Tumor diagnosis kit containing BRIX1 detection reagent

Abstract

The invention discloses a tumor diagnosis kit containing a BRIX1 detection reagent, and particularly discloses that the BRIX1 is highly expressed in breast cancer and colorectal cancer, and the higher the BRIX1 level is, the poorer the prognosis is. The invention further discloses that the expressions of BRIX1 and p21 in colorectal cancer are in negative correlation, and the combination of high-level BRIX1 and low-level p21 can better predict poorer prognosis and show a synergistic prediction effect. And the prognosis condition of the tumor can be accurately predicted by detecting the BRIX1 and the p21, so that a more reliable diagnosis basis is provided for clinic.

Description

Technical Field

[0001] The present invention belongs to the field of molecular biology and biomedicine, and in particular, the present invention relates to a tumor diagnosis kit comprising a BRIX1 detection reagent. Background Art

[0002] Tumor molecular diagnosis refers to molecular biological diagnostic technology with nucleic acid or protein as the core, which is an important method for early diagnosis of tumors. By detecting biomacromolecules related to tumor occurrence, tumor occurrence can be predicted and diagnosed, and the effect of tumor treatment can be evaluated, providing a reference for the prognosis and outcome of tumors.

[0003] Tumor heterogeneity between different individuals is very obvious, which requires understanding the characteristics of individual tumors as detailed as possible in order to develop new and more effective treatments in a small but clearly divided population. The traditional classification and staging system based on clinical pathological characteristics is difficult to truly and comprehensively reflect the molecular change characteristics and biological characteristics of tumors at different stages of development. The occurrence and development of tumors is a complex multi-step, multi-stage process with the accumulation of multiple types of molecular changes. These changed molecules and their signal transduction are closely related to the sensitivity of clinical tumor detection, and molecular diagnostic technology helps to clearly diagnose and identify difficult cases, which can further improve the clinical treatment effect of tumors. Molecular markers are also used for survival analysis of tumor patients.

[0004] Therefore, technicians in this field are committed to finding molecular markers that can accurately reflect tumor progression and prognosis, in order to provide more sufficient evidence for clinical diagnosis and identification. Summary of the invention

[0005] The object of the present invention is to provide a tumor diagnosis kit comprising a BRIX1 detection reagent.

[0006] In a first aspect of the present invention, a reagent set is provided for use in preparing a tumor detection kit, wherein the reagent set comprises a reagent for detecting BRIX1 and a reagent for detecting p21.

[0007] In another preferred embodiment, the tumor is breast cancer or colorectal cancer.

[0008] In another preferred embodiment, the reagent for detecting BRIX1 is an antibody against BRIX1 protein, preferably a monoclonal antibody.

[0009] In another preferred embodiment, the reagent for detecting BRIX1 is a reagent for specifically detecting the BRIX1 gene, such as a primer for specifically amplifying the BRIX1 gene, or a probe for specifically binding to the BRIX1 gene.

[0010] In another preferred embodiment, the reagent for detecting p21 is an antibody against p21 protein, preferably a monoclonal antibody.

[0011] In another preferred embodiment, the reagent for detecting p21 is a reagent for specifically detecting the p21 gene, such as a primer for specifically amplifying the p21 gene, or a probe for specifically binding to the p21 gene.

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

[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 replacing, deleting or adding one or more nucleotides of 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% to the polynucleotide sequence shown in SEQ ID NO.: 1;

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

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

[0019] In another preferred embodiment, the amino acid sequence of the BRIX1 protein is shown in SEQ ID NO.2.

[0020] In another preferred embodiment, the tumor is a p53 wild-type tumor.

[0021] In another preferred embodiment, the tumor is a p53 mutant tumor.

[0022] In another preferred embodiment, the kit is an in situ hybridization detection kit, a fluorescent in situ hybridization detection kit, a PCR detection kit, a Southern hybridization detection kit, a Northern hybridization detection kit, or a comparative genomic hybridization detection kit.

[0023] In a second aspect of the present invention, a tumor detection reagent set is provided, the reagent set comprising a reagent for detecting BRIX1 and a reagent for detecting p21.

[0024] In another preferred embodiment, the reagent for detecting BRIX1 is an antibody against BRIX1 protein, preferably a monoclonal antibody.

[0025] In another preferred embodiment, the reagent for detecting BRIX1 is a reagent for specifically detecting the BRIX1 gene, such as a primer for specifically amplifying the BRIX1 gene, or a probe for specifically binding to the BRIX1 gene.

[0026] In another preferred embodiment, the reagent for detecting p21 is an antibody against p21 protein, preferably a monoclonal antibody.

[0027] In another preferred embodiment, the reagent for detecting p21 is a reagent for specifically detecting the p21 gene, such as a primer for specifically amplifying the p21 gene, or a probe for specifically binding to the p21 gene.

[0028] In another preferred embodiment, the BRIX1 gene is selected from the following group:

[0029] (A) a polynucleotide sequence encoding the polypeptide shown in SEQ ID NO.2;

[0030] (B) the polynucleotide sequence shown in SEQ ID NO.1;

[0031] (C) a polynucleotide sequence formed by replacing, deleting or adding one or more nucleotides of the polynucleotide sequence shown in SEQ ID NO.1;

[0032] (D) a polynucleotide sequence having a homology of ≥90%, preferably ≥95%, more preferably ≥98%, and most preferably ≥99% to the polynucleotide sequence shown in SEQ ID NO.: 1;

[0033] (E) A polynucleotide sequence complementary to the polynucleotide sequence described in any one of (A) to (D).

