Brix1 inhibitors and their use in anti-tumor

Abstract

This invention discloses BRIX1 inhibitors and their use in anti-tumor therapy. Specifically, this invention discloses that nucleolar protein BRIX1 is a key regulator of homeostasis between ribosome biogenesis and p53 activation. The results of this invention show that inhibiting BRIX1 can induce nucleolar stress activation of p53, thereby inhibiting tumor growth. Furthermore, this invention constructs engineered exosomes with iRGD surface decoration and loaded with BRIX1-specific siRNA for cancer treatment.

Description

Technical Field

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

[0002] Ribosome biogenesis is a multi-step process involving three basic steps: the synthesis of ribosomal RNA (rRNA) and ribosomal proteins (RPs), the processing of rRNA precursors, and the assembly of 40S and 60S ribosomal subunits in the nucleolus. An abnormal increase in the size and number of nucleoli is considered a key marker of malignant cells and is associated with poor prognosis in cancer patients. This is because cancer cells, during rapid growth and proliferation, require a faster rate of ribosome production than normal cells to meet the demands of protein synthesis. 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 is involved in essential factors in ribosomal 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 disruption of any key step in ribosomal biogenesis can lead to nucleolar stress, also known as ribosomal stress. Under this stress, some ribosomal proteins, particularly RPL5 and RPL11, translocate from the nucleolus to the nucleoplasm, interact with MDM2, and inhibit the ubiquitination and degradation of p53 by its E3 ligase, thereby activating p53. p53 is located on the short arm of human chromosome 17 (17p13.1) and consists of 11 exons, 10 introns, and 393 amino acid residues. p53 is an important tumor suppressor gene and also one of the most commonly mutated genes in human cancers.

[0004] Furthermore, nucleolar stress may lead to tumor suppression through p53-independent signaling pathways. Several 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. Various strategies have been shown to trigger nucleolar stress; for example, many chemotherapeutic drugs can induce ribosomal DNA (rDNA) damage or inhibit pre-rRNA processing, thereby impairing ribosome biosynthesis. Some compounds, such as CX-5461 and BMH-21, limit rRNA synthesis by inhibiting RNA polymerase (Pol)I activity. However, these drugs have also been found to cause DNA 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 dedicated to developing the potential of inducing nucleolar stress as a cancer treatment strategy in order to obtain more effective anti-tumor drugs. Summary of the Invention

[0006] The purpose of this invention is to provide BRIX1 inhibitors and their use in antitumor therapy.

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

[0008] (1) To prepare drugs for the prevention or treatment of tumors;

[0009] (2) Preparation of reagents to activate p53;

[0010] (3) To prepare drugs that inhibit tumors through a p53-dependent mechanism; or

[0011] (4) Prepare drugs that inhibit 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 the polypeptide shown in SEQ ID NO.2;

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

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

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

[0017] (E) A polynucleotide sequence complementary to any of the polynucleotide sequences described in (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 of the BRIX1 gene or its complementary sequence.

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

[0023] In another preferred embodiment, the inhibitor of the BRIX1 gene-encoded protein is selected from the group consisting of: antibodies to the BRIX1 gene-encoded protein and binding proteins to the BRIX1 gene-encoded protein.

[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 invention, a pharmaceutical composition is provided 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 a protein encoded thereon.

[0027] In another preferred embodiment, the pharmaceutical composition is used for the prevention or treatment of tumors.

[0028] In another preferred embodiment, the inhibitor of the BRIX1 gene-encoded protein is selected from the group consisting of: antibodies that specifically target the BRIX1 gene-encoded protein, binding proteins that specifically bind to the BRIX1 gene-encoded protein, and compounds that inhibit the activity of the BRIX1 gene-encoded protein.

[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 that inhibits 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 or its complementary sequence in the BRIX1 gene.

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

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

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

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

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

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

[0038] In another preferred embodiment, 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 embodiment, the inhibition of tumor cells is to inhibit the growth of tumor cells or to inhibit tumor cell tumorigenesis.

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

[0041] In another preferred embodiment, compared with control tumor cells, the activity of the BRIX1 gene-encoded protein 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 by completely eliminating 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 by no expression of the BRIX1 gene at all.

[0043] In a fourth aspect of the invention, a siRNA targeting the BRIX1 gene is provided, said siRNA being selected from the group consisting of:

[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 invention, a siRNA precursor (shRNA) is provided, which is a precursor of the siRNA described in the fourth aspect of the invention.

[0047] In another preferred embodiment, 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, wherein the first and second sequence units are anti-complementary, causing the siRNA precursor to form a hairpin structure, and the first sequence unit is selected from the group consisting of:

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

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

[0050] In a sixth aspect of the invention, an exosome is provided, the exosome being loaded with the siRNA or a precursor thereof as described in the fourth aspect of the invention.

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

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

[0053] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (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 described in detail here. Attached Figure Description

[0054] The following figures are used to illustrate specific embodiments of the invention and are not intended to limit the scope of the invention as defined by the claims.

[0055] Figure 1 Knockdown of BRIX1 activates p53. (A) The Venn diagram illustrates the screening of potential regulators of ribosomal biogenesis. (B) The heatmap shows that BRIX1 deficiency significantly inhibited 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 increased in BRIX1-deficient CAL-51 cells. (D) KEGG pathway enrichment analysis results show that target genes in the p53 pathway are enriched in BRIX1-deficient cells. (EG) Knockdown of BRIX1 can increase the mRNA levels of p53 target genes. CAL-51 (E), MCF-7 (F), and HCT116 cells were transfected with control or BRIX1 siRNA. p53 + / + (G) Cells were cytosed for 48 hours, followed by RT-qPCR analysis. (H) BRIX1 knockdown on HCT116 cells. p53- / - The levels of p53 target gene mRNA in cells were not affected. IK knockdown of BRIX1 increased the protein levels of p53 and its target genes. CAL-51(I), MCF-7(J), and HCT116 cells were transfected with control or BRIX1 siRNA. p53+ / + (K) Cells were analyzed for 48 hours, followed by IB analysis. (L) BRIX1 knockdown on HCT116 p53- / - Protein levels of p53 target genes in cells were not affected. (M) Knockdown of BRIX1 did not increase phosphorylation levels of γ-H2AX. Cells were treated with the drug for 24 hours or transfected with siRNA for 48 hours before IB analysis. ***p<0.001.

