WO2023147790A1 - 一种脑胶质瘤生物标记物mlkl基因及其应用 - Google Patents

一种脑胶质瘤生物标记物mlkl基因及其应用 Download PDF

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WO2023147790A1
WO2023147790A1 PCT/CN2023/085516 CN2023085516W WO2023147790A1 WO 2023147790 A1 WO2023147790 A1 WO 2023147790A1 CN 2023085516 W CN2023085516 W CN 2023085516W WO 2023147790 A1 WO2023147790 A1 WO 2023147790A1
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mlkl
glioma
sirna
gene
seq
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French (fr)
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李继喜
高文青
李媛媛
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复旦大学
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Definitions

  • the invention belongs to the technical field of biomedicine, and specifically relates to a potential glioma biomarker MLKL gene and its application, including the application of MLKL in the diagnosis, treatment and prognosis of malignant glioma.
  • Glioma is the most common primary tumor of the central nervous system in adults, accounting for about 50% of malignant brain and other central nervous system tumors. It has the characteristics of high malignancy, rapid progression, and easy recurrence. According to the traditional World Health Organization (WHO) classification, gliomas are roughly divided into four grades according to histological features, including lower grade glioma (LGG) and grade IV glioblastoma (Glioblastoma, GBM). ). The annual incidence of glioma in my country is 5-8/100,000, and the 5-year mortality rate is second only to pancreatic cancer and lung cancer among systemic tumors.
  • WHO World Health Organization
  • GBM is the most malignant type of glioma, with a comprehensive incidence peak at the age of 30-40, an average progression time of 6.9 months, an average overall survival of only 14.6 months, and a 5-year survival rate of less than 3%. Family and society bring a heavy burden.
  • TMZ Temozolomide
  • MLKL substrate mixed series protein kinase-like domain protein
  • RIPK1 and RIPK3 are two key proteins that initiate necroptosis, and the substrate mixed series protein kinase-like domain protein (MLKL) of RIPK3 is a specific execution protein of programmed cell necrosis.
  • MLKL phosphorylation undergoes a constitutive change from After dissociation in the necrotic complex, the plasma membrane is transferred, resulting in the instability of the plasma membrane and the increase of intracellular osmotic pressure, which leads to the release of intracellular substances from the ruptured cell membrane, and the cell finally presents a necrotic state.
  • MLKL phosphorylation inhibitors or inhibition of MLKL protein expression can inhibit the process of programmed cell necrosis.
  • Programmed necrosis was first discovered in the pathological manifestations of cerebral ischemic injury. More and more studies have shown that programmed cell necrosis is widely involved in the occurrence and development of infectious diseases, nervous system related diseases and tumors.
  • the purpose of the present invention is to provide a biomarker of glioma that can be used for auxiliary diagnosis of malignant glioma and its application.
  • the present invention unexpectedly finds that MLKL is significantly highly expressed in brain glioma, and can be used for auxiliary diagnosis of malignant glioma; highly expressed MLKL gene and protein are related to the poor prognosis of glioma, suggesting that MLKL gene and its expression products can As a molecular marker for prognostic evaluation and therapeutic effect. Furthermore, reducing the expression of MLKL can significantly inhibit the growth of malignant glioma cells, suggesting that MLKL can also be used as a target for the treatment of glioma.
  • the invention also discloses the MLKL gene expression inhibitor siRNA, which has a good interference effect on the expression of the MLKL gene and has the application prospect of clinical gene therapy.
  • the present invention provides an application of a glioma marker MLKL gene and/or an expression product of the MLKL gene in the preparation of a detection kit for glioma diagnosis, screening and/or prognosis .
  • the present invention uses bioinformatics analysis methods to detect the mRNA expression of MLKL gene in glioma tissue (Tumor) and normal tissue (Normal), showing that MLKL gene is in 33 kinds of cancer tissue (Tumor) and normal tissue (Normal). ) mRNA expression was different, and the expression level of MLKL in cancer tissues was significantly higher than that in normal tissues.
  • the Cox regression method was used to analyze the correlation between the level of MLKL mRNA and the overall survival of patients. It was found that the overall survival (OS) of glioma patients with high expression of MLKL was significantly lower than that of patients with low expression of MLKL gene, suggesting that the expression of MLKL gene
  • the product can be used as a prognostic marker for glioma. By detecting the expression of the MLKL gene, the prognosis of patients with different grades of glioma can be evaluated, the potential survival time of the patient can be indicated, and the therapeutic effect of glioma can be evaluated.
  • MLKL can be used as a new glioma prognostic marker to provide a basis for evaluating the survival of patients with different subtypes of glioma.
  • ROC curve analysis it was found that the AUC value of MLKL gene in the diagnosis of glioma was higher than 0.7, showing that As a diagnostic and prognostic marker for glioma, it has high specificity and high sensitivity, and is an ideal target for early molecular diagnosis and disease risk screening of glioma.
  • the aforementioned glioma includes low-grade glioma (LGG) and high-grade glioma (GBM).
  • the tumor is a high-grade glioma, ie glioblastoma.
  • the expression product of the glioma marker MLKL gene includes MLKL mRNA and/or MLKL protein.
  • the present invention also provides a kit for the diagnosis and prognosis of glioma, which uses the MLKL gene and/or the expression product of the MLKL gene as a detection target.
  • the kit for the diagnosis and prognosis of glioma prepared by the present invention comprises the following components:
  • a pair of primers that can be used to specifically amplify the MLKL gene can be used to specifically amplify the MLKL gene;
  • An antibody that specifically recognizes/binds the expression product of the MLKL gene is an antibody that specifically recognizes/binds the expression product of the MLKL gene.
  • the kit for the diagnosis and prognosis of glioma prepared by the present invention, contains specific A primer pair for amplifying the MLKL gene, said primer pair comprising the following sequences:
  • Upstream primer sequence 5'-TCACACTTGGCAAGCGCATGGT-3' (SEQ ID NO: 2);
  • Downstream primer sequence 5'-GTAGCCTTGAGTTACCAGGAAGT-3' (SEQ ID NO: 3);
  • Upstream primer sequence 5'-TGCAGAGGAAGACGGAAATGA-3' (SEQ ID NO: 4);
  • Downstream primer sequence 5'-CTCCTGTGTGGGTTTTAGTGAGC-3' (SEQ ID NO: 5);
  • Upstream primer sequence 5'-AGGAGGCTAATGGGGAGATAA-3' (SEQ ID NO: 6);
  • MLKL gene sequence of MLKL the expression product of MLKL gene, such as MLKL protein, is widely known.
  • the UniProt database ID: Q8NB16 the specific sequence is shown in SEQ ID NO: 8.
  • an antibody that specifically recognizes/binds MLKL protein for example, by using the full-length MLKL protein or a partial protein sequence as an antigen such as immunizing animals such as mice, rabbits or alpacas, etc., specificity can be obtained Antibodies that recognize the MLKL protein.
  • the antibody is a monoclonal antibody, a polyclonal antibody or a nanobody, preferably a monoclonal antibody.
  • Antibodies can be prepared by themselves, or commercially available antibodies that recognize MLKL can be purchased, such as the anti-MLKL antibody from Abcam (product number: ab184718).
  • the present invention prepares a kit for the diagnosis and prognosis of glioma, which includes a pair of primers that can be used to specifically amplify the MLKL gene, or a pair of primers that can specifically amplify the expression product of the MLKL gene, Or an antibody that can specifically recognize/bind with MLKL protein.
  • the antibody has the following characteristics: it can recognize the full-length human MLKL protein, and the affinity reaches 10 -9 nM or higher.
  • the primer pair can be used for detection by genetic means, such as various PCR means, such as fluorescent real-time quantitative PCR.
  • MLKL protein can also be detected by immunological means, such as Western blot, ELISA, immunohistochemistry, immunofluorescence, etc.
  • the present invention finds that reducing the expression of MLKL can significantly inhibit the growth of malignant glioma cells, suggesting that MLKL can also be used as a target for treating glioma.
  • the present inventors found that the higher the expression of MLKL gene or protein level, the lower the survival rate of glioma patients, that is, the overexpression of MLKL gene or protein clearly indicates the poor survival prognosis of patients, indicating that MLKL gene and/or its expression products can be used as Prognostic markers of glioma, used to predict the prognosis of patients. According to the inventor's research, inhibiting endogenous MLKL can significantly reduce the proliferation of malignant glioma cells, making MLKL an effective target for treating glioma.
  • the present invention discloses the use of MLKL gene as a drug or preparation targeting glioma cells in screening drugs for treating glioma;
  • the MLKL gene as a drug or preparation targeting brain glioma cells is specifically Refers to: using MLKL gene as a drug or preparation to target glioma cells to produce RNA interference, thereby reducing the expression level of MLKL gene in glioma cells; using MLKL gene as a drug or preparation to target glioma cells
  • the application of the target to the screening of drugs for the treatment of glioma specifically refers to: screening drugs or preparations using the MLKL gene as an action object, so as to find a drug that can inhibit the expression of the MLKL gene as a candidate drug for the treatment of glioma;
  • the sequence of the target is shown in SEQ ID NO:1.
  • the present invention also prepares MLKL gene expression inhibitor siRNA, which has a good interference effect on the expression of MLKL gene and has application prospects in clinical gene therapy.
  • the MLKL gene expression inhibitor siRNA can be used to prepare medicaments for treating glioma, assist clinical treatment of glioma, and improve the therapeutic effect of glioma. Therefore, the present invention also provides an expression inhibitor of MLKL gene, said expression inhibitor comprising siRNA designed based on said MLKL gene, wherein said siRNA comprises 15-30 nucleotides.
  • siRNA (Small interfering RNA; small interfering RNA) binds to the mRNA after transcription of the target gene MLKL, and mediates the degradation of mRNA through the RNA-mediated silencing complex RISC (RNA-induce silencing complex).
  • the length of the siRNA is more preferably 21-23nt. After being recognized and bound by RISC, the siRNA unwinds. Under the guidance of the antisense strand of siRNA, RISC looks for endogenous mRNA with homologous sequence, and cleaves mRNA between 10-11 bases from the 5' end, resulting in post-transcriptional gene silencing.
  • siRNA design website http://sidirect2.rnai.jp
  • DSIR design website http://biodev.extra.cea.fr/DSIR/DSIR.html
  • invivogen The design website http://www.invivogen.com/sirnawizard/siRNA.php
  • thermofisher design website https://rnaidesigner.thermofisher.com
  • the present invention also provides an expression inhibitor of MLKL gene, said expression inhibitor comprises siRNA designed based on said MLKL gene, said siRNA nucleotide sequence sense strand is as follows:
  • the above-mentioned siRNA includes at least one modified nucleotide.