[0034] In another preferred embodiment, the BRIX1 gene is derived from mammals (including humans).

[0035] In another preferred embodiment, the amino acid sequence of the BRIX1 protein is shown in SEQ ID NO.2.

[0036] In a third aspect of the present invention, a kit is provided, comprising a first detection reagent and a second detection reagent, wherein the first detection reagent is configured to detect BRIX1; and the second detection reagent is configured to detect p21.

[0037] In another preferred embodiment, the reagent for detecting BRIX1 is an antibody against BRIX1 protein, preferably a monoclonal antibody.

[0038] In another preferred embodiment, the reagent for detecting BRIX1 is a reagent for specifically detecting the BRIX1 gene, such as a primer for specifically amplifying the BRIX1 gene, or a probe for specifically binding to the BRIX1 gene.

[0039] In another preferred embodiment, the reagent for detecting p21 is an antibody against p21 protein, preferably a monoclonal antibody.

[0040] In another preferred embodiment, the reagent for detecting p21 is a reagent for specifically detecting the p21 gene, such as a primer for specifically amplifying the p21 gene, or a probe for specifically binding to the p21 gene.

[0041] In another preferred embodiment, the BRIX1 gene is selected from the following group:

[0042] (A) a polynucleotide sequence encoding the polypeptide shown in SEQ ID NO.2;

[0043] (B) the polynucleotide sequence shown in SEQ ID NO.1;

[0044] (C) a polynucleotide sequence formed by replacing, deleting or adding one or more nucleotides of the polynucleotide sequence shown in SEQ ID NO.1;

[0045] (D) a polynucleotide sequence having a homology of ≥90%, preferably ≥95%, more preferably ≥98%, and most preferably ≥99% to the polynucleotide sequence shown in SEQ ID NO.: 1;

[0046] (E) A polynucleotide sequence complementary to the polynucleotide sequence described in any one of (A) to (D).

[0047] In another preferred embodiment, the BRIX1 gene is derived from mammals (including humans).

[0048] In another preferred embodiment, the amino acid sequence of the BRIX1 protein is shown in SEQ ID NO.2.

[0049] In a fourth aspect of the present invention, a method for diagnosing a tumor is provided, comprising the steps of: detecting the BRIX1 level and the p21 level in a sample to be detected from a tumor patient, if the BRIX1 level in the sample is increased and the p21 level is decreased, it indicates a poor prognosis of the tumor.

[0050] In another preferred example, compared with the healthy control sample, the level of the BRIX1 gene or its encoded protein in the sample to be tested is increased 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%, and more preferably more than 90%.

[0051] In another preferred embodiment, compared with the healthy control sample, the level of p21 gene or its encoded protein in the sample to be tested is reduced by more than 10%, preferably reduced by more than 20%, more preferably reduced by more than 30%, more preferably reduced by more than 40%, more preferably reduced by more than 50%, more preferably reduced by more than 60%, more preferably reduced by more than 70%, more preferably reduced by more than 80%, and more preferably reduced by more than 90%.

[0052] 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 below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] The following drawings are used to illustrate specific embodiments of the present invention and are not used to limit the scope of the present invention defined by the claims.

[0054] Figure 1 : Knockdown of BRIX1 activates p53. (A) Venn diagram depicts the screening of potential regulators of ribosome biogenesis. (B) Heat map shows that BRIX1 deficiency significantly inhibits cell growth in four cancer cell lines as measured by cell viability assay. (C) Heat map analysis results showed that the expression of many p53 target genes was elevated in BRIX1-deficient CAL-51 cells. (D) KEGG pathway enrichment analysis results showed that target genes in the p53 pathway were 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 were transfected with control or BRIX1 siRNA. p53 + / + (G) The cells were cultured for 48 h and then subjected to RT-qPCR analysis. (H) Effect of knockdown of BRIX1 on HCT116 p53- / - There was no effect on the mRNA levels of p53 target genes in cells. (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) The cells were cultured for 48 hours and then subjected to IB analysis. (L) Effect of knockdown of BRIX1 on HCT116 p53- / - There was no effect on the protein levels of p53 target genes in cells. (M) Knockdown of BRIX1 did not increase the phosphorylation level of γ-H2AX. Cells were treated with drugs for 24 hours or transfected with siRNA for 48 hours and then subjected to IB analysis. ***p<0.001.

[0055] Figure 2:BRIX1 promotes pre-rRNA processing through the PeBoW complex. (A, B) Knockdown of BRIX1 reduces 28S rRNA levels. CAL-51 (A) and HCT116 cells were transfected with control or BRIX1 siRNA p53+ / + (B) Cells were subjected to agarose gel electrophoresis and quantitative analysis by ImageJ at 48 h. (C,D) Knockdown of BRIX1 reduced 28S rRNA levels as detected by RT-qPCR. (EH) BRIX1 interacts 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 of BRIX1 with BOP1. HCT116 cells were treated with MG132 p53+ / + Cells were treated for 6 hours and subjected to co-IP-IB analysis. (J,K) Knockdown of BRIX1 impairs the interaction between BOP1 and PES1. Cells were transfected with the indicated siRNA and plasmids and then subjected to co-IP-IB analysis using the indicated antibodies. (L) Knockdown of BRIX1 impairs pre-rRNA processing. Cells were transfected with control or BRIX1 siRNA and then subjected to Northern blot analysis, with schematics of the ITS-2 probe (orange) used in the experiments indicated. ***p<0.001.