[0056] Figure 2 BRIX1 promotes pre-rRNA processing via the PeBoW complex. (A, B) Knockdown of BRIX1 reduces 28S rRNA levels. CAL-51 (A) and HCT116 were transfected with control or BRIX1 siRNA. p53+ / +(B) Cells were subjected to agarose gel electrophoresis and ImageJ quantitative analysis after 48 hours. (C,D) RT-qPCR showed that knockdown of BRIX1 reduced 28S rRNA levels. (EH) BRIX1 interacts with BOP1 and PES1. Cells were transfected with plasmids for 48 hours, followed by co-IP-IB analysis using the specified antibody. (I) Endogenous interaction between BRIX1 and BOP1. HCT116 cells were treated with MG132. p53+ / + Cells were treated for 6 hours and co-IP-IB analysis was performed. (J,K) BRIX1 knockdown inhibits the interaction between BOP1 and PES1. Cells were transfected with the specified siRNA and plasmid, followed by co-IP-IB analysis using the specified antibody. (L) BRIX1 knockdown impairs pre-rRNA processing. Cells were transfected with control or BRIX1 siRNA, followed by Northern blotting analysis. A schematic diagram of the ITS-2 probe (orange) in the experiment is shown in the figure. ***p<0.001.

[0057] Figure 3 Knockdown of BRIX1 activates the nucleolar stress-p53 pathway, inhibiting tumor growth. Knockdown of RPL5 or RPL11 in (AD) inhibits p53 activation following BRIX1 loss. CAL-51(A,B) and HCT116 were transfected with siRNA. p53+ / + (C,D) Cells were examined for 48 hours before IB analysis. (E,F) BRIX1 knockdown increased the interaction between MDM2 and RPL5 and RPL11, respectively. Co-IP-IB analysis was performed using cells stably expressing shNC or shBRIX1 with antibodies. Cells were treated with the proteasome inhibitor MG132 for 5 hours before harvesting. (G) BRIX1 knockdown reduced MDM2-induced p53 ubiquitination. HCT116 cells stably expressing control or BRIX1 shRNA were transfected with plasmids. p53- / -Cells were treated with MG132 for 5 hours after 48 hours, followed by in vivo ubiquitination assays and IB analysis. (H) BRIX1 knockdown prolonged the half-life of p53 protein. Cells were transfected with control or BRIX1 siRNA. Cells were treated with cycloheximide (CHX) (100 mg / ml) for the specified time before IB analysis. (I,J) BRIX1 knockdown 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, followed by seeding in 96-well plates for cell viability assays. (K,L) BRIX1 knockdown inhibited the clonogenic 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, followed by seeding in 6-well plates for approximately 14 days. Clones were fixed with methanol and stained with crystal violet. (M,N) In breast cancer cells carrying wild-type p53, BRIX1 knockdown induced G1 cell cycle arrest. CAL-51(M) and MCF-7(N) cells were transfected with control or BRIX1 siRNA for 48 hours, followed by flow cytometry analysis. (O,P) BRIX1 knockdown 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, followed by cell migration assays. ***p<0.001.

[0058] Figure 4 Overexpression of BRIX1 impairs p53 activation under nucleolar stress. (AD) BRIX1 overexpression inhibits the expression of p53 and its target genes under nucleolar stress. CAL-51(A,C) and HCT116 were transfected with plasmids as instructed. p53+ / + (B,D) Cells were treated with 10 nM Act D for 24 h, followed by IB or RT-qPCR analysis. (E) BRIX1 translocated from the nucleolus to the nucleoplasm in the presence of Actin D (10 nM), but did not translocate in the presence of Nutlin-3 (20 μM). Cells were transfected with plasmids and treated with the drugs as instructed, followed by immunofluorescence staining. (F) BRIX1 interacts 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, followed by co-IP-IB analysis. (G)BRIX1 inhibited the interaction between MDM2 and RPL5 / RPL11. HCT116 cells were transfected with plasmids as instructed. 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 p53 ubiquitination. Stable overexpression controls or BRIX1-containing HCT116 cells were transfected with the specified plasmid. p53- / - Cells were treated with Act D (10 nM) for 48 h, then with MG132 (20 μM) for 24 h, followed by in vivo ubiquitination experiments. (I,J) plasmid transfected HCT116 p53+ / + CAL-51 cells 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 plasmid. p53+ / + Cells were treated with Act D (10 nM) and CHX (100 mg / ml) for 24 h at different time points, and then analyzed by IB. Overexpression of BRIX1 increased CAL51 (L, M) and HCT116. p53+ / + (M) Cell proliferation. Cells were transfected with plasmids and treated with Act D (10 nM), followed by cell viability assays. (N,O) Overexpression of BRIX1 enhanced CAL51(N) and HCT116. p53+ / + (O) Cell colony formation ability. Stable overexpression of control or BRIX1 cells was treated with 10 nM Act D and then subjected to colony formation assays. (P, Q) BRIX1 overexpression increased the migration of CAL51 (P) and HCT116 p53+ / + (Q) cells. Stable overexpression of control or BRIX1 cells was treated with 10 nM Act D and then subjected to cell migration assays. *p<0.05, **p<0.01, ***p<0.001. (RU) BRIX1 overexpression promoted HCT116 cell migration. p53+ / + The growth rate (R), weight (S), and size (T) of cell-derived transplanted tumors were measured, but mouse body weight (U) was not affected. Mice were intraperitoneally injected with 30 μg / kg Act D on the designated day after inoculation. Data are presented as mean ± SD, n = 5. **p < 0.01, **p < 0.001.

[0059] Figure 5 The tumor-suppressive effect of BRIX1 knockdown is largely dependent on p53. (A,B) and HCT116 p53- / - Compared to cells (B), knockdown of BRIX1 had a significant impact on HCT116 cells. p53+ / + The growth inhibition effect was more significant in cells (A). Knockdown of BRIX1 in cells (C,D) reduced HCT116. p53+ / + Cell (C) compared to HCT116 p53- / -Cells (D) showed a significantly reduced colony-forming ability. Cells were transfected with siRNA and colony-forming assays were performed. (E, F) and HCT116 p53- / - Compared to cells (F), BRIX1 knockdown on HCT116 cells... p53 + / + Cell migration (E) was more significantly affected. Cells were transfected with siRNA and cell migration assays were performed as instructed. (G,H) were in HCT116 p53+ / + In (G) cells, knockdown of BRIX1 induces G1 phase arrest, while in HCT116 cells... p53- / - This effect is absent in cells (H). Cells were transfected with siRNA as shown in the diagram, followed by flow cytometry analysis. (IP) BRIX1 deletion significantly inhibited HCT116. p53 + / + The growth rate (I), weight (J), and size (K) of cellular tumors, but for HCT116 p53- / - Cellular tumors (MOs) had minimal impact. Mouse body weight was unaffected (L, P). Data are expressed as mean ± SD, n = 6. *p < 0.05, **p < 0.01, ***p < 0.001.