  • the modified siRNA generally has less immunostimulatory effect than the corresponding unmodified siRNA sequence, and maintains the RNAi activity against the target gene of interest.
  • the modified siRNA contains at least one 2'OMe purine or pyrimidine nucleotide such as 2'OMe-guanosine, 2'OMe-uridine, 2'OMe-adenosine, and/or 2'OMe- Cytosine nucleotides (reference M.M. Zhang et al., Biochemical Pharmacology 189 (2021) 114432).
  • modified nucleotides may be present in one strand (ie, sense or antisense) or both strands of the siRNA.
  • siRNA sequences may have overhangs (eg, 3' or 5' overhangs) or may lack overhangs (ie, have blunt ends).
  • the phosphodiester bond connecting the RNA phosphate backbone is the chemical bond for nuclease action, and the phosphorus atom is the center of nuclease attack.
  • the modification of this atom can affect the degradation of the enzyme, thereby improving the ability of small interfering nucleic acids to resist nucleases and increase its stability.
  • the phosphorothioate modification is the substitution of a sulfur atom for the non-bridging oxygen atom of the phosphodiester bond by exchanging the phosphate bond (PO) for a phosphorothioate (PS), i.e. P-S Bonds replace P-O bonds, a modification that generally increases the stability of nucleic acids against nucleases.
  • the preparation method of the siRNA is not particularly limited, and can be obtained by chemical synthesis, or by expression of plasmids and/or viral vectors.
  • modified nucleotides can be used to replace unmodified nucleotides in corresponding positions, or phosphorothioate modification of phosphodiester bonds can be performed.
  • the method for synthesizing siRNA includes the following four processes: (1) synthesis of oligoribonucleotides; (2) deprotection; (3) purification and separation; (4) desalting and so on.
  • siRNA has a lot of negative charges because it contains multiple phosphate bonds, and it is difficult for such a polar molecule to pass through the cell membrane by itself; in addition, siRNA exposure to blood will cause stability problems and cause immunogenicity. Therefore, in another preferred embodiment, the above-mentioned siRNA needs to be wrapped with a carrier (such as LNP) or linked to a specific ligand (such as GalNac) to effectively avoid renal elimination, and can also be enriched in specific tissues in a targeted manner. middle.
  • a carrier such as LNP
  • a specific ligand such as GalNac
  • the present invention has prepared a kind of siRNA nucleic acid-lipid particle, comprises:
  • siRNA nucleic acid the positive strand of the siRNA nucleotide sequence is shown in SEQ ID NO.9, SEQ ID NO.10 or SEQ ID NO.11;
  • non-cationic lipids comprising a mixture of phospholipids and cholesterol or derivatives thereof, wherein the phospholipids account for 3mol% to 15mol% of the total lipids present in the particle and the cholesterol or derivatives account for 30 mol% to 40 mol% of the total lipid present in the particle;
  • a conjugated lipid that inhibits particle aggregation comprising from 0.5 mol% to 2 mol% of the total lipids present in the particle; wherein, wherein the cationic lipid comprises 1,2-dilinoleyloxy -N,N-Dimethylaminopropane (DLinDMA), 1,2-Dilinenyloxy-N,N-Dimethylaminopropane (DLenDMA) or mixtures thereof;
  • DLinDMA 1,2-dilinoleyloxy -N,N-Dimethylaminopropane
  • DLenDMA 1,2-Dilinenyloxy-N,N-Dimethylaminopropane
  • the phospholipids include dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC) or a mixture thereof;
  • DPPC dipalmitoylphosphatidylcholine
  • DSPC distearoylphosphatidylcholine
  • conjugated lipids that inhibit particle aggregation include polyethylene glycol (PEG)-lipid conjugates, selected from PEG-diacylglycerol (PEG-DAG) conjugates, PEG-dialkoxypropane PEG-DAA conjugates or mixtures thereof.
  • PEG polyethylene glycol
  • PEG-DAG PEG-diacylglycerol
  • the present invention has prepared a GalNAc-siRNA conjugate, that is, siRNA as shown in SEQ ID NO.9, SEQ ID NO.10 or SEQ ID NO.11 is conjugated to N-acetyl Galactosamine (N-Acetylgalactosamine, GalNAc) forms a GalNAc-siRNA conjugate.
  • a GalNAc-siRNA conjugate that is, siRNA as shown in SEQ ID NO.9, SEQ ID NO.10 or SEQ ID NO.11 is conjugated to N-acetyl Galactosamine (N-Acetylgalactosamine, GalNAc) forms a GalNAc-siRNA conjugate.
  • the GalNAc ligand can bind to the asialoglycoprotein receptor (ASGPR) expressed by hepatocytes and deliver siRNA to hepatocytes. It is a huge advantage of GalNAc-siRNA that it can be administered subcutaneously and in a long cycle, and it is more suitable for subcutaneous administration.
  • ASGPR asialoglycoprotein receptor
  • the present invention also provides a method for preparing GalNAc-siRNA conjugates, that is, in the process of solid-phase oligonucleotide synthesis, GalNAc is used to load a solid support, so that GalNAc and the 3' end of the sense strand of siRNA pass through a three-element spacer Phase connection; followed by deprotection of nucleotides and deprotection of galactosamine acetate, removing the solid support, the GalNAc-siRNA conjugate can be obtained.
  • the siRNA can be attached to one or more (eg, two, three, four or more) GalNAc derivatives.
  • the attachment may be via no linkers, or via one or more linkers (eg, two, three, four or more linkers).
  • the linkers described herein are multivalent (eg, bivalent, trivalent, or tetravalent) branched linkers.
  • the one or more GalNAc derivatives are attached to the 3' end of the sense strand, the 3' end of the antisense strand, the 5' end of the sense strand, and/or the antisense strand of the dsRNA. 5' end of the chain.
  • GalNAc-siRNA conjugates For the synthesis of GalNAc-siRNA conjugates, reference can be made to WO2009/073809, WO2011/104169, WO2012/083046, WO2014/118267, and WO2014/179620, all of which are hereby incorporated by reference.
  • CDK2 cyclin-dependent kinase 2
  • cyclin A2 cyclin A2
  • CDKs mainly combine with cyclins to form The active proteasome complex catalyzes the phosphorylation of substrate serine/threonine residues and promotes the continuous progression of the cell cycle.
  • CDK2 regulates the transition from G1 phase to S phase and the progress of S phase by forming a proteasome complex with cyclin A.
  • CDK2-cyclin A2 The decrease in the expression of CDK2-cyclin A2 suggests that knocking down MLKL may inhibit the proliferation rate of malignant glioma cells, suggesting that siRNA, an inhibitor of MLKL gene expression, can be used to prepare drugs for the treatment of glioma, assist in the clinical treatment of glioma, and improve the growth rate of malignant glioma cells. Tumor treatment effect.
  • the present invention also provides the use of the above-mentioned MLKL expression inhibitor siRNA in the preparation of medicaments for treating glioma.
  • the present invention provides a new theoretical basis and a new target for the treatment of glioma, and the present invention also provides a new auxiliary diagnosis and prognosis diagnosis method and a new therapeutic drug for glioma.
  • Figure 1 shows the comparison of the expression of MLKL mRNA provided by Example 1 of the present invention in 33 cancer tissues (Tumor) and normal tissues (Normal).
  • Fig. 2 shows the survival analysis forest plot of the effect of MLKL gene expression level on the overall survival (OS) of 29 cancer patients provided by Example 2 of the present invention.
  • Figure 3 shows the relationship between the expression level of MLKL gene and the survival rate of high-grade glioma (GBM) patients provided by Example 3 of the present invention compared with other glioma markers NUSAP1, GINS2 and TRIM21 reported in the literature sex is more pronounced.
  • Fig. 4 shows is that the expression level of MLKL gene provided by the embodiment of the present invention 4 has an effect on low-grade glioma (LGG) and high-grade glioma (GBM) overall survival (OS) (a), disease-specific survival period ( Survival analysis curves affected by DSS) (b) and progression-free survival (PFI) (c).
  • LGG low-grade glioma
  • GBM high-grade glioma
  • OS overall survival
  • OS disease-specific survival period
  • PFI progression-free survival
  • Fig. 5 shows the ROC curve for the diagnosis of glioma by the expression level of MLKL gene provided in Example 5 of the present invention.
  • Fig. 6 shows the expression levels of the MLKL gene provided in Example 6 of the present invention in various molecular subtypes of glioma. Among them, (a) is WHO grade; (b) is IDH mutation; (c) is 1p/19q gene combined deletion; (d) clinicopathological type.
  • FIG. 7 shows the survival analysis curves of the influence of the expression level of MLKL gene on the survival period of various molecular subtypes of glioma provided by Example 7 of the present invention.
  • (a) is WHO grade;
  • (b) is IDH mutation;
  • (c) is clinicopathological type.
  • Figure 8 shows that in clinical glioma samples provided by Example 8 of the present invention, the higher the degree of malignancy of glioma, the higher the expression of MLKL protein (a); quantitative analysis shows that MLKL protein in high-grade glioma ( b) and IDH1 wild-type glioma (c) significantly increased expression;
  • Fig. 9 shows the correlation analysis graph between the expression level of MLKL protein and the overall survival of glioma patients (a), IDH1 mutation (b) and tumor proliferation marker Ki67 (c) provided by Example 8 of the present invention;
  • Figure 10 shows the electrophoresis results (a) and quantitative analysis diagram (b) used by the MLKL mRNA level detection kit provided by Example 9 of the present invention
  • Figure 11 shows the western blot experiment (a) and its quantitative analysis (b) of the MLKL knockdown cells and control cells (NC) provided by Example 11 of the present invention, and the cell proliferation rate detection experiment (c).
  • Fig. 12 shows the inhibitory effect of the knockout of MLKL provided in Example 12 of the present invention on the proliferation rate of glioma cell U251.
  • Fig. 13 shows the inhibitory effect of knockout MLKL provided by Example 13 of the present invention on the growth of subcutaneous tumor tissue in mice.