[0056] Figure 3 : Overexpression of BRIX1 impairs p53 activation under nucleolar stress. (AD) Overexpression of BRIX1 inhibits the expression of p53 and its target genes under nucleolar stress. CAL-51 (A, C) and HCT116 cells were transfected with plasmids as indicated. p53+ / + (B, D) Cells were treated with 10 nM Act D for 24 h and then subjected to IB or RT-qPCR analysis. (E) BRIX1 translocated from the nucleolus to the nucleoplasm under the action of actinomycin D (Act D) (10 nM), while BRIX1 did not translocate under the action of Nutlin-3 (20 μM). Cells were transfected with plasmids and treated with drugs as indicated, 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, and then subjected to co-IP-IB analysis. (G) BRIX1 inhibits the interaction between MDM2 and RPL5 / RPL11. HCT116 cells were transfected with plasmids as indicated. p53+ / +Cells were treated with Act D (10 nM) for 24 h and MG132 (20 μM) for 6 h, and then subjected to co-IP-IB analysis. (H) Overexpression of BRIX1 increases the ubiquitination of p53. HCT116 cells stably overexpressing control or BRIX1 were transfected with the indicated plasmids. p53- / - The cells were incubated for 48 hours and then treated with Act D (10 nM) for 24 hours and MG132 (20 μM) for 6 hours before in vivo ubiquitination assays. (I, J) Plasmid transfection of HCT116 p53+ / + and CAL-51 cells were treated with Act D (10 nM) for 24 h and MG132 (20 μM) for 6 h, and then subjected to IB analysis. (K) Overexpression of BRIX1 shortened the half-life of p53 protein. HCT116 cells were transfected with plasmids p53+ / + Cells were treated with Act D (10 nM) and CHX (100 mg / ml) at different time points for 24 h, and then subjected to IB analysis. (L, M) Overexpression of BRIX1 increased the expression of CAL51 (L) and HCT116 p53+ / + (M) Cell proliferation. Cells were transfected with plasmids and treated with Act D (10 nM), and then cell viability was measured. (N,O) Overexpression of BRIX1 enhanced the proliferation of CAL51 (N) and HCT116 p53+ / + (O) Cell clone formation ability. Cells stably overexpressing control or BRIX1 were treated with 10nM Act D, and then clone formation assay was performed. (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 10nM Act D, and then cell migration assay was performed. *p<0.05, **p<0.01, ***p<0.001. (RU) Overexpression of BRIX1 promoted the migration of HCT116 p53+ / + Growth rate (R), weight (S) and size (T) of cell-derived transplanted tumors, but no effect on mouse body weight (U). On the designated first day after inoculation, mice were intraperitoneally injected with 30 μg / kg Act D. Data are expressed as mean ± SD, n = 5. **p < 0.01, **p < 0.001.

[0057] Figure 4: BRIX1 is highly expressed in breast and colorectal cancer tissues and is associated with poor prognosis. (A,B) BRIX1 protein and mRNA levels are elevated in breast cancer samples compared with adjacent normal tissues. Five pairs of fresh tissues were analyzed by IB and RT-qPCR, respectively. (C,D) Elevated BRIX1 expression levels are a factor for poor prognosis in breast cancer. (EG) Representative images (E) and ten pairs of sample graphs (F,G) show that BRIX1 expression levels are higher and p21 expression levels are lower in colorectal cancer tissues compared with paired adjacent normal tissues. (H,I) In a cohort of 62 colorectal cancer patients, higher levels of BRIX1 (H) or lower levels of p21 (I) were associated with poor overall survival.

[0058] Figure 5 : High levels of BRIX1 and low levels of p21 were more significantly associated with poor overall survival.

[0059] Figure 6 : High levels of BRIX1 and low levels of p21 were more significantly associated with poor overall survival. DETAILED DESCRIPTION

[0060] After extensive and in-depth research, the inventors found that BRIX1 is highly expressed in breast cancer and colorectal cancer, and the higher the BRIX1 level, the worse the prognosis. Further analysis unexpectedly found that the expression of BRIX1 and p21 in colorectal cancer is negatively correlated, and high levels of BRIX1 combined with low levels of p21 can better predict a poor prognosis. These findings indicate that the prognosis of tumors can be accurately predicted by detecting BRIX1 and p21, thereby providing a more reliable diagnostic basis for clinical practice.

[0061] the term

[0062] BRIX1 gene and its encoded protein

[0063] BRIX (ribosome biogenesis in Xenopus laevis) was first discovered in frogs and later in yeast to be involved in rRNA processing. Expression of human BRIX1 was recently shown to be associated with rRNA synthesis and promote GLUT1 translation in colorectal cancer. However, the role and mechanism of BRIX1 in rRNA synthesis and cancer development remain largely unknown.

[0064] The human BRIX1 gene (NCBI sequence number Gene ID: 55299) is located on human chromosome 5.