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

[0061] Figure 7 Therapeutic BRIX1 siRNA delivered via iRGD-modified exosomes inhibited colorectal cancer in vitro and in vivo. (A,C) iRGD-modified exosomes transported fam-tagged siBRIX1 to cancer cells. CAL-51 and HCT116 p53+ / + Cells were incubated with FAM-siBRIX1 or iRGD-Exo-FAM-siBRIX1 for 8 hours, followed by fluorescence microscopy observation. FAM-siBRIX1, green; DAPI, blue for cell nuclei; scale bar, 10 μm. (B,D) iRGD-Exo-siBRIX1 activates p53. CAL-51(B) and HCT116 p53+ / + (D) Cells were incubated with designated exosomes for 48 hours, followed by IB analysis. (EJ) iRGD-Exo-siBRIX1 inhibited CAL-51 and HCT116. p53+ / + Cell growth (E, F), colony formation (G, H), and migration (I, J). CAL-51 and HCT116p53+ / + Cells were incubated with designated exosomes for 24 hours, followed by cell viability assays, colony formation assays, or cell migration assays. (K,L)iRGD increased the enrichment of exosomes in the tumor region. DIR-labeled exosomes were intravenously injected into cells carrying exosomes from HCT116. p53+ / + Cells were transplanted into BALB / c nude mice containing tumor cells and analyzed by in vivo fluorescence imaging. (MP)iRGD-Exo-siBRIX1 inhibits HCT116. p53+ / + Growth of cell-derived transplanted tumors. (Q)siBRIX1 combined with 5-FU synergistically inhibits cancer cell growth. HCT116 p53+ / + Cells were administered the combined drugs as directed for 48 hours, followed by cell viability assays and Chou-Talalay analysis. (RU)iRGD-Exo-siBRIX1 and 5-FU jointly inhibited HCT116. p53+ / + Growth of cell-derived transplanted tumors. Data are expressed 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 expressing iRGD on the surface. (B) Transmission electron microscopy to verify the morphology of purified exosomes. (C) Nanoparticle tracking analysis to verify the morphology of purified exosomes. (D) Detection results of exosome markers. Detailed Implementation

[0063] Through extensive and in-depth research, the inventors discovered that BRIX1 primarily promotes the processing of 12S and 32S rRNA precursors by binding to the PES1-BOP1-WDR12 (PeBoW) complex. BRIX1 deficiency induces nucleolar stress and activates p53, while BRIX1 overexpression alleviates nucleolar stress-induced p53 activation by blocking the interaction between RPL5 / RPL11 and MDM2. The findings also indicate that BRIX1 is highly expressed in breast and colorectal cancers, and higher BRIX1 levels are associated with poorer prognosis. These findings suggest that BRIX1 is a potential target for cancer therapy. This invention develops a delivery system (Exo-siBRIX1) that utilizes engineered exosomes to load siRNA targeting BRIX1. This Exo-siBRIX1 has been shown to effectively inhibit cancer growth in vivo. In summary, the research of this invention reveals the crucial role of BRIX1 in regulating the nucleolar stress-p53 pathway and provides a potential BRIX1-targeting strategy for cancer therapy.

[0064] the term

[0065] BRIX1 gene and its encoded protein

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

[0067] The human BRIX1 gene (NCBI sequence 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 shown in SEQ ID NO.2:

[0070] MAATKRKRRGGFAVQAKKPKRNEIDAEPPAKRHATAEEVEEEERDRIPGPVC

[0071] KGKWKNKERILIFSSSRGINFRTRHLMQDLRMLMPHSKADTKMDRKDKLFVINE

[0072] VCEMKNCNKCIYFEAKKKQDLYMWLSNSPHGPSAKFLVQNIHTLAELKMTGN

[0073] CLKGSRPLSFDPAFDELPHYALLKELLIQIFSTPRYHPKSQPFVDHVFTFTILDN

[0074] RIWFRNFQIIEEDAALVEIGPRFVLNLIKIFQGSFGGPTLYENPHYQSPNMHRRVI

[0075] RSITAAKYREKQQVKDVQKLRKKEPKTLLPHDPTADVFVTPAEEKPIEIQWVKPEPKVDLKARKKRIYKRQRKMKQRMDSGKTK(SEQ ID NO.2)

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

[0077] BRIX1 gene inhibitors

[0078] As used herein, the term "substance that inhibits gene expression" refers to any substance that inhibits the transcription of the mRNA of a target gene, breaks down transcribed mRNA, or inhibits the translation of mRNA into protein, without particular limitation. Examples of such substances include nucleic acids, such as siRNA and miRNA. siRNA is preferred. In addition to the above, "substances that inhibit gene expression" may also include proteins or peptides, or other small molecules. It should be noted that in this invention, the target gene is the BRIX1 gene.

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

[0080] As used in this article, the term "RNAi" (RNA interference) refers to the highly conserved evolutionary phenomenon of efficient and specific degradation of RNA with complementary sequences induced by double-stranded RNA (dsRNA). Because RNAi technology can specifically shut down the expression of specific genes, it has been widely used in areas such as gene function research and gene therapy for infectious diseases and tumors.

[0081] As used in this article, the term "siRNA" (Small interfering RNA) refers to a small RNA molecule (approximately 21-25 nucleotides) that can be processed from its precursors (such as dsRNA, shRNA, etc.) by Dicer (an enzyme in the RNase III family that is specific to double-stranded RNA), or it can be synthesized chemically or produced by processing other proteins. siRNA is a major member of siRISC, stimulating the rapid cleavage and degradation of its complementary target RNA, leading to the silencing of the target gene, thus becoming 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, that is, selectively processed by Dicer or other similar proteins to produce mature siRNA, thereby enabling RNAi.

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

[0084] In a preferred embodiment of the present invention, an expression cassette is provided. As used herein, the term "expression cassette" refers to an expression cassette containing the coding sequence of the siRNA precursor of the present invention, as well as a promoter and a termination signal operatively linked to said coding sequence, said expression cassette producing the siRNA precursor of the present invention post-transcriptionally.

[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, this hairpin sequence is expressed, forming a double-stranded RNA (shRNA) with a terminal loop structure. This shRNA is then recognized and processed by the Dicer protein in the cell, producing a functional siRNA.

[0086] As used herein, the term "shRNA" refers to a specific type of shRNA constructed using the precursor of human miR-26b as its backbone. The shRNA comprises, from 5′ to 3′, the following: (a) a 5′ flanking sequence region; (b) a 5′ paired siRNA region; (c) a apical loop region; (d) a 3′ paired siRNA region, wherein the 5′ paired siRNA region and the 3′ paired siRNA region form a double-stranded region; and (e) a 3′ flanking sequence region. The shRNA produces siRNA, and the nucleotide sequence of the siRNA corresponds to either the 3′ paired siRNA region or the 5′ paired siRNA region.