  • MLKL antibody was purchased from abcam company, Name: recombinant Anti-MLKL antibody (ab184718). ⁇ -actin antibody was purchased from Proteintech (No.66009). Anti-rabbit IgG antibody was purchased from CellSignalling (#7074). Enhanced CCK-8 kit, purchased from Beyontian Biotechnology Company, product number: C0042. ECL chemiluminescence substrate was purchased from Tianneng Company (No. 180-5001) and Lipofectamine 2000 transfection reagent was purchased from ThermoFisher Scientific Company, No. 11668-019. TRIzol reagent was purchased from ThermoFisherScientific, Cat. No. 15596026. PrimeScript 1st strand cDNA synthesis kit was purchased from TaKaRa Company, Cat. No. 6110A. ChamQ Universal SYBR qPCR master mix was purchased from Vazyme, Cat. No. Q711-03.
  • a method for detecting the mRNA expression of MLKL gene in glioma tissue (Tumor) and normal tissue (Normal) by using bioinformatics analysis method the specific content is as follows:
  • MLKL mRNA in 33 cancers and normal tissues was downloaded from TCGA database (https://portal.gdc.cancer.gov/) and GTEx database (http://gtexportal.org/home/). The difference in the expression of MLKL in normal tissues and cancer tissues was detected by Wilcoxon rank sum test.
  • Figure 1 shows the mRNA expression difference of MLKL gene in 33 cancer tissues (Tumor) and normal tissues (Normal) As can be seen from Figure 1, the expression level of MLKL in cancer tissues is significantly higher than that in normal tissues.
  • the Cox regression method was used to analyze the correlation between the MLKLmRNA level and the overall survival of patients, and the survival analysis forest plot as shown in Figure 2 was obtained.
  • genes can evaluate the prognosis of patients with different grades of glioma, indicate the potential survival time of patients, and can also be used to evaluate the therapeutic effect of glioma. More meaningfully, high expression of MLKL was not associated with poor prognosis in other 29 cancers, suggesting the specificity of MLKL as a marker for detection and prognosis of glioma.
  • MLKL, NUSAP1, GINS2 and TRIM21 information and patient information were downloaded from the TCGA database, and the COX regression method was used to compare and analyze MLKL and three discovered glioma markers, namely, the gene expression levels of NUSAP1, GINS2 and TRIM21 and the overall survival of patients Correlation between periods, the survival analysis heat map as shown in Figure 3 is obtained.
  • the Kaplan-Meier curve and log-rank detection are used to analyze the relationship between MLKL gene expression level and patient overall survival (OS), disease specificity Correlation between survival period (DSS) and progression-free survival (PFI), the survival analysis curve as shown in Figure 3 was obtained.
  • the prognosis of patients with low-grade glioma and high-grade glioma can be evaluated in time, which can be used to adjust clinical treatment strategies, further improve clinical treatment effects, and improve patient outcomes. Quality of life is instructive.
  • This example aims to provide a method for evaluating the MLKL gene expression level for the diagnostic value of glioma.
  • the specific content is: using the information of glioma patients obtained from the TCGA database in Example 2 to edit and analyze through software, Obtain a time-dependent ROC (receiver operator characteristic curve) curve.
  • the above software is R (version 3.6.3), the R package used for statistical analysis is timeROC (version 0.4), and the R package used for data visualization is ggplot2 (version 3.3.3).
  • the ROC curve is a graphical way to show the trade-off between the true positive rate (sensitivity, or sensitivity, TPR) and the false positive rate (FPR).
  • a good classification model which refers to a good cancer marker in this invention, should probably be near the upper left corner of the ROC curve.
  • the area under the curve can also be used to represent the average performance of a model. The larger the AUC, the higher the diagnostic accuracy.
  • the area under the curve (AUC) was 0.738
  • the sensitivity of MLKL gene expression in diagnosing glioma was 65.3%
  • the specificity was 75.3%.
  • the area under the curve (AUC) was 0.791
  • the sensitivity of MLKL gene expression in diagnosing glioma was 64.1%
  • the specificity was 84.2%. It is shown that it has high specificity and high sensitivity as a glioma diagnostic and prognostic marker, and is an ideal target for early molecular diagnosis and disease risk screening of glioma.
  • the Kruskal-Wallis test was used to detect the expression of MLKL gene and the different clinical types of glioma, that is, WHO grade, IDH mutation, and 1p/19q co-occurrence. Missing and the correlation between different tissue types, resulting in the histogram shown in Fig. 6(a-b).
  • Fig. 6(a) shows that the expression level of MLKL gene in grade III glioma is higher than that in grade II, and the expression in grade IV glioma is higher than that in grade II and grade III.
  • Figure 6(b) shows that the expression level of MLKL gene in IDH wild type is significantly higher than that in IDH mutant type.
  • Figure 6(c) shows that the expression level of MLKL gene without 1p/19 joint deletion is significantly higher than its expression level in 1p/19q joint deletion.
  • Figure 6(d) shows that MLKL gene expression in oligodendroglioma (oligodendroglioma), oligoastrocytoma (oligoastrocytoma), astrocytoma (astrocytoma), glioblastoma (glioblastoma)
  • the expression level gradually increased.
  • the above results together indicate that the higher the degree of malignancy of glioma, the higher the expression level of MLKL gene. It can be seen that the expression of MLKL is correlated with the grade of glioma, IDH mutation, 1p/19 co-deficient MLKL gene and its expression products can be used as markers for clinical judgment of glioma grade and histological type.
  • MLKL is significantly higher in patients with higher-grade glioma (G3 and G4) (a), in patients with IDH wild-type (b), and in 3 more severe histological types (including oligoastrocytic Glioblastoma, astrocytoma, and neuroblastoma) (c) have prognostic significance. Therefore, MLKL can be used as a new glioma prognostic marker to provide a basis for evaluating the survival of patients with different subtypes of glioma.
  • MLKL protein expression in high-grade glioma clinical samples was higher than that in low-grade glioma tissues.
  • the research subjects selected 81 clinical samples of glioma patients, including 17 cases of WHO grade II, 19 cases of grade III, and 52 cases of grade IV. This study was reviewed and approved by the hospital ethics committee, and all patients signed a written informed notice before enrollment Book.
  • the clinical glioma tissue was broken into tissue homogenate with a tissue breaker, and then the cells of each component were broken with RIPA cell lysate, and the protein was dissolved. After the protein was quantitatively adjusted under the same conditions, the western blot analysis was performed.
  • the specific steps are as follows: first, the protein was separated by SDS-PAGE electrophoresis, and the protein was transferred to a nitrocellulose membrane, and then 5% skimmed milk powder was used for blocking experiment.
  • the sex-binding antibody was fully combined with it, further combined with a secondary antibody that can specifically bind to the primary antibody, and then photographed by a protein chromogenic imager, and finally the quantitative analysis of the colored protein bands was completed by Image J software.
  • survival analysis curves were drawn by the Kalpan-Meier method, and the statistical significance was detected by the log-rank method. The detection coefficient P ⁇ 0.05 has a statistically significant difference.
  • R package survival package (version 3.2-10) for statistical analysis of survival data, the relationship between the expression of MLKL protein and the overall survival rate (Overall survival, OS) of glioma patients. MLKL Protein overexpression and underexpression were demarcated according to the median of MLKL protein expression level.
  • the t test of GraphPad Prism 7 software was used to analyze the correlation between MLKL protein expression, IDH1 mutation and Ki67 index.
  • Ki67 is a nuclear protein related to cell proliferation, which can be used to judge the tumor proliferation index. Generally speaking, the higher the value of Ki-67 positive marker, the worse the tissue differentiation, the higher the degree of malignancy, and the worse the prognosis. In conclusion, the high expression level of MLKL protein can be used as a marker of poor prognosis in glioma.
  • RT-qPCR was used to detect the mRNA level of MLKL in glioma tissue samples.
  • a glioma MLKL diagnosis and detection method disclosed in this example uses normal brain tissue or tumor tissue from a glioma patient, extracts total RNA according to the operating steps in the instructions, and uses TaKaRa reverse transcriptase to reverse transcribe the extracted total RNA into cDNA , and the mRNA level of MLKL was analyzed by PCR.
  • the specific conditions are as follows:
  • Trizol lysate was added to each 100 mg of tissue, and placed on ice after low-temperature ultrasonication for lysis for 5 min; then 200 ⁇ L of chloroform was added, shaken vigorously several times, and placed in ice bath for 10 min. After centrifugation at 12,000 rpm at 4°C for 10 minutes, carefully transfer the top transparent solution to an RNase-free centrifuge tube, add 500 ⁇ L of isopropanol to shake several times, place on ice for 20 minutes, and then centrifuge at 12,000 rpm at 4°C for 15 minutes.
  • the reverse transcription product amplification method refers to the instruction manual of SYBR qPCR enzyme (product number: Q711-03) of Vazyme Company, the primer concentration is 10 ⁇ M, and GAPDH is used as the internal reference, and the MLKL mRNA level is quantitatively analyzed by ImageJ software.
  • the primer sequences for the copy number of MLKL gene level are:
  • R 5'-GTAGCCTTGAGTTACCAGGAAGT-3', (SEQ ID NO: 3);
  • the primer sequence for the copy number of GAPDH gene level is:
  • R 5'-GGCTGTTGTCATACTTCTCATGG-3', (SEQ ID NO: 13).
  • the method for diagnosing and detecting MLKL in glioma disclosed in this embodiment uses the Western Blot method to detect the protein expression level of MLKL in glioma tissue samples, and quantitatively analyzes the protein expression level of MLKL.
  • the kit contains antibodies capable of specifically recognizing human MLKL and the internal reference ⁇ -Actin, and the schematic diagram of antibody binding is shown in Figure 8a.
  • MLKL protein promotes malignant proliferation of glioma cells.
  • MLKL in U87 malignant glioma cells was specifically knocked down by siRNA technology.
  • siNC interfering sequences siNC(5'–UUCUCCGAACGUGUCACGUTT–3'(SEQ ID NO.14) and siMLKL(#1:5'-GAAGCAUAUUAUCACCCUUTT-3'(SEQ ID NO.9), #2:5'-GCAAUAGAUCCAAUAUCUGTT-3' (SEQ ID NO.10)) after transfecting cells
  • immunoblotting experiments showed that the expression level of MLKL was significantly reduced (Fig. The expression level decreases with the decrease of MLKL ( Figure 11(b)).
  • CDKs mainly combine with cyclin to form an active proteasome complex, thereby catalyzing the phosphorylation of substrate serine/threonine residues , to promote the continuous progress of the cell cycle.
  • CDK2 regulates the transition from G1 phase to S phase and the progress of S phase by forming a proteasome complex with cyclin A.
  • the expression of CDK2-cyclin A2 decreases, suggesting that knocking down MLKL may inhibit U87 cells growth rate.