[0065]

[0066] (SEQ ID NO.1)

[0067] 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:

[0068] MAATKRKRRGGFAVQAKKPKRNEIDAEPPAKRHATAEEVEEEERDRIPGPVCKGKWKNKERILIFSSRG

[0069] INFRTRHLMQDLRMLMPHSKADTKMDRKDKLFVINEVCEMKNCNKCIYFEAKKKQDLYMWLSNSPHGPSAK

[0070] FLVQNIHTLAELKMTGNCLKGSRPLLSFDPAFDELPHYALLKELLIQIFSTPRYHPKSQPFVDHVFTFTIL

[0071] DNRIWFRNFQIIEEDAALVEIGPRFVLNLIKIFQGSFGGPTLYENPHYQSPNMHRRVIRSITAAKYREKQQ

[0072] VKDVQKLRKKEPKTLLPHDPTADVFVTPAEEKPIEIQWVKPEPKVDLKARKKRIYKRQRKMKQRMDSGKTK(SEQ ID NO.2)

[0073] p21 gene and its encoded protein

[0074] The p21 gene (NCBI sequence number Gene ID: 1026) is a member of the Clp family. It is a cyclin-dependent kinase inhibitor located downstream of the p53 gene. p21 and p53 can form the cell cycle G1 checkpoint together. Since DNA damage cannot be passed without repair, it reduces the replication and accumulation of damaged DNA, thereby playing a role in suppressing cancer.

[0075]

[0076] In a preferred embodiment of the present invention, the sequence of the protein encoded by the p21 gene is shown in SEQ ID NO.4:

[0077] MSEPAGDVRQNPCGSKACRRLFGPVDSEQLSRDCDALMAGCIQEARERWNFDFVTETPLEGDFAWERVRGLGLPKLYLPTGPRRGRDELGGGRRPGTSPALLQGTAEEDHVDLSLSCTLVPRSGEQAEGSPGGPGDSQGRKRRQTSMTDFYHSKRRLIFSKRKP (SEQ ID NO. 4).

[0078] Reagent Sets and Kits

[0079] The present invention provides a reagent set (tumor detection reagent set), which comprises a reagent for detecting BRIX1 and a reagent for detecting p21. Preferably, the detection reagent is a specific antibody or a nucleic acid-specific amplification primer.

[0080] In another preferred embodiment, the reagent for detecting BRIX1 is an antibody against BRIX1 protein, preferably a monoclonal antibody, for example: the anti-BRIX1 antibody with the catalog number 17295-1-AP of Proteintech (Cat No. 17295-1-AP, Proteintech).

[0081] In another preferred embodiment, the reagent for detecting BRIX1 is a reagent for specifically detecting the BRIX1 gene, such as a primer for specifically amplifying the BRIX1 gene, or a probe for specifically binding to the BRIX1 gene.

[0082] In another preferred embodiment, the reagent for detecting p21 is an antibody against p21 protein, preferably a monoclonal antibody, such as: Anti-p21 antibody from abcam (Cat. No. ab227443); anti-p21 antibody from Cell Signaling Technology (Cat. No. 2947).

[0083] In another preferred embodiment, the reagent for detecting p21 is a reagent for specifically detecting the p21 gene, such as a primer for specifically amplifying the p21 gene, or a probe for specifically binding to the p21 gene.

[0084] In the present invention, antibodies for detecting specific targets can be purchased through conventional commercial channels; PCR amplification primers and probes for detecting specific nucleic acid targets can be designed in a manner well known to those skilled in the art, and can also be purchased through conventional commercial channels.

[0085] According to a preferred embodiment of the present invention, the kit may be an ELISA kit (enzyme-linked immunosorbent assay kit).

[0086] According to a preferred embodiment of the present invention, the kit may be a PCR detection kit; preferably a quantitative PCR detection kit.

[0087] The main advantages of the present invention are:

[0088] (1) It was discovered that the BRIX1 gene and its encoded protein can serve as a new type of tumor diagnostic target.

[0089] (2) For the first time, it was proposed that high levels of BRIX combined with low levels of p21 can better and more accurately predict the prognosis of tumors and provide more sufficient basis for clinical diagnosis.

[0090] The present invention is further described in detail below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually carried out according to conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, parts and percentages are by weight.

[0091] Example

[0092] (I) Materials and methods

[0093] Cell culture and transient transfection

[0094] 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 (Basal Media, Shanghai, China). All cell lines were obtained from ATCC and were mycoplasma-free by PCR. Cells were plated at optimal density 12-24 h before transfection. Transfection was performed using Hieff trans-lipid transfection reagent (Yeasen) according to the manufacturer's protocol.

[0095] Plasmids and antibodies

[0096] Primers were designed to amplify BRIX1, BOP1, PES1, and WDR12 cDNAs, and the amplified sequences were subsequently 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. 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), and anti-coxⅣ (Cat; No.11242-1-AP, Proteintech) were purchased commercially.The secondary antibodies were enzyme-labeled affinity pure goat anti-rabbit IgG (Cat; No. SA00001-2, Proteintech) and anti-mouse IgG (Cat; No. SA00001-1 Proteintech). The images were developed using ECL chemiluminescence reagent (Yeasen).

[0097] Reverse transcription and real-time quantitative PCR

[0098] 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 mRNAs were calculated using the comparative Ct method normalized to GAPDH.