[0087] In a broad sense, shRNA is an abbreviation for short hairpin RNA. shRNA consists of two short, inversely complementary sequences separated by a terminal loop sequence, forming a hairpin structure. Transcription is typically controlled by the promoter of endogenous RNA polymerase III, with 5-6 T-termini attached to the end of the shRNA sequence as a transcription terminator for RNA polymerase III. shRNA can also be transcribed from the promoters of other RNA polymerases.

[0088] As used in this article, the term "miRNA" (microRNA) refers to a class of non-coding single-stranded RNA molecules, approximately 20-24 nucleotides in length, encoded by endogenous genes, which participate in the regulation of expression of a large number of genes in plants and animals. To date, more than four thousand miRNA molecules have been discovered in plants, animals, and viruses. Most miRNA genes exist in the genome as single copies, multiple copies, or gene clusters. Each miRNA can regulate multiple target genes, and several miRNAs can also work together to regulate the same gene, forming a complex regulatory network. It is estimated that miRNAs regulate the expression of more than half of the genes in humans. miRNAs exist in various forms, the most primitive being pri-miRNA; after processing by Drosha, pri-miRNA becomes pre-miRNA, i.e., miRNA precursor, approximately 50-90 nucleotides in length; pre-miRNA is then cleaved by the Dicer enzyme to become mature miRNA, approximately 20-24 nucleotides in length. miRNAs mainly inhibit target gene expression by inhibiting translation and accelerating mRNA deadenylation, a mechanism different from siRNA-mediated mRNA degradation.

[0089] exosomes

[0090] As used in this article, exosomes are tiny vesicles secreted by cells, approximately 30-200 nm in diameter, with a density of 1.13-1.21 g / ml. They have 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 media. Almost all types of cells (immune cells, nerve cells, stem cells), including tumor cells, can produce and release exosomes.

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

[0092] This invention constructs engineered exosomes with iRGD (a tumor-targeting peptide) on their surface and loaded with BRIX1-specific siRNA for cancer treatment. Notably, these engineered exosomes, iRGD-Exo-siBRIX1, significantly inhibit the growth of colorectal cancer in vivo and improve the efficacy of 5-FU chemotherapy.

[0093] Pharmaceutical Composition

[0094] The present invention provides a pharmaceutical 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” means an amount that is functional or active 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 formulation 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 solutions, glucose, water, glycerol, ethanol, powders, and combinations thereof. The pharmaceutical formulation should be matched to the route of administration.

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

[0099] The pharmaceutical compositions of the present invention can be formulated into liquid preparations, which can be carried out by conventional methods and should be manufactured under aseptic conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 1 microgram / kg body weight to about 50 mg / kg body weight daily, about 5 micrograms / kg body weight to about 10 mg / kg body weight daily, and about 10 micrograms / kg body weight to about 5 mg / kg body weight daily. Furthermore, the formulations of the present invention can also be used with other therapeutic agents.

[0100] When using the formulation of this invention, a safe and effective amount of the drug is administered to mammals, wherein the safe and effective amount is generally at least about 10 micrograms per kilogram of body weight, and in most cases does not exceed about 50 milligrams per kilogram of body weight, preferably about 10 micrograms per kilogram of body weight to about 20 milligrams per kilogram of body weight. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health condition, which are all within the scope of the skill of a skilled physician.

[0101] As used herein, a "pharmaceuticalally acceptable" ingredient is a substance suitable for human and / or mammalian use without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., a substance with a reasonable benefit / risk ratio. The term "pharmaceuticalally acceptable carrier" refers to a carrier used for the administration of therapeutic agents, including various excipients and diluents.

[0102] The pharmaceutical compositions of the present invention contain 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 solutions, glucose, water, glycerol, ethanol, and combinations thereof. Generally, pharmaceutical formulations should be matched to the route of administration; the dosage forms of the pharmaceutical compositions of the present invention are injections, oral formulations (tablets, capsules, oral liquids), transdermal formulations, and sustained-release formulations. They are prepared, for example, using physiological saline or an aqueous solution containing glucose and other excipients by conventional methods. The pharmaceutical compositions are preferably manufactured under aseptic conditions.

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

[0104] The pharmaceutically acceptable carriers described in this invention include (but are not limited to): water, saline, liposomes, lipids, proteins, protein-antibody conjugates, peptides, cellulose, nanogels, or combinations thereof. The choice of carrier should be matched to the route of administration, as is well known to those skilled in the art.

[0105] application

[0106] The present invention provides a method for treating tumors, comprising the steps 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-therapeutic inhibition of tumor cells in vitro, comprising the steps of: culturing tumor cells in the presence of an inhibitor of the BRIX1 gene or its encoded protein, thereby inhibiting 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 BRIX1 gene-encoded protein inhibitor inhibits the activity of the BRIX1 gene-encoded protein in the tumor cells; or the BRIX1 gene inhibitor 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 tumor formation of tumor cells.

[0111] Preferably, compared with control tumor cells, the activity of the BRIX1 gene-encoded protein in the tumor cells is reduced by more than 10%, more 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 by completely eliminating the activity of the BRIX1 gene-encoded protein.

[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%, more 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 by no expression of the BRIX1 gene at all.

[0113] The main advantages of this invention are:

[0114] (1) It was verified that the BRIX1 gene and its encoded protein can serve as novel tumor therapeutic targets.

[0115] (2) A highly efficient siRNA for inhibiting the BRIX1 gene was obtained.

[0116] (3) Exosomes with excellent anti-tumor effects targeting the BRIX1 gene were obtained.

[0117] The present invention will be further described in detail below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions as described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, parts and percentages are by weight.

[0118] Example

[0119] (I) Materials and Methods

[0120] Cell culture and transient transfection

[0121] Human colorectal cancer cell line HCT116 p53+ / + HCT116 p53- / -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 derived from ATCC and were mycoplasma-free as confirmed by PCR. Cells were seeded at optimal density 12–24 h prior to transfection. Transfection was performed using Hieff transliposome transfection reagent (Yeasen) according to the manufacturer's protocol.

[0122] plasmids and antibodies

[0123] Primers were designed to amplify BRIX1, BOP1, PES1, and WDR12 cDNAs, and the amplified sequences were then cloned 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. The following 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), and anti-coxⅣ (Cat; No. 11242-1-AP, Proteintech) were purchased commercially.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). Development was performed using ECL chemiluminescence reagent (Yeasen).

[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 Ct method with normalized GAPDH.

[0126] Transcriptome sequencing

[0127] 48 hours after transfection of CAL-51 cells with siNC or siBRIX1, 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 benzyl sulfonyl fluoride, and a mixture of 10% protease inhibitors], and quantified using BCA reagent (Yeasen). Equal volumes of clear cell lysis buffer (20–80 μg) were used for Western blot (IB) analysis.