  • CCK8 kit detects the proliferation rate of malignant glioma cells
  • the enhanced CCK8 kit was used to count the number of cells in different groups, and the changes in the growth rate of glioma cells knocked down MLKL protein were detected.
  • the results are shown in Figure 11(c), in the cell proliferation curve detection experiment, the growth of U87 was significantly inhibited after knocking down MLKL.
  • the above experiments show that MLKL can be used as a new type of tumor-promoting molecule and participate in the malignant growth and development of glioma.
  • MLKL knockdown inhibits the proliferation rate of glioma cell U251.
  • MLKL knockout U251 cells In order to determine the effect of MLKL on the proliferation of glioma, CRISPR-Cas9 technology was used to construct MLKL knockout U251 cells.
  • the DNA sequence corresponding to the gRNA is 5'-GAAGCTGAGTGATGTCTGGA-3' (SEQ ID NO.15), and the gene editing results are shown in Figure 12a.
  • Collect wild-type or MLKL -/- U251 cells in the logarithmic growth phase add 2000 cells/well to a 6-well plate, shake gently to make the cells evenly distributed in the plate, and culture them in an incubator for 2 weeks before crystallization Violet stain.
  • the results are shown in Figure 12b, the proliferation rate of MLKL -/- U251 cells was significantly slowed down.
  • the cells were added to a 96-well plate at 2000 cells/well, and the number of cells per day was recorded using the CCK8 kit, and the results are shown in Figure 12c.
  • the cell proliferation curve showed that the growth of MLKL -/- U251 cells was significantly inhibited.
  • MLKL knockdown inhibits the formation of subcutaneous tumor tissue.
  • mice were subcutaneously injected with U251 cells and MLKL ⁇ / ⁇ U251 cells prepared in Example 12. On day 21 and day 27 after injection, the subcutaneous tumor volume was measured and calculated with a caliper. The photograph of the mice 27 days after injection is shown in Figure 13a, and the tumor volume is shown in Figure 13b. The results showed that the subcutaneous tumorigenesis rate of MLKL -/- U251 cells was poor, and the cell growth slowed down significantly.
  • MLKL is highly expressed in gliomas, especially high-grade gliomas, through bioinformatics analysis, and its high expression level is closely related to poor survival of glioma patients.
  • LGG low-grade glioma
  • GBM high-grade glioma
  • siRNA interference technology was used to knock down MLKL in human glioma cell U87, and then the western blot experiment and cell proliferation rate detection experiment were performed.
  • the results showed that interference with MLKL may reduce cell cycle
  • the proteins CDK2 and cyclinA2 inhibited the proliferation rate of U87 cells; further, through in vitro and in vivo experiments, it was proved that MLKL knockout significantly inhibited the proliferation of tumor cells and tumor tissues.
  • MLKL gene or its expression product MLKL protein as a target in the preparation of antitumor drugs is also within the protection scope of the present invention.

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Abstract

本发明公开一种脑胶质瘤生物标记物MLKL基因及其应用。MLKL在脑胶质瘤中显著高表达；高表达的MLKL基因和蛋白与胶质瘤的不良预后相关；降低如KL表达可明显抑制恶性胶质瘤细胞的生长；因而MLKL基因和其表达产物可作为预后评估和治疗效果的分子标志物；MLKL还可作为治疗胶质瘤的靶标。本发明还公开脑胶质瘤诊断、筛查和/或预后的检测试剂盒，siRNA核酸-脂质颗粒，MLKL基因表达抑制剂siRNA,GalNAc-siRNA缀合物，以及它们在脑胶质瘤诊断、筛查和/或预后的检测中的应用。

Description

一种脑胶质瘤生物标记物MLKL基因及其应用 技术领域
本发明属于生物医药技术领域，具体涉及一种潜在的脑胶质瘤生物标记物MLKL基因及其应用，包括MLKL在恶性胶质瘤诊断、治疗以及预后中的应用。
背景技术
胶质瘤是成年人最常见的中枢神经***原发性肿瘤，约占恶性的脑及其他中枢神经***肿瘤的50％，具有恶性程度高、进展快、易复发等特点。根据传统的世界卫生组织(WHO)分级，胶质瘤根据组织学特征大致分为四级，包括较低级别胶质瘤(Lower Grade Glioma,LGG)及IV级胶质母细胞瘤(Glioblastoma,GBM)。我国脑胶质瘤的年发病率为5-8/10万，5年病死率在全身肿瘤中仅次于胰腺癌和肺癌。GBM是胶质瘤中恶性程度最高的类型，综合发病高峰在30-40岁，平均进展时间6.9个月，患者总生存期平均仅有14.6个月，5年生存率不足3％，给个人、家庭、社会带来沉重的负担。
目前的临床治疗手段仍然仅限于手术切除后放疗与化疗。手术切除是治疗脑胶质瘤的首要策略，但因胶质瘤浸润性生长特点，与正常脑组织边界模糊，几乎难以切除干净，手术切除后的复发概率仍高达95％以上，并最终造成患者死亡。替莫唑胺(temozolomide,TMZ)是一种口服烷化剂，具有广谱的抗肿瘤活性，在安全有效切除原发胶质瘤后，其可以穿过血脑屏障直接作用于病灶，进一步抑制肿瘤复发率，但TMZ耐药问题严峻。近年来，新的治疗方法陆续出现，包括免疫治疗和分子靶向治疗，但这些治疗方法并没有明显提高胶质瘤患者的生存率，主要是由于胶质瘤的遗传异质性。因此，寻找有效的生物标志物和治疗靶点对胶质瘤患者的诊断和治疗十分重要。
细胞程序性坏死(necroptosis)在人类免疫反应中起着重要作用。受体相互作用蛋白RIPK1和RIPK3是启动necroptosis的两个关键蛋白，而RIPK3的作用底物混合系列蛋白激酶样结构域蛋白(MLKL)，是细胞程序性坏死的特异性执行蛋白。MLKL磷酸化后发生构建变化，从 坏死复合体中解离后转移质膜，造成质膜的不稳定，胞内渗透压升高，导致胞内物质从破裂的细胞膜中释放，细胞最终呈现坏死状态。研究发现，使用选择性MLKL磷酸化抑制剂或抑制MLKL蛋白的表达均可抑制程序性细胞坏死的进程。程序性细胞坏死最先被发现于脑缺血损伤的病理表现中。越来越多的研究表明，程序性细胞坏死广泛参与感染类疾病、神经***相关疾病以及肿瘤的发生发展。
发明内容
本发明的目的在于提供一种能够用于恶性胶质瘤的辅助诊断的脑胶质瘤的生物标志物及其应用。