[0099] The preferred quantitative PCR (qPCR) primer sequences are as follows:

[0100] BRIX1-F 5'-CGGAATTCGAATGGCGGCAACCAA-3'(SEQ ID NO.5)

[0101] BRIX1-R 5'-CCGCTCGAGTTATTTTGTTTTCCCACTGT-3' (SEQ ID NO. 6);

[0102] p21-F 5'-CTGGACTGTTTTCTCTCGGCTC-3'(SEQ ID NO.7)

[0103] p21-R 5'-TGTATATTCAGCATTGTGGGAGGA-3' (SEQ ID NO. 8).

[0104] Transcriptome sequencing

[0105] After 48 h of 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).

[0106] Immunoblotting

[0107] 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 cocktail] and quantified using BCA reagent (Yeasen). Equal amounts of clear cell lysate (20-80 μg) were used for immunoblot (IB) analysis.

[0108] Northern blotting

[0109] Northern blot analysis 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 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 of 1.5J cm2, pre-hybridized at 65°C for 30 minutes, and hybridized with the probe at 42°C overnight. 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, and washed with detection buffer for 5 minutes. Finally, the membrane was prepared with CDP-Star (Roche, USA). The digoxigenin (DIG) labeled probe was synthesized by GENEWIZ (Suzhou, China).

[0110] Immunofluorescence staining

[0111] Cells transfected with siRNA were fixed with methanol overnight at -20°C, washed three times with PBS, and incubated with blocking buffer (8% BSA and 0.3% Triton X-100) for 1 hour at room temperature. They were then incubated with primary antibodies (anti-BRIX1, 1:100; anti-NPM1, 1:50 dilution) overnight at 4°C. The next day, cells were washed with PBS and incubated with fluorescent secondary antibodies (Yeasen) and DAPI (Sigma-Aldrich). Images were acquired using an inverted fluorescence microscope (Leica, Wetzlar, Germany).

[0112] Protein co-immunoprecipitation

[0113] 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 cocktail] and immunoprecipitated (IP) with antibodies, as described in Figure 3 Briefly, proteins (0.5-1 mg) were incubated with the indicated antibodies for 5 h at 4 °C and then incubated with protein A / G magnetic beads (Santa Cruz Biotechnology) for 2 h. The beads containing the immunoprecipitated proteins were washed 6-8 times with lysis buffer and analyzed by IB.

[0114] In vivo ubiquitination assay

[0115] As shown in the figure, plasmids of p53, HA-MDM2, His-Ub and BRIX1 siRNA were transfected into HCT116 p53- / - Cells were transfected with MG132 for 4-6 hours and then harvested for in vivo ubiquitination assays. Briefly, 48 hours after transfection, cells were harvested and split into two portions, 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, and 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 perform IB experiments with the indicated antibodies.

[0116] RNA interference and generation of stable cell lines

[0117] Small interfering RNAs (siRNAs) targeting BRIX1, RPL5, and RPL11 were synthesized and purified by GenePharma (Shanghai, China). Transfection of siRNAs was performed using Hieff transfectant liposome transfection reagent according to the manufacturer's protocol (Yeasen). All transfections were verified by qPCR or western blot. Specific sequences of shRNA targeting BRIX1 were obtained from Sigma-Aldrich and cloned into the PLKO.1 vector (Supplementary Table 8). Plasmids containing shRNAs were transformed into HEK293T cells along with packaging plasmids psPAX2 and pMD2G. Lentiviral particles were collected 48 h after transfection and used for cell infection. Stably infected cells were selected with 1 μg / ml puromycin.

[0118] Cell viability assay

[0119] To assess cell viability, 2-4 × 10 3 Cells were seeded in each well of a 96-well culture plate. Cell counting kit-8 (CCK-8) reagent (Yeasen) was added to each well at a final concentration of 10%. The cells were incubated at 37°C for 2 hours, and the absorbance at 450 nm was measured using an ELISA reader. CompuSyn software was used for the analysis of combined drug use, and the relationship between the combined drug index (CI) and the impact fraction (FA) was calculated using the Chou-Talalay method. When the CI value is less than, equal to, or greater than 1, it indicates synergy, additiveness, or antagonism.

[0120] Colony formation assay

[0121] 6-18 hours after cells were transfected with siRNA, 1×10 3 Cells were plated on 6 cm plates and cultured for 14 days. The 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 number of cell colonies was determined by ImageJ.

[0122] Cell cycle assay

[0123] 48 hours after siRNA transfection, cells were fixed in 70% ethanol in 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, cell cycle was analyzed by flow cytometry (CytoFLEX S, Beckman Coulter, Indianapolis, IN, USA).

[0124] Transwell cell migration assay

[0125] In the cell migration experiment, 200 μl serum-free medium was inoculated in the chamber, 800 μl 20% FBS medium was inoculated in the lower well plate, and 5-10×10 4 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 minutes. The number of migrated cells was counted under a microscope and quantified using ImageJ.

[0126] Apoptosis detection

[0127] Cell apoptosis experiments were performed according to the manufacturer's protocol of the Cell Apoptosis Detection Kit (Vazyme). Briefly, cells were washed twice with cold PBS and then incubated with 1 × 10 6 Cells were resuspended in 1× Binding Buffer at a concentration of 10 cells / ml. Cells were incubated with Annexin V-PE and 7-aminoactinomycin D (7AAD) at room temperature for 15 min. Finally, the level of cell apoptosis was detected by flow cytometry (CytoFLEX S, Beckman Coulter, Indianapolis, IN, USA).