[0130] Northern Imprint

[0131] Northern blotting was performed using the NorthernMax-Gly kit (Thermo Fisher Scientific, USA). RNA (30–60 μg) denatured with glyoxal-loaded dye was separated by electrophoresis on a 1% agarose gel and transferred to BrightStar gel. TMRNA was cross-linked onto a Plus nylon membrane (Thermo Fisher Scientific, USA) under 1.5 J cm² UV irradiation, pre-hybridized at 65°C for 30 min, and hybridized with the probe overnight at 42°C. The next day, the membrane was washed with Low Stringency Wash Solution for 10 min at room temperature, followed by washing at 42°C for 2 min, and then blocked with blocking buffer for 30 min. Subsequently, the membrane was incubated with anti-DIG at room temperature for 1 h (Universal Biotech Co., Shanghai), washed twice with washing buffer for 15 min each time, and washed with detection buffer for 5 min each time. Finally, the membrane was prepared using 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 with methanol at -20°C, washed three times with PBS, and incubated for 1 hour at room temperature with blocking buffer (8% BSA and 0.3% Triton X-100). They were then incubated overnight at 4°C with primary antibodies (anti-BRIX1, 1:100; anti-NPM1, 1:50 dilution). The next day, 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 using 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 a 10% protease inhibitor mixture], followed by immunoprecipitation (IP) with antibodies. Figure 4 As shown. In short, the protein (0.5-1 mg) was incubated with the specified antibody at 4°C for 5 hours, followed by incubation 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 using IB.

[0136] In vivo ubiquitination assay

[0137] As shown in the figure, plasmids containing 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. In short, 48 hours post-transfection, cells were collected and divided into two aliquots, one for IB and the other for the ubiquitination assay. Cell globules were lysed in buffer I [8M urea, 0.1M Na2HPO4 / NaH2PO4 (pH 8.0)), 10mM Tris-HCl (pH 8.0), 10mM β-mercaptoethanol, and 5mM imidazole] and incubated with Ni-NTA beads (Takara) at room temperature for 4 hours. The Ni-NTA beads (Takara) captured his-labeled proteins / complexes. The beads were washed twice with buffer I, and then twice with buffer II [8M urea, 0.1M Na2HPO4 / NaH2PO4 (pH 6.3), 10mM Tris-HCl (pH 6.3), and 10mM β-mercaptoethanol] to elute the captured protein, and then subjected to IB experiments with the specified antibody.

[0138] RNA interference and the generation of stable cell lines

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

[0140] We designed siRNA targeting the BRIX1 gene and tested its knockdown efficiency and effectiveness.

[0141] siRNA knockdown efficiency screening:

[0142] After the designed siRNA was synthesized, cellular RNA was extracted after transfection into cells, and after reverse transcription, the knockdown efficiency of the BRIX1 gene was detected by qPCR.

[0143] The following are some representative siRNAs designed:

[0144] 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％ Comparison siNC / 0

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

[0146] siRNA transfection was performed using Hieff transliposome transfection reagent according to the manufacturer's protocol (Yeasen). All transfections were validated by qPCR or Western blot. A BRIX1-targeting shRNA-specific sequence was obtained from Sigma-Aldrich and cloned into the PLKO.1 vector. The plasmid containing the shRNA, along 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 using 1 μg / ml puromycin.

[0147] Cell viability assay

[0148] To assess cell viability, 2-4 × 10⁶ cells were injected with water 6-12 hours after transfection. 3 Cells were seeded in 96-well plates. Cell counting kit-8 (CCK-8) reagent (Yeasen) was added to each well to a final concentration of 10%. Cells were incubated at 37°C for 2 hours, and absorbance was measured at 450 nm using a microplate reader. Combination therapy analysis was performed using CompuSyn software, and the Chou-Talalay method was used to calculate the relationship between the combination index (CI) and the effect fraction (FA). A CI value less than, equal to, or greater than 1 indicated a synergistic, additive, or antagonistic effect.

[0149] Cloning assay

[0150] 6-18 hours after cell transfection with siRNA, 1×10 3 Cells were placed in 6 cm plates and cultured for 14 days. The culture medium was changed every 3 days until colonies were visible, and then stained with a methanol solution containing 0.2% crystal violet for 30 minutes at room temperature. The cell clone count was determined using ImageJ.

[0151] Cell cycle assay

[0152] Forty-eight hours after siRNA transfection, cells were fixed overnight in 70% ethanol containing PBS. They were then incubated for 30 minutes at 37°C in PBS with 50 μg / ml RNase A (Sangon) and 0.1% Triton X-100 (Sangon), followed by staining in the dark for 30 minutes with 50 μg / ml propidium iodide (PI) (Vazyme). Finally, cell cycle analysis was performed by flow cytometry (CytoFLEX S, Beckman Coulter, Indianapolis, IN, USA).

[0153] Transwell cell migration assay

[0154] In cell migration experiments, 200 μl of serum-free medium was inoculated into each well chamber, and 800 μl of 20% FBS medium was inoculated into the lower well plate. Inoculation was performed at 5-10 × 10⁶ cells / wells. 4 Cells were cultured at 37°C for 36-48 hours, and 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 minutes. Migrating cells were counted under a microscope and quantified using ImageJ.

[0155] Apoptosis detection

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

[0157] In vivo transplantation experiment

[0158] Five-week-old female BALB / c nude mice were obtained from the Laboratory Animal Science Center of Shanghai Cancer Center, Fudan University. To study BRIX1 function, 5 × 10⁶ mice stably expressing shNC or shBRIX1 were used. 6 HCT116 p53+ / + and HCT116 p53- / - Cells, along with PCDH or PCDH-BRIX1 resuspended in 100 μL of serum-free medium, were injected into the flank region of mice. Tumor volume and weight were measured, as shown in the figure. The formula for calculating tumor volume is: Tumor volume (mm²) 3 = (length × width) 2×0.52. To study the antitumor effect of iRGD-Exo-siBRIX1, exosomes were injected into tumor-bearing mice three times a week via tail vein injection. To investigate the combined effect of iRGD-Exo-siBRIX1 and chemotherapy, tumor-bearing mice were randomly divided into four groups, receiving 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), respectively. Saline and 5-FU were administered intraperitoneally, while engineered exosomes were administered intravenously. When the tumor reached an appropriate volume, the nude mice were euthanized, the tumor was dissected, weighed, and photographed. All studies were approved by the Animal Welfare Committee of Shanghai Cancer Center, Fudan University, and all animals were handled according to institutional guidelines.