本发明意外发现MLKL在脑胶质瘤中显著高表达，能够用于恶性胶质瘤的辅助诊断；高表达的MLKL基因和蛋白与胶质瘤的不良预后相关，提示MLKL基因和其表达产物能够作为预后评估和治疗效果的分子标志物。进一步地，降低MLKL表达可明显抑制恶性胶质瘤细胞的生长，提示MLKL还可作为治疗胶质瘤的靶标。本发明还公开了MLKL基因表达抑制剂siRNA，对MLKL基因的表达具有良好的干扰效果，具有临床基因治疗的应用前景。
在第一方面，本发明提供了一种脑胶质瘤标志物MLKL基因和/或MLKL基因的表达产物在制备用于脑胶质瘤诊断、筛查和/或预后的检测试剂盒中的应用。
本发明通过生物信息学分析方法，检测MLKL基因在胶质瘤组织(Tumor)及正常组织(Normal)中的mRNA表达量的方法，显示MLKL基因在33种癌组织(Tumor)与正常组织(Normal)的mRNA表达存在差异，MLKL在癌症组织的表达水平均显著高于正常组织。
采用Cox回归方法分析MLKL mRNA水平与患者总生存期的相关性，发现MLKL高表达的胶质瘤患者总生存期(OS)显著低于MLKL基因低表达患者的总生存期，提示MLKL基因的表达产物能作为胶质瘤的预后标志物，通过检测MLKL基因的表达，可以评估不同级别胶质瘤患者的预后、提示患者的潜在生存时间，同时可以用于评估胶质瘤的治疗效果。更有意义的是，MLKL的高表达与其它29种癌症的不良预后无关，提示MLKL作为胶质瘤检测与预后标志物的专一性特点。通过检测MLKL基因表达，能够及时评估低级胶质瘤和高级别胶质瘤患者的预后，对调整临床治疗策略，进一步提高临床治疗效果，改善患者生存质量具有指导意义。
因此，MLKL可作为一种新的胶质瘤预后标志物，为不同亚型的胶质瘤患者的生存期提供评估依据。结合ROC曲线分析发现，MLKL基因应用于胶质瘤诊断的AUC值高于0.7，显示 其作为胶质瘤诊断和预后标志物具有高特异性和高灵敏度，是胶质瘤早期分子诊断与患病分险筛查的理想靶点。
可选择地，上述的脑胶质瘤包括低级别胶质瘤(LGG)与高级别胶质瘤(GBM)。优选地，所述肿瘤为高级别胶质瘤，即胶质母细胞瘤。
所述脑胶质瘤标志物MLKL基因的表达产物包括MLKL mRNA和/或MLKL蛋白。
在第二方面，本发明还提供用于胶质瘤诊断和预后的试剂盒，所述试剂盒使用MLKL基因和/或MLKL基因的表达产物作为检测靶标。
本发明制备的用于胶质瘤诊断和预后的试剂盒，所述试剂盒包含如下的组分：
可用于特异性扩增MLKL基因的引物对；和/或
特异识别/结合MLKL基因的表达产物的抗体。
人源MLKL基因的序列可参考美国国家生物技术信息中心Gene ID:197259(NM_152649.4)，具体序列为SEQ ID NO：1所示。
本领域技术人员通过已知的引物设计方法即可设计出适用于扩增MLKL的特异性引物。
更优选的，本发明制备的用于胶质瘤诊断和预后的试剂盒，所述试剂盒包含可用于特异性 扩增MLKL基因的引物对，所述引物对包含如下的序列：
(1)上游引物序列：5’-TCACACTTGGCAAGCGCATGGT-3’(SEQ ID NO：2)；
下游引物序列5’-GTAGCCTTGAGTTACCAGGAAGT-3’(SEQ ID NO：3)；
(2)上游引物序列：5’-TGCAGAGGAAGACGGAAATGA-3’(SEQ ID NO：4)；
下游引物序列5’-CTCCTGTGTGGGTTTTAGTGAGC-3’(SEQ ID NO：5)；
(3)上游引物序列：5′-AGGAGGCTAATGGGGAGATAA-3′(SEQ ID NO：6)；
下游引物序列5′-TGGCTTGCTGTTAGAAACCTG-3′(SEQ ID NO：7)。
根据MLKL的基因序列，MLKL基因的表达产物如MLKL蛋白已广为人知，具体可参考UniProt数据库ID:Q8NB16，具体序列为SEQ ID NO：8所示。
此外，本领域技术人员掌握如何获得特异识别/结合MLKL蛋白的抗体的方法，例如通过全长的MLKL蛋白或其部分蛋白序列作为抗原如免疫动物如老鼠、兔子或羊驼等，可以获得特异性识别MLKL蛋白的抗体。所述抗体是单克隆抗体、多克隆抗体或纳米抗体，优选单克隆抗体。抗体可以自行制备，或者可以购买商用的识别MLKL抗体，例如Abcam公司的anti-MLKL抗体(货号：ab184718)。
更优选的，本发明制备了用于胶质瘤诊断和预后的试剂盒，所述试剂盒包含可以用于特异扩增MLKL基因的引物对，或特异扩增MLKL基因的表达产物的引物对，或能特异识别/结合MLKL蛋白的抗体。所述抗体具有如下的特征：可识别人源全长MLKL蛋白，亲和力达到10-9nM或更高。
在更优选的实施方案中，可以通过基因手段，如多种PCR手段，例如荧光实时定量PCR，使用所述引物对进行检测。也可以通过免疫手段，如蛋白质印迹、酶联免疫反应、免疫组化、免疫荧光等检测MLKL蛋白。
第三方面，本发明发现降低MLKL表达可明显抑制恶性胶质瘤细胞的生长，提示MLKL还可作为治疗胶质瘤的靶标。
本发明人发现MLKL基因或蛋白水平表达越高，胶质瘤患者的生存率越低，即MLKL基因或蛋白过表达明确指示患者较差的生存预后，说明MLKL基因和/或其表达产物能够作为胶质瘤的预后标志物，用于预测患者的预后情况。根据本发明人的研究，抑制内源性MLKL可显著降低恶性胶质瘤细胞的增殖，促使MLKL可成为治疗胶质瘤的有效靶标。
因此，本发明公开了MLKL基因作为药物或制剂针对脑胶质瘤细胞的作用靶标在筛选脑胶质瘤治疗药物中的用途；MLKL基因作为药物或制剂针对脑胶质瘤细胞的作用靶标具体是指：将MLKL基因作为药物或制剂针对脑胶质瘤细胞产生RNA干扰作用的靶标，从而能降低脑胶质瘤细胞中MLKL基因的表达水平；将MLKL基因作为药物或制剂针对脑胶质瘤细胞的作用靶标应用于筛选脑胶质瘤治疗药物具体是指：将MLKL基因作为作用对象对药物或制剂进行筛选，以找到可以抑制MLKL基因表达的药物作为脑胶质瘤治疗备选药物；所述靶标的序列如SEQ ID NO：1所示。
基于这种认识，本发明还制备了MLKL基因表达抑制剂siRNA，对MLKL基因的表达具有良好的干扰效果，具有临床基因治疗的应用前景。所述MLKL基因表达抑制剂siRNA可用于制备治疗胶质瘤的药物，辅助胶质瘤的临床治疗，提高胶质瘤的治疗效果。因此，本发明还提供一种MLKL基因的表达抑制剂，所述表达抑制剂包括基于所述MLKL基因设计的siRNA，其中所述siRNA包含15-30个核苷酸。siRNA(Small interfering RNA；小干扰RNA)与靶基因MLKL转录后的mRNA结合，通过RNA介导的沉默复合体RISC(RNA-induce siliencing complex)介导mRNA的降解。siRNA长度更优选为21-23nt，被RISC识别结合之后，siRNA发生解旋。RISC在siRNA的反义链指导下，寻找具有同源序列的内源性的mRNA，并在距离5’端10-11位碱基之间切割mRNA，从而导致转录后基因沉默。已知siRNA有多个在线设计工具，例如siDirect设计网站(http://sidirect2.rnai.jp)、DSIR设计网站(http://biodev.extra.cea.fr/DSIR/DSIR.html)、invivogen设计网站(http://www.invivogen.com/sirnawizard/siRNA.php)和thermofisher设计网站(https://rnaidesigner.thermofisher.com)等，可供参考。
优选的，本发明还提供一种MLKL基因的表达抑制剂，所述表达抑制剂包括基于所述MLKL基因设计的siRNA，所述siRNA核苷酸序列正义链如：
SEQ ID NO.9(5’-GAAGCAUAUUAUCACCCUUTT-3’)或
SEQ ID NO.10(5’-GCAAUAGAUCCAAUAUCUGTT-3’)或
SEQ ID NO.11(5’-GAACCUGCCCGAUGACAUU-3’)等所示。
在另一优选实施方案中，上述siRNA包括至少一个修饰的核苷酸。所述修饰的siRNA通常具有比相应未修饰siRNA序列更小的免疫刺激性，且保持针对目的靶基因的RNAi活性。在一些实施方案中，修饰的siRNA含有至少一个2’OMe嘌呤或嘧啶核苷酸诸如2’OMe-鸟苷、2’OMe-尿苷、2’OMe-腺苷，和/或2’OMe-胞嘧啶核苷酸(参考M.M.Zhang et al.，Biochemical Pharmacology 189(2021)114432)。在优选的实施方案中，修饰的核苷酸可以存在于siRNA的一条链(即，有义或反义)或两条链中。siRNA序列可以具有突出端(例如3’或5’突出端)或可以缺少突出端(即具有平端)。
连接RNA磷酸骨架的磷酸二酯键是核酸酶作用的化学键，而磷原子是核酸酶攻击的中心，对该原子的修饰能够影响酶的降解作用，从而提高小干扰核酸抗核酸酶的能力，增加其稳定性。在另一优选实施方案中，通过将磷酸酯键(PO)换成硫代磷酸酯(PS)，所述硫代磷酸修饰是用一个硫原子取代磷酸二酯键的非桥氧原子，即P-S键替代P-O键，这种修饰通常会增加核酸对核酸酶的稳定性。
本发明中，所述siRNA的制备方法没有特别的限制，可以通过化学合成得到，或者通过质粒和/或病毒载体的表达而得到。根据不同的修饰方式，可以使用经过修饰核苷酸替代相应位置中未修饰的核苷酸，或者进行磷酸二酯键的硫代磷酸修饰等。选择性地，也可以委托专门从事核酸合成的生物技术公司合成本发明的siRNA。一般来说，用于合成siRNA的方法包括以下四个过程：(1)寡聚核糖核苷酸的合成；(2)脱保护；(3)纯化分离；(4)脱盐等。
siRNA因为含有多个磷酸酯键而带有大量的负电荷，这样的极性分子很难自行穿过细胞膜；此外siRNA暴露血液会有稳定性问题并造成免疫原性。因此，在另一优选实施方案中，需要将上述siRNA用载体(如LNP)包裹或者和特定配体(如GalNac)连接以有效避免肾消除，而且还能靶向性地富集在特定的组织中。
在另一优选实施方案中，本发明制备了一种siRNA核酸-脂质颗粒，包含：
(a)siRNA核酸，所述siRNA核苷酸序列正义链如SEQ ID NO.9、SEQ ID NO.10或SEQ ID NO.11所示；
(b)阳离子脂质，其占所述颗粒中存在的总脂质的50mol％-65mol％；
(c)非-阳离子脂质，其包括磷脂和胆固醇或其衍生物的混合物，其中所述磷脂占所述颗粒中存在的总脂质的3mol％-15mol％，所述胆固醇或其衍生物占所述颗粒中存在的总脂质的30mol％-40mol％；和