[0128] In vivo transplantation assay

[0129] Five-week-old BALB / c female nude mice were obtained from the Experimental Animal Science Center of Shanghai Cancer Center, Fudan University. To study the function of BRIX1, 5×10 mice stably expressing shNC or shBRIX1 were randomly divided into 6 HCT116 p53+ / + and HCT116 p53- / - Cells, and PCDH or PCDH-BRIX1 resuspended in 100 μL serum-free medium were injected into the flank region of mice. The tumor volume and weight of mice were measured as shown in the figure. The tumor volume calculation formula is: Tumor volume (mm 3 )=(length×width 2)×0.52. In order to study the anti-tumor effect of iRGD-Exo-siBRIX1, the present invention injected exosomes into tumor-bearing mice through the tail vein three times a week. To explore the combined effect of iRGD-Exo-siBRIX1 and chemotherapy, tumor-bearing mice were randomly divided into 4 groups and given 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). Saline and 5-FU were injected intraperitoneally, and engineered exosomes were injected intravenously. When the tumor reached an appropriate volume, the nude mice were killed, the tumor was removed, 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.

[0130] Human breast and colorectal cancer specimens

[0131] 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. Paraffin sections of 91 breast cancers 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 Committee of the First Affiliated Hospital of China Medical University and the First Affiliated Hospital of Nanchang University.

[0132] Immunohistochemical staining

[0133] Paraffin-embedded breast cancer or colorectal cancer tissue samples were dehydrogenated, rehydrated, and treated with sodium citrate-EDTA antigen retrieval solution for antigen retrieval. Subsequently, the tissue was incubated with the primary antibody for 1-2 hours at room temperature. After extensive washing with PBS, the tissue was 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 5m to show the staining signal, and then counterstained with hematoxylin. The Leica DMIRE2 microscope (Leica Microsystems Imaging Solutions Ltd.) was used for photography.

[0134] Isolation and purification of exosomes

[0135] Exosomes expressing a plasmid of iRGD-LAMP2B fusion protein were collected from the culture medium of HEK293T cells (preparation method reference PMID: 24345736). The sample was centrifuged at 4500rpm for 20 minutes to collect the supernatant, filtered through a 0.45μm membrane, and then filtered through a 0.22μm membrane. The exosome sample was concentrated using a hollow fiber column. The concentrated sample was chromatographically purified and then passed through a hollow fiber column again. Ultrafiltration concentration: The sample was added to an ultrafiltration tube and centrifuged at 3000rpm for 10-15 minutes at 4°C. The retentate was sterilized by filtration through a 0.22μm filter and stored at -80°C. Shanghai Youmi Biotechnology Co., Ltd. provided transmission electron microscopy and nanoparticle tracking analysis (NTA) services.

[0136] siRNA loading into exosomes

[0137] 2'-methoxy-modified siRNA was prepared by GenePharma (Shanghai, China). The siRNA solution was combined with the exosome suspension (the ratio of siRNA to exosome was 1 μg / 4 μg) in the electroporation dish. The voltage, pulse length, and number of pulses were optimized to effectively take up siRNA without destroying the exosomes. After electroporation, the cells were incubated at room temperature for 60 min, and 1 × 10 4 The free siRNA was removed and the exosomes were isolated by centrifugation at 4 °C for 60 min.

[0138] Statistical analysis

[0139] All in vitro experiments were performed in triplicate. Animals for in vivo experiments were randomly divided into different groups. Differences between two or more groups were analyzed by Student's t-test or one-way analysis of variance. Statistical analysis was performed using GraphPad Prism8.0, and the mean ± standard deviation (SD) was expressed. Kaplan-Meier plots and log-rank tests were used to analyze significant differences in patient survival. Multivariate Cox proportional hazard models produced hazard ratios with 95% confidence intervals. Asterisks indicate statistically significant differences: *p<0.05; **p<0.01; ***p<0.001.

[0140] (II) Results

[0141] Identification of the nucleolar protein BRIX1 as a regulator of p53

[0142] Due to the important role of ribosome homeostasis in controlling p53 activity and promoting cancer development, the present invention studies nucleolar proteins that may be critical for ribosome biogenesis. Currently, 286 nucleolar proteins involved in yeast pre-rRNA processing have been identified.

[0143] These nucleolar proteins were selected based on the following criteria:

[0144] (i) Proteins that directly control pre-rRNA processing in yeast;

[0145] (ii) proteins with homologs in humans and yeast;

[0146] (iii) proteins that may have similar functions in ribosome biogenesis in humans and yeast;

[0147] (iv) Proteins whose roles in cancer are still unclear.

[0148] Therefore, the present invention obtained six nucleolar proteins: BRIX1, DHX35, EXOSC6, EXOSC7, LSM6 and PPAN for further analysis ( Figure 1 A).

[0149] To test whether these nucleolar proteins are essential for cancer cell growth, 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 present invention's results showed that compared with 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).