[0159] Human breast cancer and colorectal cancer specimens

[0160] Five pairs of fresh breast cancer and adjacent normal tissues from the First Affiliated Hospital of China Medical University were selected to detect BRIX1 protein and mRNA levels. Ninety-one paraffin-embedded sections of breast cancer tissue were obtained from the First Affiliated Hospital of China Medical University for immunohistochemical analysis. In addition, ten pairs of colorectal cancer and adjacent normal tissues and 62 paraffin-embedded sections of colorectal cancer tissue 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 or colorectal cancer tissue samples were dehydrogenated, rehydrated, and treated with sodium citrate-EDTA antigen retrieval solution for antigen extraction. Subsequently, the tissues were incubated with primary antibody at room temperature for 1–2 hours. After extensive washing with PBS, the tissues were treated with secondary antibody (Cat; No. GK500705, GeneTech) at room temperature for 1 hour. The sections were then stained with 3'-diaminobenzidine (Cat; No. GK500705, GeneTech) for 5 minutes to reveal the staining signal, followed by counterstaining with hematoxylin. Images were then taken using a Leica DMIRE2 microscope (Leica Microsystems Imaging Solutions Ltd.).

[0163] Isolation and purification of exosomes

[0164] Exosomes expressing the iRGD-LAMP2B fusion protein plasmid were collected from HEK293T cell culture medium (preparation method reference PMID: 24345736). The sample was centrifuged at 4500 rpm for 20 min, and the supernatant was collected, filtered through a 0.45 μm membrane, and then through a 0.22 μm membrane. The exosome samples were concentrated using a hollow fiber column. The concentrated samples were purified by chromatography and then passed through a hollow fiber column again. Ultrafiltration concentration: The sample was added to an ultrafiltration tube, centrifuged at 3000 rpm for 10-15 min at 4 °C, and the residue was sterilized by filtration 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] siRNA loaded into exosomes

[0166] 2'-methoxylated siRNA was prepared by GenePharma (Shanghai, China). The siRNA solution was bound to an exosome suspension (siRNA / exosome ratio of 1 μg / 4 μg) in electroporation dishes. Voltage, pulse length, and pulse number were optimized to efficiently take up siRNA without damaging the exosomes. After electroporation, the mixture was incubated at room temperature for 60 minutes at 1 × 10⁻⁶ pulses. 4 Centrifuge for 60 minutes to remove free siRNA and isolate exosomes.

[0167] Statistical analysis

[0168] All in vitro experiments were performed in triplicate. In vivo experimental animals were randomly assigned to different groups. Differences between two or more groups were analyzed using Student's t-test or one-way ANOVA. Statistical analysis was performed using GraphPad Prism 8.0, and results are expressed as mean ± standard deviation (SD). Significant differences in patient survival rates were analyzed using Kaplan-Meier plots and log-rank tests. Multivariate Cox proportional hazards models were used to generate hazard ratios with 95% confidence intervals. An asterisk indicates statistical significance: *p < 0.05; **p < 0.01; ***p < 0.001.

[0169] (II) Results

[0170] Identification of nucleolar protein BRIX1 as a regulator of p53

[0171] Because ribosome homeostasis plays a crucial role in controlling p53 activity and promoting cancer development, this invention investigates nucleolar proteins that may be essential for ribosome biogenesis. To date, 286 nucleolar proteins involved in yeast pre-rRNA processing have been identified.

[0172] These nucleolar proteins were selected based on the following criteria:

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

[0174] (ii) Proteins homologous in human and yeast;

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

[0176] (iv) Proteins whose role in cancer is unclear.

[0177] Therefore, this invention yielded six nucleolar proteins: BRIX1, DHX35, EXOSC6, EXOSC7, LSM6, and PPAN for further analysis. Figure 1 A).

[0178] To test whether these nucleolar proteins are essential for cancer cell growth, this 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 results showed that, compared to knocking down other nucleolar proteins, the loss of BRIX1 had a more significant inhibitory effect on the growth of all cell lines. Figure 1 B).

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

[0180] To verify this result, this invention knocked down BRIX1 expression using two independent siRNAs and found that BRIX1 deficiency 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, BRIX1 knockdown also induces 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 affects HCT116. p53- / - Expression in cells was not affected. Figure 1 H and 1L).

[0183] Interestingly, compared to treatment with cisplatin (DDP), 5-fluorouracil (5-FU), or actinomycin D (Act D), BRIX1 knockdown did not lead to an increase in γ-H2AX phosphorylation. Figure 1 M) indicates that no DNA damage stress was induced.

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

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

[0186] Brix has been reported to be involved in the processing of pre-rRNA and the synthesis of the large ribosomal subunit in yeast. However, it remains unclear whether BRIX1 has a conserved function in human cells. To clarify this, this invention relates to CAL-51 and HCT116. p53+ / + Total RNA in cells was analyzed by gel electrophoresis, and the results showed that the level of 28S rRNA was significantly reduced when BRIX1 was knocked down. Figure 2 A and 2B). These results were also confirmed by RT-qPCR analysis of the two cell lines (A and 2B). Figure 2 (C and 2D).

[0187] To investigate the underlying mechanisms, this invention used the STRING database to predict potential interacting proteins with BRIX1, and found that BOP1, PES1, and WDR12 have potential interaction relationships with BRIX1 (supplementary). Figure 1 A). These three proteins constitute the PeBoW complex, which is crucial for the processing of pre-rRNA and the maturation of the 60S ribosomal subunit in mammalian cells. Therefore, this invention hypothesizes that BRIX1 may promote rRNA synthesis through interaction with the PeBoW complex.

[0188] To verify this hypothesis, this invention conducted a series of protein co-immunoprecipitation (co-IP) experiments, which revealed that exogenous BRIX1 interacts with exogenous BOP1 and PES1. Figure 2 E-2H), but does 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, this invention further tested whether BRIX1 binds to endogenous BOP1 and modulates its level.

[0189] Although an endogenous interaction between BRIX1 and BOP1 has been observed in cancer cells ( Figure 2I), but knocking out BRIX1 does not affect the protein level of BOP1. Notably, the results of this study show that knocking down BRIX1 significantly reduces the interaction between BOP1 and PES1 (I). Figure 2 (J and 2K), indicating that BRIX1 is essential for the formation of the PeBoW complex. Since the PeBoW complex facilitates the synthesis and processing of 32S pre-rRNA, this invention used a digoxigenin-labeled ITS2 probe for Northern blot analysis, which specifically detects 47S, 32S, and 12S pre-rRNA.

[0190] The results of this study show that BRIX1 deletion primarily impairs the processing of 32S pre-rRNA, leading to a significant reduction in 12S pre-rRNA, and the processing of 47S pre-rRNA may also be slightly affected. Figure 2 In summary, these results indicate that BRIX1 is essential for pre-rRNA processing by modulating the stability of the PeBoW complex.