(d)抑制颗粒聚集的缀合脂质，其占所述颗粒中存在的总脂质的0.5mol％-2mol％；其中，其中所述阳离子脂质包括1,2-二亚油基氧基-N,N-二甲基氨基丙烷(DLinDMA)、1,2-二亚麻基氧基-N,N-二甲基氨基丙烷(DLenDMA)或其混合物；
其中，所述磷脂包括二棕榈酰磷脂酰胆碱(DPPC)、二硬脂酰磷脂酰胆碱(DSPC)或其混合物；
其中，所述抑制颗粒聚集的缀合脂质包括聚乙二醇(PEG)-脂质缀合物，选自PEG-二酰甘油(PEG-DAG)缀合物、PEG-二烷氧基丙基(PEG-DAA)缀合物或其混合物。
在另一优选实施方案中，本发明了制备了一种GalNAc-siRNA缀合物，即将如SEQ ID NO.9、SEQ ID NO.10或SEQ ID NO.11所示siRNA缀合至N-乙酰半乳糖胺(N-Acetylgalactosamine，GalNAc)形成GalNAc-siRNA缀合物。
其中GalNAc配体可以结合肝细胞表达的去唾液酸糖蛋白受体(Asialoglycoprotein receptor，ASGPR)并将siRNA靶向递送至肝细胞。可以皮下长循环给药是GalNAc-siRNA的一个巨大优势，更适合用于皮下给药。
本发明还提供一种制备GalNAc-siRNA缀合物的方法，即在固相寡核苷酸合成过程中，使用GalNAc负载固体载体，从而将GalNAc与siRNA正义链的3'末端通过三元间隔子相连接；随后进行核苷酸脱保护和乙酸半乳糖胺脱保护，除去固体载体，可得到GalNAc-siRNA缀合物。在一些实施方案中，所述siRNA可附接至一个或多个(例如，两个、三个、四个或更多个)GalNAc衍生物。所述附接可以不经由接头，或经由一个或多个接头(例如，两个、三个、四个或更多个接头)。在一些实施方案中，本文所述的接头是多价(例如，二价、三价或四价)支化接头。在一些实施方案中，所述一个或多个GalNAc衍生物附接至所述dsRNA的有义链的3'端、反义链的3′端、有义链的5′端和/或反义链的5′端。有关GalNAc-siRNA缀合物的合成可参考WO2009/073809、WO2011/104169、WO2012/083046、WO2014/118267和WO2014/179620等，在此全部引入作为参考。
利用本发明制备的MLKL表达抑制剂siRNA转染恶性胶质瘤细胞后，免疫印迹实验显示MLKL的表达水平显著降低，同时细胞周期蛋白依赖激酶2(CDK2)和细胞周期蛋白A2(cyclin A2)表达量伴随MLKL的下降而降低。在机体，CDKs主要与周期蛋白(cyclin)结合形成激 活态的蛋白酶体复合物，从而催化底物丝氨酸/苏氨酸残基磷酸化，促进细胞周期不断进行。其中CDK2通过与cyclin A形成蛋白酶体复合物，调控细胞G1期到S期的转变和S期的进行。CDK2-cyclin A2表达量降低，提示敲低MLKL可能抑制恶性胶质瘤细胞的增殖速率，提示MLKL基因表达抑制剂siRNA可用于制备治疗胶质瘤的药物，辅助胶质瘤的临床治疗，提高胶质瘤的治疗效果。
因此，本发明还提供了上述MLKL表达抑制剂siRNA在制备治疗脑胶质瘤药物中的用途。
综上，本发明为胶质瘤治疗提供新的理论依据和全新的靶标，本发明还为胶质瘤提供了新的辅助诊断和预后诊断的方法以及新的治疗药物。
附图说明
图1显示的是本发明实施例1提供的MLKL mRNA在33种癌症组织(Tumor)和正常组织(Normal)中表达对比。
图2显示的是本发明实施例2提供的MLKL基因表达水平对29种癌症患者总生存期(OS)影响的生存分析森林图。
图3显示的是本发明实施例3提供的与其它3个文献报道的胶质瘤标志物NUSAP1、GINS2和TRIM21相比，MLKL基因表达水平与高级别胶质瘤(GBM)患者的生存率相关性更显著。
图4显示的是本发明实施例4提供的MLKL基因表达水平对低级别胶质瘤(LGG)和高级别胶质瘤(GBM)总生存期(OS)(a)、疾病特异性生存期(DSS)(b)以及无进展生存期(PFI)(c)影响的生存分析曲线。
图5显示的是本发明实施例5提供的MLKL基因表达水平用于诊断胶质瘤的ROC曲线。
图6显示的是本发明实施例6提供的MLKL基因在各个分子亚型胶质瘤中的表达水平。其中，(a)为WHO级别；(b)为IDH突变；(c)为1p/19q基因联合缺失；(d)临床病理分型。
图7显示的是本发明实施例7提供的MLKL基因表达水平对各个分子亚型胶质瘤生存期影响的生存分析曲线。其中，(a)为WHO级别；(b)为IDH突变；(c)为临床病理分型。
图8显示的是本发明实施例8提供的在临床胶质瘤样本中，胶质瘤恶性程度越高的MLKL蛋白表达量越高(a)；定量分析显示MLKL蛋白在高级别胶质瘤(b)和IDH1野生型胶质瘤中(c)表达量显著升高；
图9显示的是本发明实施例8提供的MLKL蛋白表达水平与胶质瘤病人总生存期(a)；IDH1突变(b)以及肿瘤增殖标志物Ki67(c)相关性分析图；
图10显示的是本发明实施例9提供的MLKL mRNA水平检测试剂盒使用的电泳结果(a)以及定量分析图(b)；
图11显示的是本发明实施例11提供的利用siRNA敲减MLKL的细胞与对照细胞(NC)的免疫印迹实验(a)及其定量分析(b)，以及细胞增殖率检测实验(c)。
图12显示的是本发明实施例12提供的敲除MLKL对胶质瘤细胞U251增殖速率的抑制作用。
图13显示的是本发明实施例13提供的敲除MLKL在小鼠体内对皮下肿瘤组织生长的抑制作用。
具体实施方式
下面结合具体实施例和附图对本发明内容进一步阐述。下列实施例中未注明具体条件的实验方法，通常按照本技术领域常规技术，所述siRNA合成工作由上海吉玛制药技术有限公司完成，所有引物合成由金唯智生物科技有限公司完成，下述实施例中所涉及的细胞均购于上海生命科学研究所，所用到的试剂和原料均可由市场购得。
下述实施例中所涉及的试剂如下所示：
MLKL抗体购自abcam公司，Name:重组Anti-MLKL抗体(ab184718)。β-actin抗体购自Proteintech公司(No.66009)。抗兔IgG抗体购自CellSignalling公司(#7074)。增强型CCK-8试剂盒，购自碧云天生物技术公司，产品编号：C0042。ECL化学发光底物购自天能公司(编号：180-5001)Lipofectamine 2000转染试剂购自ThermoFisherScientific公司，货号11668-019。TRIzol试剂购自ThermoFisherScientific公司，货号15596026。PrimeScript 1st strand cDNA合成试剂盒购自TaKaRa公司，货号6110A。ChamQ Universal SYBR qPCR预混液购自Vazyme公司，货号Q711-03。
实施例1
供利用生物信息学分析方法，检测MLKL基因在胶质瘤组织(Tumor)及正常组织(Normal)中的mRNA表达量的方法，具体内容如下：
从TCGA数据库(https://portal.gdc.cancer.gov/)和GTEx数据库(http://gtexportal.org/home/)上下载33种癌症与正常组织中MLKLmRNA表达情况。通过Wilcoxon rank sum test检测MLKL在正常组织和癌组织中表达量的差异。
图1显示MLKL基因在33种癌组织(Tumor)与正常组织(Normal)的mRNA表达差异 水平，由图1可知，MLKL在癌症组织的表达水平均显著高于正常组织。
实施例2
通过TCGA数据库获取的30种癌症组织中MLKLmRNA信息和病人信息，采用Cox回归方法分析MLKLmRNA水平与患者总生存期的相关性，得到如图2所述的生存分析森林图。
由图2可知，MLKL基因高表达的低级胶质瘤(LGG)和高级胶质瘤(GBM)患者，其总生存率的风险比(HR)明显高于MLKL基因地表达的***患者(P<0.01)，即MLKL高表达的胶质瘤患者总生存期(OS)显著低于MLKL基因低表达患者的总生存期，提示MLKL基因的表达产物能作为胶质瘤的预后标志物，通过检测MLKL基因的表达，可以评估不同级别胶质瘤患者的预后、提示患者的潜在生存时间，同时可以用于评估胶质瘤的治疗效果。更有意义的是，MLKL的高表达与其它29种癌症的不良预后无关，提示MLKL作为胶质瘤检测与预后标志物的专一性特点。
实施例3
从TCGA数据库分别下载MLKL、NUSAP1、GINS2和TRIM21信息以及病人信息，采用COX回归方法对比分析MLKL和3个已发现的胶质瘤标志物，即NUSAP1、GINS2、TRIM21的基因表达水平与患者总生存期的相关性，得到如图3所述的生存分析热图。
由图3可知，虽然MLKL、NUSAP1、GINS2和TRIM21基因的高表达与低级别胶质瘤(LGG)的不良预后均存在显著相关性(P<0.001)，但仅有MLKL的高表达可以作为评估高级别胶质瘤(GBM)预后的分子标志物，进一步验证MLKL在不同级别的胶质瘤中均具有准确的诊断意义。
实施例4
利用实施案例2中从TCGA数据库获取的胶质瘤组织中MLKLmRNA信息和病人信息，通过Kaplan-Meier曲线和log-rank检测用于分析MLKL基因表达水平与患者总生存期(OS)、疾病特异性生存期(DSS)以及无进展生存期(PFI)的相关性，得到如图3所述的生存分析曲线。
由图4a-c可知，MLKL基因高表达的低级别胶质瘤患者(LGG)和高级别胶质瘤患者(GBM)，在同一生存时间下的患者总生存率(OS)、疾病特异性生存率(DSS)以及肿瘤无进展生存率(PFI)明显低于MLKL低表达的胶质瘤患者(P<0.01)，进一步验证MLKL基因表达的产物能够作为胶质瘤的预后标志物。通过检测MLKL基因表达，能够及时评估低级胶质瘤和高级别胶质瘤患者的预后，对调整临床治疗策略，进一步提高临床治疗效果，改善患者 生存质量具有指导意义。
实施例5
本实施例旨在提供一种评估MLKL基因表达水平用于胶质瘤诊断价值的的方法，具体内容为：采用实施例2中从TCGA数据库获取的胶质瘤病人的信息通过软件进行编辑分析，获得时间依赖型ROC(receiver operator characteristic curve)曲线。
上述的软件是R(3.6.3版本)，其中统计分析所用的R包为timeROC(0.4版本)，数据可视化所用的R包为ggplot2(3.3.3版本)。