[0150] To explore the potential signaling pathways regulated by BRIX1, we performed RNA sequencing (RNA-seq) analysis by knocking down BRIX1 in CAL-51 breast cancer cells. Heat map and KEGG pathway enrichment analysis showed that knocking down BRIX1 activated the p53 pathway ( Figure 1 C and 1D).

[0151] To verify this result, the present invention knocked down the expression of BRIX1 by 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).

[0152] Consistent with this, knockdown of BRIX1 also induced the expression of p53 and p21 at the protein level ( Figure 1 I-1K).

[0153] Upregulation of p21, BTG2, and MDM2 is dependent on p53, as knockdown of BRIX1 has an adverse effect on HCT116 p53- / - There was no effect on the expression in cells ( Figure 1 H and 1L).

[0154] Interestingly, in contrast to the effects of cisplatin (DDP), 5-fluorouracil (5-FU), or actinomycin D (Act D) treatment, knockdown of BRIX1 did not result in an increase in γ-H2AX phosphorylation ( Figure 1 M), indicating that no DNA damage stress was induced.

[0155] Taken together, these results suggest that the nucleolar protein BRIX1 may function as a regulator of ribosome biogenesis and the p53 pathway.

[0156] BRIX1 promotes pre-rRNA processing via the PeBoW complex

[0157] It has been reported that Brix1 is involved in the processing of pre-rRNA 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 investigated the expression of CAL-51 and HCT116 p53+ / + Gel electrophoresis analysis of total RNA in cells 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 both cell lines ( Figure 2 C and 2D).

[0158] In order to study 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 interactions with BRIX1 (Supplementary Figure 1 A). These three proteins constitute the PeBoW complex, which is essential for the processing of pre-rRNA and the maturation of the 60S ribosomal subunit in mammalian cells. Therefore, the present invention speculates that BRIX1 may promote rRNA synthesis by interacting with the PeBoW complex.

[0159] To verify this hypothesis, the present invention conducted a group of protein co-immunoprecipitation (co-IP) experiments 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 essential for the formation of the PeBoW complex, the present invention further tested whether BRIX1 binds to and regulates the level of endogenous BOP1.

[0160] Although endogenous interaction between BRIX1 and BOP1 has been observed in cancer cells ( Figure 2I), but knocking out BRIX1 did not affect the protein level of BOP1. It is worth noting that the results of the present invention show that knocking down BRIX1 greatly reduces the interaction between BOP1 and PES1 ( Figure 2 J and 2K), indicating that BRIX1 is required 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.

[0161] The results of the present invention show that BRIX1 deficiency mainly 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 L). Together, these results indicate that BRIX1 is required for pre-rRNA processing by regulating the stability of the PeBoW complex.

[0162] Overexpression of BRIX1 inhibits nucleolar stress-induced p53 activation

[0163] Next, the present invention continued to explore the potential effect of overexpression of BRIX1 on p53 activity. Surprisingly, under normal growth conditions, overexpression of BRIX1 in cancer cells did not affect the expression level of p53. Therefore, the present invention attempted to detect whether overexpression of BRIX1 regulates p53 under cell stress conditions.

[0164] Interestingly, the results of the present invention showed that overexpression of BRIX1 significantly reduced p53 protein in cancer cells when treated with low doses of Act D and DDP or 5-FU ( Figure 3 A and B) and their target genes ( Figure 3 C and D). However, when overexpressed BRIX1 was treated with Nutlin-3, an inducer that induces p53 by antagonizing MDM2, it failed to regulate p53 levels in cancer cells. It is known that low concentrations of Act D preferentially inhibit RNA Pol I activity, while both DDP and 5-FU may impair ribosome biogenesis by inducing rDNA damage or inhibiting pre-rRNA processing. Therefore, these results suggest that BRIX1 inhibits the activation of p53 caused by nucleolar stress.

[0165] In order 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 showed that BRIX1 was mainly distributed in the nucleolus under non-stress conditions or under Nultin-3 treatment, but under nucleolar stress caused by Act D, BRIX1 could migrate from the nucleolus to the nucleoplasm ( Figure 3 E).

[0166] Then, the present invention wanted to know whether overexpression of BRIX1 prevented 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 3 F).

[0167] In addition, overexpression of BRIX1 reduced the interaction of MDM2 with RPL5 and RPL11 ( Figure 3 G), which may be due to the competitive binding between BRIX1 and these two nucleolar proteins and MDM2. In addition, the present invention found that overexpression of BRIX1 enhanced MDM2-induced p53 ubiquitination ( Figure 3 H), while p53 degradation caused by BRIX1 overexpression can be blocked by the proteasome inhibitor MG132 ( Figure 3 I and J).

[0168] Finally, cycloheximide tracking analysis showed that BRIX1 overexpression shortened the half-life of p53 protein ( Figure 3 K). These results suggest that BRIX1 translocates to the nucleoplasm to prevent the interaction of MDM2 with RPL5 / RPL11, thereby inhibiting the stabilization and activation of p53 induced by nucleolar stress.

[0169] Next, the present invention examined 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 the action of low doses of Act d, overexpression of BRIX1 significantly increased the proliferation of cancer cells ( Figure 3 L and M) and colony formation ( Figure 3 N and O), reducing the apoptosis of cancer cells.

[0170] In addition, overexpression of BRIX1 promoted the migration of cancer cells ( Figure 3 P and Q).