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

[0192] Next, this invention further investigates the potential impact of BRIX1 overexpression on p53 activity. Surprisingly, under normal growth conditions, BRIX1 overexpression in cancer cells did not affect p53 expression levels. Therefore, this invention attempts to examine whether BRIX1 overexpression regulates p53 under cellular stress.

[0193] Interestingly, the results of this invention show that overexpression of BRIX1 significantly reduces p53 protein in cancer cells during low-dose Act D treatment and DDP or 5-FU treatment. Figure 4 A and 4B) and their target genes ( Figure 4 The expression of C and 4D was inhibited. However, when BRIX1 was overexpressed, it failed to modulate p53 levels in cancer cells when treated with Nutlin-3 (an inducer of p53 by antagonizing MDM2). It is well known that low concentrations of Act D preferentially inhibit RNA Pol I activity, while both DDP and 5-FU can impair ribosome biogenesis by inducing rDNA damage or inhibiting pre-rRNA processing. Therefore, these results indicate that BRIX1 inhibits nucleolar stress-induced p53 activation.

[0194] To elucidate how BRIX1 inhibits p53 expression, this invention first examined the subcellular localization of BRIX1. The results showed that BRIX1 is primarily distributed in the nucleolus under non-stress conditions or Nultin-3 treatment, but under Act D-induced nucleolar stress, BRIX1 can migrate from the nucleolus to the nucleoplasm. Figure 4 E).

[0195] The present invention then sought to determine whether overexpression of BRIX1 prevented the interaction between RPs and MDM2 in the nucleoplasm under nucleolar stress. Co-IP analysis showed that BRIX1 bound to RPL5 and RPL11 when cancer cells were treated with Act D. Figure 4 F).

[0196] Furthermore, overexpression of BRIX1 reduced the interaction between MDM2 and RPL5 and RPL11. Figure 4 G), possibly because BRIX1 and these two nucleolar proteins competitively bind to MDM2. Furthermore, this invention found that overexpression of BRIX1 enhances MDM2-induced p53 ubiquitination (G). Figure 4 H), while p53 degradation induced by BRIX1 overexpression can be blocked by the proteasome inhibitor MG132 (H). Figure 4 I and 4J).

[0197] Finally, cycloheximine tracking analysis showed that BRIX1 overexpression shortened the half-life of p53 protein. Figure 4 These results indicate that BRIX1 translocates into the nucleoplasm to prevent the interaction of MDM2 with RPL5 / RPL11, thereby inhibiting nucleolar stress-induced stabilization and activation of p53.

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

[0199] Furthermore, overexpression of BRIX1 promotes cancer cell migration. Figure 4 P and 4Q).

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

[0201] In summary, these results indicate that excessive BRIX1 counteracts nucleolar stress-mediated antitumor effects by inactivating p53, suggesting that BRIX1 is involved in promoting chemotherapy resistance.

[0202] The tumor-suppressive effect of BRIX1 knockdown largely depends on p53.

[0203] Since BRIX1 plays a crucial role in regulating p53 activity, this invention aims to determine whether BRIX1 deficiency inhibits colorectal cancer in a p53-dependent manner. To this end, this invention utilizes the isogenetic colorectal cancer cell line HCT116. p53+ / + and HCT116 p53- / - .

[0204] The results of this study show that BRIX1 knockdown significantly inhibits HCT116. p53+ / + Cell proliferation was inhibited, while HCT116 was only slightly suppressed. p53- / - Cell proliferation ( Figure 5 (A and 5B). Similarly, with HCT116 p53- / - Compared to other cells, BRIX1 knockdown significantly reduced HCT116 p53+ / + The effect on cell clonogenic ability is more significant. Figure 5 C and 5D).

[0205] Furthermore, the inhibitory effect of BRIX1 knockdown on colorectal cancer cell migration is also partly dependent on the p53 status. Figure 5 E and 5F).

[0206] These results indicate that BRIX1 deficiency, in the presence of p53, moderately inhibits cancer cell growth and migration by interfering with ribosome biogenesis. However, the induction of G1 cell cycle arrest by BRIX1 knockdown is entirely 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, this invention tested in vivo whether BRIX1 knockdown inhibited tumor growth via p53. The results showed that BRIX1 deficiency significantly reduced HCT116. p53+ / + The growth rate, weight, and size of cell-derived transplanted tumors ( Figure 5 I-5L), while for HCT116 p53- / - The growth of cell-derived transplanted tumors is minimally affected. Figure 5These results indicate that the lack of BRIX1 has a more significant impact on colorectal cancer cells with wild-type p53 compared to those lacking p53.

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

[0209] Given that BRIX1 is essential for the survival and proliferation of breast and colorectal cancer cells, this invention investigates the clinical significance of BRIX1 in these two cancers.

[0210] First, this invention evaluated the expression of BRIX1 in breast cancer and matched normal tissues using IB and RT-qPCR analyses. The results showed that the levels of BRIX1 protein and mRNA in breast cancer tissues were higher than those in normal tissues. Figure 6 A and 6B). Furthermore, immunohistochemical (IHC) analysis of 91 breast cancer tissue samples showed that high expression of BRIX1 was significantly associated with higher tumor / lymph node / metastasis (TNM) stage and poorer overall survival. Figure 6 C).

[0211] Univariate and multivariate Cox regression analyses further confirmed that high BRIX1 expression is a factor contributing to poor prognosis in breast cancer. Figure 6 D). This invention uses 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. Immunohistochemical analysis of this invention shows that BRIX1 expression levels are higher in colorectal cancer than in normal tissue.

[0212] Furthermore, both univariate and multivariate analyses indicated that BRIX1 is a prognostic factor for colorectal cancer. Finally, this invention validated the clinical relevance of BRIX1 using the TCGA database. Compared to normal tissues, BRIX1 mRNA and protein levels were upregulated in various cancer tissues, and higher BRIX1 levels were associated with poorer prognosis across different cancer types. In summary, these findings suggest that high levels of BRIX1 promote ribosome biogenesis and limit p53 activity, which is crucial for cancer progression, while reducing BRIX1 activation of p53 through nucleolar stress inhibits cancer development.

[0213] Inhibits colorectal cancer development through the delivery of therapeutic BRIX1 siRNA via iRGD-modified exosomes.

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

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

[0216] First, this 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 αv integrin and neuropilin-151. The plasmid encoding the fused iRGD-lamp2b protein is stably transfected into HEK293T cells to generate engineered exosomes expressing iRGD on the surface, serving as donor cells. Figure 8 A).

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

[0218] Furthermore, only exosome markers TSG101, CD81, and CD9 were detected in exosomes, with mitochondrial protein COX IV used as a control. Figure 8 D). Next, the fam-tagged siRNA was loaded into iRGD-modified exosomes via electroporation.

