti-tumor effect in transplanted mice, with significantly reduced tumor growth rate, weight, and size ( Figure 7 R-7T). The average body weight of the treated mice was comparable to that of the control group mice, indicating that the combined treatment had no obvious toxic effect on the mice ( Figure 7 U).

[0230] In summary, these results indicate that targeted inhibition of BRIX1 by iRGD-modified exosomes can effectively inhibit the growth of colorectal tumors and enhance their sensitivity to chemotherapy.

[0231] Discussion

[0232] Ribosome biogenesis is essential for the rapid growth and proliferation of cancer cells. Damage to this process leads to the translocation of ribosomal proteins (RPs) from the nucleolus to the nucleoplasm, thereby preventing mdm2-induced proteasomal degradation of p53. In this study, the present invention found that BRIX1 is a key regulator of ribosome biogenesis and nucleolar stress.

[0233] BRIX1 plays an oncogenic role through at least two mechanisms.

[0234] First, BRIX1 binds to BOP1 and PES1, promoting the formation of the PeBoW complex. This is mainly required for 32S pre-rRNA processing ( Figure 2 ).

[0235] In addition, BRIX1 interacts with RPL5 and RPL11, thereby preventing the interaction between RPs and MDM2. This leads to increased mdm2-induced ubiquitination and p53 degradation ( Figure 4 A-4K).

[0236] These results indicate that BRIX1 promotes cancer growth and progression by facilitating ribosome biogenesis and restricting p53 activity. More importantly, the deletion of BRIX1 triggers nucleolar stress by disrupting pre-rRNA processing ( Figure 2A-2D, 2L), leading to an increased interaction between RPL5 / RPL11 and MDM2, which in turn stabilizes and activates p53 Figure 1 C-1L and 3A-3H).

[0237] Finally, the research results of the present invention show that the targeted inhibition of BRIX1 by therapeutic exosomes effectively enhances 5-FU chemotherapy and inhibits the growth of wild-type p53 colorectal cancer Figure 7 ).

[0238] The present invention notes that BRIX1 can function in a p53-independent manner because the knockdown of BRIX1 slightly but significantly inhibits the growth, colony formation, and migration of p53-deficient colorectal cancer cells Figure 5 ). This demonstrates the anti-tumor effect of BRIX1 inhibitors in p53-deficient or mutant tumors.

[0239] The research results of the present invention show that the depletion of BRIX1 impairs pre-rRNA processing Figure 2 ), and disrupts the nucleolar structure, as shown by the relocalization of NPM1. This leads to nucleolar stress without causing DNA damage Figure 1 M), leading to RPL5 / RPL11-dependent p53 activation. Conversely, excessive BRIX1 blocks p53 activation by interacting with RPL5 and RPL11. Therefore, the research of the present invention shows that BRIX1 is another target molecule that selectively induces nucleolar stress.

[0240] In the present invention, the present invention developed iRGD-modified exosomes loaded with siRNA against BRIX1 and verified that these exosomes can transport siBRIX1 to the tumor site Figure 7 K and 7L), and increase the uptake of siBRIX1 by cancer cells Figure 7 A and 7C). Notably, the research results of the present invention show that iRGD-Exo-siBRIX1 effectively inhibits the growth of colorectal cancer in vivo and enhances the efficacy of 5-FU chemotherapy Figure 7 M-7U). Notably, the tumor-targeting potential of exosomes may be further improved.

[0241] In summary, the research of the present invention determines that BRIX1 is an oncoprotein overexpressed in colorectal cancer and breast cancer, which is associated with poor prognosis. BRIX1 promotes pre-rRNA processing by interacting with the PeBoW complex and inhibits p53 activity by sequestering RPL5 and RPL11. Conversely, the deletion of BRIX1 activates p53 and inhibits cancer growth by triggering nucleolar stress. Finally, the research results of the present invention indicate that using the delivery system of exosomes to load BRIX1-specific siRNA may be a promising cancer treatment method.

[0242] All documents mentioned in the present invention are cited herein by reference as if each individual document was cited by reference. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims

1. A siRNA targeting the BRIX1 gene, characterized in that: The siRNA is selected from the group consisting of: siBRIX1-1:5'-CGTGTTTACTTTCACCATT-3', and siBRIX1-2: 5'-GCATCGGCGTGTCATAAGA-3'.

2. A siRNA precursor (shRNA), characterized in that The siRNA precursor is the siRNA precursor of claim 1; preferably, the 5' to 3' ends of the siRNA precursor sequentially include: a first sequence unit, a stem-loop sequence unit and a second sequence unit, the first sequence unit and the second sequence unit are complementary so that the siRNA precursor forms a hairpin structure, and the first sequence unit is selected from the following group: siBRIX1-1:5'-CGTGTTTACTTTCACCATT-3', and siBRIX1-2: 5'-GCATCGGCGTGTCATAAGA-3'.

3. An exosome, characterized in that: The exosomes are loaded with the siRNA or its precursor according to claim 1.

4. A use of an inhibitor of the BRIX1 gene or its encoded protein, characterized in that: Used for: (1) Preparation of drugs for preventing or treating tumors; (2) preparing reagents for activating p53; (3) preparing drugs that inhibit tumors through p53-dependent mechanisms; or (4) Prepare drugs that inhibit tumors through p53-independent mechanisms.

5. The use according to claim 1, characterized in that The BRIX1 gene is selected from the group consisting of: (A) a polynucleotide sequence encoding the polypeptide shown in SEQ ID NO. 2; (B) the polynucleotide sequence shown in SEQ ID NO. 1; (C) a polynucleotide sequence formed by replacing, deleting or adding one or more nucleotides of the polynucleotide sequence shown in SEQ ID NO. 1; (D) a polynucleotide sequence having a sequence homology of ≥90%, preferably ≥95%, more preferably ≥98%, and most preferably ≥99% compared to the polynucleotide sequence shown in SEQ ID NO.: 1; (E) A polynucleotide sequence complementary to the polynucleotide sequence described in any one of (A) to (D).

6. The use according to claim 1, characterized in that The BRIX1 gene inhibitor is: BRIX1 gene-specific siRNA or its precursor, BRIX1 gene-specific microRNA or its precursor, an inhibitor that inhibits the BRIX1 gene promoter, or a combination thereof.

7. The use according to claim 1, characterized in that The tumor is a p53 wild-type tumor, or the tumor is a p53 mutant tumor.

8. A pharmaceutical composition, characterized in that The invention comprises a pharmaceutically acceptable carrier and an effective amount of active ingredients, wherein the active ingredients are inhibitors of BRIX1 gene or its encoded protein.

9. A method for non-therapeutic inhibition of tumor cells in vitro, characterized in that: The method comprises the steps of: culturing tumor cells in the presence of an inhibitor of the BRIX1 gene or its encoded protein, thereby inhibiting the tumor cells.

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