ROC曲线是显示真阳性率(灵敏度，或称敏感性，TPR)和假阳性率(FPR)之间折中的一种图形化方法。一个好的分类模型，在本发明中指一个好的癌症标志物，应该进可能靠近ROC曲线的左上角。同时，也可以采用曲线下面积(AUC)来表示一个模型的平均表现，AUC越大，诊断准确度越高。
图5所示MLKL基因表达水平用于诊断胶质瘤的ROC曲线，如图所示，预测年限为1年时，曲线下面积(AUC)为0.753，MLKL基因表达用于诊断胶质瘤的灵敏度为66.4％，特异性为74.8％。预测年限为5年时，曲线下面积(AUC)为0.738，MLKL基因表达用于诊断胶质瘤的灵敏度为65.3％，特异性为75.3％。预测年限为10年时，曲线下面积(AUC)为0.791，MLKL基因表达用于诊断胶质瘤的灵敏度为64.1％，特异性为84.2％。显示其作为胶质瘤诊断和预后标志物具有高特异性和高灵敏度，是胶质瘤早期分子诊断与患病分险筛查的理想靶点。
实施例6
利用实施案例2中从TCGA数据库获取的胶质瘤组织中MLKLmRNA信息和病人信息，通过Kruskal-Wallis test检测MLKL基因表达与胶质瘤临床不同分型，即WHO分级、IDH突变、1p/19q共缺失以及不同组织类型之间的相关性，得到如图6(a-b)所示的柱形图。
WHO分级越高指向越差的生存率，而IDH突变和1p/19q联合缺失突变与较好的预后具有强相关性。图6(a)显示MLKL基因在III级胶质瘤中的表达量高于II级，而在IV级胶质瘤中表达量高于II级与III级。图6(b)显示MLKL基因在IDH野生型中的表达量显著高于其在IDH突变型中的表达量。图6(c)显示MLKL基因在不发生1p/19联合缺失的表达量显著高于其在1p/19q联合缺失的表达量。图6(d)显示MLKL基因在少突胶质细胞瘤(oligodendroglioma)、少突星型胶质细胞瘤(oligoastrocytoma)、星形胶质细胞瘤(astrocytoma)、胶质母细胞瘤(glioblastoma)的表达量逐步升高。以上结果共同说明胶质瘤的恶性程度越高，MLKL基因的表达量越高。由此可知，MLKL的表达与胶质瘤的级别、IDH突变、1p/19共缺 失以及病理组织分型具有相关性，MLKL基因及其表达产物可作为临床判断胶质瘤级别和组织分型的标志物。
实施例7
利用实施案例2中从TCGA数据库获取的胶质瘤组织中MLKLmRNA信息和病人信息，通过Kaplan-Meier生存曲线检测MLKL表达在胶质瘤患者在不同亚型中患者总生存(OS)的预后价值。结果如图7a-c所示，MLKL在较高级别胶质瘤患者(G3和G4)(a)、IDH野生型患者(b)、以及3种较严重的组织分型(包括少突星形胶质细胞瘤、星形胶质细胞瘤、以及神经母细胞瘤)(c)患者中均具有预后意义。因此，MLKL可作为一种新的胶质瘤预后标志物，为不同亚型的胶质瘤患者的生存期提供评估依据。
实施例8
MLKL蛋白在高级别胶质瘤临床样本中蛋白表达高于低级别胶质瘤组织。
1.研究对象
研究对象选取81例胶质瘤患者临床样本，其中WHO分级II级17例，III级19例，IV级52例，本研究通过医院伦理委员会审查和批准，所有患者入组前均签署书面知情通知书。
2.免疫印迹实验检测MLKL蛋白在临床胶质瘤样本中的表达量。
用组织破碎仪将临床胶质瘤组织破碎为组织匀浆，进而通过RIPA细胞裂解液使各组分细胞破碎，蛋白溶解，在相同条件下对蛋白质进行定量调整后，进行western blot检测分析。具体步骤如下：首先通过SDS-PAGE电泳分离蛋白，并将蛋白转到硝酸纤维素膜上，然后采用5％的脱脂奶粉进行封闭实验，封闭结束后对膜进行清洗，并用分别与MLKL和Actin特异性结合的抗体与其充分结合，进一步用能与一抗特异性结合的二抗与其结合后，通过蛋白显色成像仪进行拍照，最后通过Image J软件对所显色的蛋白条带完成定量分析。
所获得的结果如图8a所示，对Western Blot定量分析结果表明MLKL在高级别胶质瘤(GBM)中的蛋白表达量显著高于在低级别胶质瘤中(LGG)的表达量(图8b)，并在IDH1野生型中的表达量显著高于其在IDH1突变型胶质瘤组织中的表达量(图8c)，说明MLKL在恶性程度高的胶质瘤中高表达，提示其蛋白表达量与病患的不良预后相关。
3.MLKL蛋白过表达的临床意义。
采用Kalpan-Meier方法绘制生存分析曲线，并通过log-rank方法检测其统计学意义。检测系数P<0.05统计学上有显著性差异。并且通过R包：survival包(3.2-10版本)用于生存资料的统计分析MLKL蛋白的表达与胶质瘤患者总生存率(Overall survival,OS)的关系。MLKL 蛋白过表达和低表达按照MLKL蛋白表达水平的中位数来划定。同时结合临床胶质瘤病患的IDH1突变信息和免疫组化Ki67染色结果，应用GraphPad Prism 7软件的t检验，分析MLKL蛋白表达量与IDH1突变与Ki67指标之间的相关性。
结果如图9a所示，在81例胶质瘤组织中，MLKL蛋白过表达的胶质瘤病人比MLKL低表达的胶质瘤患者的总生存率(OS)明显降低。MLKL在2种分子亚型(即Ki67与IDH1)胶质瘤中的表达水平图显示，MLKL蛋白在野生型IDH1分子亚型(b)，以及高Ki67(>＝30)(c)的胶质瘤中特异性高表达。IDH1野生型与不良预后相关，提示MLKL蛋白水平与胶质瘤恶性程度正相关。Ki67是一种与细胞增殖相关的核内蛋白,可用于判断肿瘤增生指数。总体来说，Ki-67阳性标记数值越高，则组织分化越差，恶性程度越高，预后越差。综上，MLKL蛋白表达水平高，可作为胶质瘤不良预后的标志物。
实施例9
MLKL mRNA水平检测试剂盒使用示例
方法：采用RT-qPCR，在胶质瘤组织样本中检测MLKL的mRNA水平。
本实施例公开的一种胶质瘤MLKL诊断检测方法，采取正常脑组织或胶质瘤患者肿瘤组织，按照说明书操作步骤提取总RNA，使用TaKaRa逆转录酶将提好的总RNA逆转录为cDNA，并通过PCR分析MLKL的mRNA水平。具体条件如下：
1.胶质瘤组织总RNA的提取方法：
将组织称重后，每100mg组织加入1mLTrizol裂解液，低温超声破碎后放置在冰上，裂解5min；随后加入200μL氯仿，剧烈震荡数次后，冰浴10min。12000rpm 4℃低温离心10min后，小心转移最上层透明溶液至无RNA酶的离心管中，加入500μL异丙醇震荡数次，冰上放置20min后，12000rpm 4℃低温离心15min。小心吸去上清，加入1mL冰的75％乙醇，颠倒洗涤，不超过7500rpm离心5min，随后重复洗涤一次。小心吸去上清，加入20μL预冷的DEPC水，吹打混匀并测定总RNA浓度。
2.逆转录反应按Takara逆转录酶说明书进行操作，最终获得cDNA产物-80℃。
3.逆转录产物扩增方法参考Vazyme公司SYBR qPCR酶(货号：Q711-03)说明书，引物浓度为10μM，以GAPDH为内参，通过ImageJ软件定量分析MLKL mRNA水平。
MLKL基因水平拷贝数的引物序列为：
F:5’-TCACACTTGGCAAGCGCATGGT-3’，(SEQ ID NO：2)；
R:5′-GTAGCCTTGAGTTACCAGGAAGT-3’，(SEQ ID NO：3)；
GAPDH基因水平拷贝数的引物序列为：
F:5′-GGAGCGAGATCCCTCCAAAAT-3’,(SEQ ID NO：12)；
R:5′-GGCTGTTGTCATACTTCTCATGG-3’,(SEQ ID NO：13)。
结果：RT-PCR产物电泳结果及分析定量结果如图11所示。
实施例10
MLKL蛋白水平检测试剂盒使用示例
本实施例公开的一种胶质瘤MLKL诊断检测方法，采用WesternBlot方法，在胶质瘤组织样本中检测MLKL的蛋白表达水平，并通过定量分析MLKL的蛋白表达水平。
该试剂盒中包含能够特异性识别人源MLKL以及内参β-Actin的抗体，抗体结合示意图如图8a所示。
实施例11
MLKL蛋白促进胶质瘤细胞的恶性增殖。
1.免疫印迹实验检测敲低MLKL后细胞周期蛋白的表达量
为探索MLKL在胶质瘤中的生物学功能，利用siRNA技术特异性地敲低恶性胶质瘤细胞U87的MLKL。使用干扰序列siNC(5’–UUCUCCGAACGUGUCACGUTT–3’(SEQ ID NO.14)和siMLKL(#1：5’-GAAGCAUAUUAUCACCCUUTT-3’(SEQ ID NO.9)，#2：5’-GCAAUAGAUCCAAUAUCUGTT-3’(SEQ ID NO.10))转染细胞后，免疫印迹实验显示MLKL的表达水平显著降低(图11(a))，同时细胞周期蛋白依赖激酶2(CDK2)和细胞周期蛋白A2(cyclin A2)表达量伴随MLKL的下降而降低(图11(b))。在机体，CDKs主要与周期蛋白(cyclin)结合形成激活态的蛋白酶体复合物，从而催化底物丝氨酸/苏氨酸残基磷酸化，促进细胞周期不断进行。其中CDK2通过与cyclin A形成蛋白酶体复合物，调控细胞G1期到S期的转变和S期的进行。CDK2-cyclin A2表达量降低，提示敲低MLKL可能抑制U87细胞的增殖速率。
2.CCK8试剂盒检测恶性胶质瘤细胞的增殖速率
利用增强型CCK8试剂盒对不同组的细胞进行细胞数目统计，检测敲低MLKL蛋白的胶质瘤细胞生长速率的变化。结果如图11(c)显示，在细胞增殖曲线检测实验中，敲低MLKL后U87的生长收到了显著的抑制。以上实验说明MLKL可作为一种新型的促癌分子，参与胶质瘤的恶性生长及发展。
实施例12
MLKL敲除抑制胶质瘤细胞U251的增殖速率。
为了确定MLKL对胶质瘤增殖情况的影响，利用CRISPR-Cas9技术构建MLKL基因敲除型U251细胞。gRNA对应的DNA序列为5’-GAAGCTGAGTGATGTCTGGA-3’(SEQ ID NO.15)，基因编辑结果如图12a所示。收集对数生长期的野生型或MLKL-/-U251细胞，以2000个细胞/孔加入6孔板中，轻轻摇晃使细胞在板内分布均匀，放入培养箱内培养2周后进行结晶紫染色。结果如图12b所示，MLKL-/-U251细胞增殖速率明显变慢。将细胞以2000个细胞/孔加入96孔板中，使用CCK8试剂盒记录每天细胞数量，结果如图12c所示。细胞增殖曲线显示，MLKL-/-U251细胞生长受到显著抑制。
实施例13
在小鼠体内，MLKL敲除抑制皮下肿瘤组织的形成。