[0219] CAL-51 and HCT116 were treated with these exosomes. p53+ / + Cells were observed under a fluorescence microscope after 8-12 hours. The results of this invention indicate that 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] Furthermore, iRGD-Exo-siBRIX1 significantly increased the levels of p53 and p21 by reducing BRIX1 expression in both cell lines. Figure 7 B and 7D).

[0221] Furthermore, this invention tested whether iRGD-Exo-siBRIX1 inhibited the growth and migration of cancer cells. As expected, iRGD-Exo-siBRIX1 treatment of CAL-51 and HCT116 p53+ / + cells significantly reduced their proliferation ( Figure 7 E and 7F), clonal formation ( Figure 7 G and 7H) and migration ( Figure 7 These results indicate that engineered exosomes can serve as vectors for BRIX1 siRNA to enter cancer cells.

[0222] To test whether iRGD-modified exosomes targeted tumors, this invention administered intravenous injections of exosomes labeled with DiR (a lipophilic fluorescent dye) to transplanted mice. Although exosomes were primarily located in the liver and spleen, iRGD significantly increased the accumulation of exosomes in the transplanted tumor. Figure 7 K and 7L).

[0223] Next, this invention investigates in vivo whether iRGD-Exo-siBRIX1 inhibits tumor growth. The siRNAs used in subsequent experiments underwent 2'-methoxy modification, which enhanced their stability and binding affinity, and reduced immune activation. Methods for 2'-methoxy modification of siRNAs are known in the art. BALB / c nude mice were subcutaneously inoculated with HCT116. p53+ / + Cells, then iRGD-Exo-siBRIX1 or iRGD-Exo-siNC is administered intravenously 3 times a week.

[0224] The results of this invention show that iRGD-Exo-siBRIX1 significantly inhibits the growth rate of transplanted tumors. Figure 7 M), weight ( Figure 7 N) and size (Fig. 70), while no significant weight loss was observed ( Figure 7 P).

[0225] Furthermore, this invention aims to investigate whether iRGD-Exo-siBRIX1 combined with chemotherapy can synergistically inhibit colorectal cancer. To verify this hypothesis, this invention first determined whether BRIX1 knockdown enhanced HCT116. p53+ / + Cellular sensitivity to 5-FU.

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

[0227] Furthermore, the combined use of siBRIX1 and 5-FU exhibits a strong synergistic effect on cell viability, with a combination index (CI) of approximately 0.5 or less. Figure 7 Q).

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

[0229] The results showed that 5-FU inhibited the growth of graft 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 a significant reduction in tumor growth rate, weight, and size. Figure 7 R-7T was detected in mice, and the average body weight of the treated mice was comparable to that of the control group, indicating that the combined treatment had no significant toxic effects on the mice. Figure 7 U).

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

[0231] discuss

[0232] Ribosome biogenesis is essential for the rapid growth and proliferation of cancer cells. Damage to this process leads to the translocation of ribosomal proteasomes (RPs) from the nucleolus to the nucleoplasm, thereby preventing MDM2-induced p53 proteasome degradation. In this study, we found that BRIX1 is a key regulator of ribosomal biogenesis and nucleolar stress.

[0233] BRIX1 exerts its carcinogenic effect through at least two mechanisms.

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

[0235] Furthermore, BRIX1 interacts with RPL5 and RPL11, thereby preventing the interaction of RPs with 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 enhancing ribosome biogenesis and restricting p53 activity. More importantly, the loss of BRIX1 triggers nucleolar stress by disrupting pre-rRNA processing. Figure 2A-2D, 2L), leading to increased interaction between RPL5 / RPL11 and MDM2, thereby stabilizing and activating p53 ( Figure 1 C-1L and 3A-3H).

[0237] Finally, the results of this invention demonstrate that the targeted inhibition of BRIX1 by therapeutic exosomes effectively enhanced 5-FU chemotherapy and inhibited the growth of wild-type p53 colorectal cancer. Figure 7 ).

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

[0239] The results of this invention show that depletion of BRIX1 impairs the processing of pre-rRNA. Figure 2 This disrupts the nucleolar structure, as demonstrated by the repositioning of NPM1. This leads to nucleolar stress without causing DNA damage. Figure 1 RPL5 / RPL11-dependent p53 activation is caused by excess BRIX1. Conversely, excess BRIX1 prevents p53 activation by interacting with RPL5 and RPL11. Therefore, this study demonstrates that BRIX1 is another target molecule for selectively inducing nucleolar stress.

[0240] In this invention, iRGD-modified exosomes were developed, loaded with siRNA targeting BRIX1, and it was verified that these exosomes could transport siBRIX1 to tumor sites. Figure 7 K and 7L), and increase the uptake of siBRIX1 by cancer cells ( Figure 7 A and 7C). It is noteworthy that the research results of this invention demonstrate 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). It is worth noting that the tumor-targeting potential of exosomes may be further enhanced.

[0241] In summary, this study identifies BRIX1 as an oncoprotein overexpressed in colorectal and breast cancer, associated with poor prognosis. BRIX1 promotes pre-rRNA processing through interaction with the PeBoW complex and inhibits p53 activity by isolating RPL5 and RPL11. Conversely, BRIX1 deficiency activates p53, suppressing cancer growth by triggering nucleolar stress. Finally, the results of this study suggest that loading BRIX1-specific siRNA using an exosome delivery system may be a promising cancer treatment approach.

[0242] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

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

2. A siRNA precursor, characterized by, The siRNA precursor is the siRNA precursor described in claim 1.

3. The siRNA precursor of claim 2, wherein 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 and second sequence units are complementary, causing the siRNA precursor to form a hairpin structure. The first sequence unit is selected from the following group: siBRIX1-1:5'-CGTGTTTACTTTCACCATT-3', and siBRIX1-2: 5'-GCATCGGCGTGTCATAAGA-3'.

4. An exosome, characterized in that, The exosomes are loaded with the siRNA or its precursor as described in claim 1.

5. Use of the siRNA or precursor thereof targeting the BRIX1 gene according to claim 1, characterized in that, This is used to prepare a drug for the prevention or treatment of tumors, specifically colorectal cancer or breast cancer.

6. Use according to claim 5, characterized in that, The tumor is either a p53 wild-type tumor or a p53 mutant tumor.

7. A pharmaceutical composition, characterized by, It includes a pharmaceutically acceptable carrier and an effective amount of the active ingredient, wherein the active ingredient is the siRNA targeting the BRIX1 gene as described in claim 1.

8. A pharmaceutical composition, characterized by, It includes a pharmaceutically acceptable carrier and an effective amount of the active ingredient, wherein the active ingredient is the siRNA precursor of claim 2.

9. A pharmaceutical composition, characterized by, It includes a pharmaceutically acceptable carrier and an effective amount of the active ingredient, wherein the active ingredient is the exosome as described in claim 3.

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