[0171] In addition, the present invention established a transplant tumor model to determine the role of BRIX1 in vivo. p53+ / + Stable overexpression of BRIX1 in these cells significantly promoted the growth rate, weight, and size of transplanted tumors derived from these cells ( Figure 3 RT) without affecting the average body weight of mice ( Figure 3 U).

[0172] Taken together, these results suggest that excess BRIX1 counteracts nucleolar stress-mediated antitumor effects by inactivating p53, indicating that BRIX1 is involved in promoting chemoresistance.

[0173] High expression of BRIX1 is associated with poor prognosis in breast and colorectal cancer

[0174] Considering that BRIX1 is essential for the survival and proliferation of breast cancer and colorectal cancer cells, the present invention investigated the clinical significance of BRIX1 in these two cancers.

[0175] First, the present invention evaluated the expression of BRIX1 in breast cancer and matched normal tissues by IB and RT-qPCR analysis. The results of the present invention showed that the levels of BRIX1 protein and mRNA in breast cancer tissues were higher than those in normal tissues ( Figure 4 A and B).

[0176] In addition, immunohistochemistry (IHC) analysis of 91 breast cancer tissues showed that high expression of BRIX1 was significantly associated with higher tumor / node / metastasis (TNM) stage and poor overall survival of patients ( Figure 4 C).

[0177] Univariate and multivariate Cox regression analysis further confirmed that high expression of BRIX1 was a factor for poor prognosis in breast cancer ( Figure 4 D).

[0178] The present invention uses colorectal cancer samples with low p53 expression to determine the clinical relevance of BRIX1 levels, because samples with low p53 expression are likely to contain wild-type p53. Immunohistochemical analysis of the present invention showed that BRIX1 expression levels in colorectal cancer were higher than those in normal tissues, while p21 expression levels in colorectal cancer were lower than those in normal tissues ( Figure 4 EG).

[0179] This finding is partially consistent with the cell-based results of the present invention ( Figure 3 AD). By evaluating the expression of BRIX1 and p21 in 62 colorectal cancer samples, the present invention found that elevated BRIX1 levels were associated with advanced TNM stage and poor prognosis ( Figure 4 H), while elevated p21 levels are associated with a good prognosis ( Figure 4 I).

[0180] In addition, the analysis of the present invention showed that the expression of BRIX1 and p21 in colorectal cancer was negatively correlated ( Figure 5 ). High levels of BRIX1 combined with low levels of p21 are more predictive of a poor prognosis ( Figure 6). High levels of BRIX1 and low levels of p21 were more significantly associated with poor overall survival, and using high levels of BRIX1 and low levels of p21 as diagnostic indicators showed significant synergy.

[0181] In addition, both univariate and multivariate analyses showed that BRIX1 and p21 were prognostic factors for colorectal cancer. High levels of BRIX1 combined with low levels of p21 significantly improved the accuracy of prediction.

[0182] Finally, the present invention verifies the clinical relevance of high levels of BRIX1 combined with low levels of p21 through the TCGA database. Compared with normal tissues, higher levels of BRIX1 and lower levels of p21 are strongly correlated with poor prognosis in different types of cancer.

[0183] In summary, the above research results show that high levels of BRIX combined with low levels of p21 can better and more accurately predict the prognosis of tumors and provide more sufficient basis for clinical diagnosis.

[0184] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. 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 claims attached to this application.

Claims

1. Use of a reagent set in preparing a tumor detection kit, characterized in that: The reagent set includes a reagent for detecting BRIX1 and a reagent for detecting p21.

2. The use according to claim 1, characterized in that The tumor is breast cancer or colorectal cancer.

3. The use according to claim 1, characterized in that The reagent for detecting BRIX1 is an antibody against BRIX1 protein; or, the reagent for detecting BRIX1 is a reagent for specifically detecting BRIX1 gene.

4. The use according to claim 1, characterized in that The reagent for detecting p21 is an antibody against p21 protein; or, the reagent for detecting p21 is a reagent for specifically detecting p21 gene.

5. A tumor detection reagent set, characterized in that: The reagent set includes a reagent for detecting BRIX1 and a reagent for detecting p21.

6. The tumor detection reagent set according to claim 5, characterized in that: The reagent for detecting BRIX1 is an antibody against BRIX1 protein; preferably a monoclonal antibody.

7. The tumor detection reagent set according to claim 6, characterized in that: The reagent for detecting BRIX1 is a reagent for specifically detecting the BRIX1 gene, such as a primer for specifically amplifying the BRIX1 gene, or a probe for specifically binding to the BRIX1 gene.

8. A kit, characterized in that: The kit comprises a first detection reagent and a second detection reagent, wherein the first detection reagent is configured to detect BRIX1; and the second detection reagent is configured to detect p21.

9. The kit according to claim 8, characterized in that The reagent for detecting BRIX1 includes an antibody against BRIX1 protein, and the reagent for detecting p21 includes an antibody against p21 protein; or The reagent for detecting BRIX1 is a reagent for specifically detecting the BRIX1 gene, and the reagent for detecting p21 is a reagent for specifically detecting the p21 gene.

10. A method for diagnosing a tumor, characterized in that: The method comprises the steps of: detecting the BRIX1 level and the p21 level in the sample to be detected from the tumor patient; if the BRIX1 level in the sample is increased and the p21 level is decreased, it indicates that the tumor prognosis is poor.

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