为了确定MLKL对U251细胞荷瘤组织在体内生长的影响，使用U251细胞和由实施例12制备的MLKL-/-U251细胞对7周龄雌性BALB/c小鼠进行皮下注射。在注射后第21天和第27天，用游标卡尺测量并计算皮下肿瘤体积。注射27天后小鼠照片如图13a所示，肿瘤体积如图13b所示。结果显示MLKL-/-U251细胞皮下成瘤率差，细胞生长明显变慢。
总结：本发明通过生物信息学分析发现了MLKL在胶质瘤，尤其是高级别胶质瘤中高表达，且其高表达水平与较差的胶质瘤患者生存期密切相关。同时在临床水平对比检测了MLKL在低级别胶质瘤(LGG)和高级别胶质瘤(GBM)组织中的表达情况，发现MLKL在GBM中显著高表达，且其蛋白表达量与Ki67和胶质瘤患者的总生存率相关，结果与生物信息学分析。为进一步研究MLKL是否参与调控胶质瘤细胞的增殖，利用siRNA干扰技术敲低人胶质瘤细胞U87的MLKL后，进行免疫印迹实验和细胞增殖速率检测实验，结果显示干扰MLKL可能通过降低细胞周期蛋白CDK2和cyclinA2抑制U87细胞的增殖速率；进一步地，通过体外和体内实验，证明了MLKL敲除显著抑制肿瘤细胞和肿瘤组织的增殖。
另外，MLKL基因或其表达产物MLKL蛋白作为靶点在制备抗肿瘤药物中的用途也在本发明的保护范围之内。
本领域的普通技术人员可以理解，上述各实施方式是实现本发明的具体实施例，而在实际应用中，可以在形式上和细节上对其作各种改变，而不偏离本发明的精神和范围。

Claims (38)

  1. 脑胶质瘤标志物MLKL基因和/或MLKL基因的表达产物在制备用于脑胶质瘤诊断、筛查和/或预后的检测试剂盒中的应用。
  2. 用于脑胶质瘤的诊断、筛查和预后的试剂盒，所述试剂盒包括：特异性扩增MLKL基因的引物对、特异扩增MLKL基因的表达产物的引物对，和/或特异性识别/结合MLKL基因的表达产物的抗体或其结合片段。
  3. 根据权利要求2所述的试剂盒，其中，所述引物对包含如下序列：
    (1)上游引物序列：SEQ ID NO：2，
    下游引物序列：SEQ ID NO：3；
    (2)上游引物序列：SEQ ID NO：4，
    下游引物序列：SEQ ID NO：5；和/或
    (3)上游引物序列：SEQ ID NO：6，
    下游引物序列：SEQ ID NO：7。
  4. 根据权利要求2所述的试剂盒，其中，所述抗体包括单克隆抗体、多克隆抗体和/或纳米抗体。
  5. 根据权利要求2所述的试剂盒，其中，所述抗体识别人源全长MLKL蛋白。
  6. 一种脑胶质瘤的诊断、筛查和/或预后方法，所述方法包括检测受试者MLKL基因和/或MLKL基因的表达产物的水平。
  7. 根据权利要求1所述的应用、权利要求2-5所述的试剂盒和权利要求6所述的方法，其中，所述脑胶质瘤包括低级别胶质瘤和/或高级别胶质瘤。
  8. 根据权利要求1所述的应用、权利要求2-5所述的试剂盒和权利要求6所述的方法，其中，所述脑胶质瘤为胶质母细胞瘤。
  9. 根据权利要求1所述的应用、权利要求2-5所述的试剂盒和权利要求6所述的方法，其中，所述MLKL基因的表达产物包括MLKL mRNA和/或MLKL蛋白。
  10. 根据权利要求9所述的应用、试剂盒和方法，所述MLKL蛋白包括如SEQ ID NO：8所示的序列。
  11. 根据权利要求9所述的应用、试剂盒和方法，其中，检测MLKL mRNA的表达水平， 优选地，通过荧光实时定量PCR检测MLKL mRNA的表达水平，优选地，使用如下引物对进行检测：
    (1)上游引物序列：SEQ ID NO：2，
    下游引物序列：SEQ ID NO：3；
    (2)上游引物序列：SEQ ID NO：4，
    下游引物序列：SEQ ID NO：5；和/或
    (3)上游引物序列：SEQ ID NO：6，
    下游引物序列：SEQ ID NO：7。
  12. 根据权利要求9所述的应用、试剂盒和方法，其中，通过蛋白免疫检测MLKL蛋白的表达水平，优选地，所述蛋白免疫包括蛋白质印迹、酶联免疫反应、免疫组化或免疫荧光。
  13. 根据权利要求6所述的方法，其中，使用权利要求2-5所述的试剂盒检测MLKL基因和/或MLKL基因的表达产物的水平。
  14. 一种抑制MLKL基因表达的抑制剂，其中，所述抑制剂包括基于MLKL基因序列制备的siRNA，所述siRNA包含15-30个核苷酸。
  15. 根据权利要求14所述的抑制剂，其中，所述siRNA的长度为21-23个核苷酸。
  16. 根据权利要求14所述的抑制剂，其中，所述siRNA的核苷酸序列正义链如SEQ ID NO.9、SEQ ID NO.10或SEQ ID NO.11所示。
  17. 根据权利要求14所述的抑制剂，其中，所述siRNA包括修饰的siRNA，所述修饰的siRNA包含至少一个修饰的核苷酸。
  18. 根据权利要求17所述的抑制剂，其中，所述修饰的siRNA包含至少一个2’OMe嘌呤或2’OMe嘧啶核苷酸。
  19. 根据权利要求17所述的抑制剂，其中，所述修饰的核苷酸位于siRNA的一条链或两条链中。
  20. 根据权利要求14所述的抑制剂，其中，所述siRNA序列具有突出端或不具有突出端。
  21. 根据权利要求14所述的抑制剂，其中，所述siRNA包含硫代磷酸修饰，所述硫代磷酸修饰是将连接RNA磷酸骨架的磷酸二酯键换成硫代磷酸酯，用一个硫原子取代磷酸二酯键的非桥氧原子。
  22. 一种siRNA核酸-脂质颗粒，其包含：
    (a)siRNA核酸，所述siRNA的核苷酸序列正义链如SEQ ID NO.10或SEQ ID NO.11所示；
    (b)阳离子脂质，所述阳离子脂质占所述颗粒中存在的总脂质的50mol％-65mol％；
    (c)非阳离子脂质，所述非阳离子脂质包含磷脂和胆固醇或胆固醇衍生物，其中，所述磷脂占所述颗粒中存在的总脂质的3mol％-15mol％，所述胆固醇或胆固醇衍生物占所述颗粒中存在的总脂质的30mol％-40mol％；和
    (d)抑制颗粒聚集的缀合脂质，所述缀合脂质占所述颗粒中存在的总脂质的0.5mol％-2mol％。
  23. 根据权利要求22所述的siRNA核酸-脂质颗粒，其中，所述阳离子脂质包括1,2-二亚油基氧基-N,N-二甲基氨基丙烷、1,2-二亚麻基氧基-N,N-二甲基氨基丙烷或它们的混合物。
  24. 根据权利要求22所述的siRNA核酸-脂质颗粒，其中，所述磷脂包括二棕榈酰磷脂酰胆碱、二硬脂酰磷脂酰胆碱或它们的混合物。
  25. 根据权利要求22所述的siRNA核酸-脂质颗粒，其中，所述抑制颗粒聚集的缀合脂质包括聚乙二醇-脂质缀合物，所述聚乙二醇-脂质缀合物选自PEG-二酰甘油缀合物、PEG-二烷氧基丙基缀合物或它们的混合物。
  26. 一种GalNAc-siRNA缀合物，所述GalNAc-siRNA缀合物由siRNA附接至GalNAc或GalNAc衍生物形成，所述siRNA的序列如SEQ ID NO.10或SEQ ID NO.11所示。
  27. 根据权利要求26所述的GalNAc-siRNA缀合物，其中，所述siRNA附接至一个以上GalNAc衍生物。
  28. 根据权利要求27所述的GalNAc-siRNA缀合物，其中，所述附接不使用接头或通过一个以上接头进行。
  29. 根据权利要求28所述的GalNAc-siRNA缀合物，其中，所述接头是多价支化接头。
  30. 根据权利要求26-29中任一项所述的GalNAc-siRNA缀合物，其中，一个以上GalNAc衍生物附接至所述siRNA的有义链的3'端、反义链的3′端、有义链的5′端和/或反义链的5′端。
  31. 制备权利要求26-30中任一项所述的GalNAc-siRNA缀合物的方法，所述方法包括：在固相寡核苷酸合成过程中，使用GalNAc加载固体载体，从而通过三元间隔子将GalNAc与siRNA正义链的3'末端相连接；随后进行核苷酸脱保护和乙酸半乳糖胺脱保护，除去固体载体，得到GalNAc-siRNA缀合物。
  32. MLKL基因的抑制剂在制备治疗脑胶质瘤药物中的用途。
  33. 一种治疗脑胶质瘤的方法，所述方法包括向受试者施用MLKL基因的抑制剂，以抑制MLKL基因的表达和/或抑制MLKL基因的表达产物的活性。
  34. 根据权利要求32所述的用途和权利要求33所述的方法，其中，所述MLKL基因的抑制剂包括权利要求14-21中任一项所述的抑制剂、权利要求22-25中任一项所述的siRNA核酸-脂质颗粒、权利要求26-30中任一项所述的GalNAc-siRNA缀合物、CRISPR/Cas***和/或抑制MLKL蛋白活性的小分子化合物、抗体和/或抗体偶联物；优选地，所述CRISPR/Cas***为CRISPR/Cas9***。
  35. 根据权利要求33所述的方法，其中，通过RNA干扰、基因编辑抑制MLKL基因的表达；优选地，所述基因编辑使用CRISPR/Cas***，更优选地，CRISPR/Cas9***；优选地，所述RNA干扰使用权利要求14-21中任一项所述的抑制剂、权利要求22-25中任一项所述的siRNA核酸-脂质颗粒和/或权利要求26-30中任一项所述的GalNAc-siRNA缀合物。
  36. MLKL基因在筛选治疗脑胶质瘤的药物中的用途，其中，MLKL基因或其表达产物作为治疗脑胶质瘤细胞的药物的靶标。
  37. 根据权利要求36所述的用途，其中，MLKL基因或其表达产物作为治疗脑胶质瘤细胞的药物的靶标包括：将MLKL基因作为药物针对脑胶质瘤细胞产生RNA干扰作用的靶标，从而能降低脑胶质瘤细胞中MLKL基因的表达水平，以及，将MLKL蛋白作为药物结合的靶标，从而能降低脑胶质瘤细胞中MLKL蛋白的活性；将MLKL基因或其表达产物作为治疗脑胶质瘤细胞的药物的靶标用于筛选治疗脑胶质瘤的药物包括：将MLKL基因或其表达产物作为靶标对药物进行筛选，以获得抑制MLKL基因表达或MLKL蛋白活性的药物作为治疗脑胶质瘤药物。
  38. 根据前述任一权利要求所述的应用、试剂盒、诊断、筛查和/或预后方法、抑制剂、siRNA核酸-脂质颗粒、GalNAc-siRNA缀合物、制备方法、制备治疗脑胶质瘤药物的用途、治疗方法、筛选治疗脑胶质瘤的药物的用途，其中，所述MLKL基因的序列如SEQ ID NO.1所示。
PCT/CN2023/085516 2022-02-02 2023-03-31 一种脑胶质瘤生物标记物mlkl基因及其应用 WO2023147790A1 (zh)

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