CN111635946A - Molecular biomarker for diagnosis and treatment of glioma and application thereof - Google Patents

Abstract

The invention provides a molecular biomarker for diagnosing and treating glioma and application thereof, belonging to the technical field of crude drug medicines and molecular biology. The invention proves that the expression of SNORA8 is reduced along with the increase of the malignancy degree of glioma for the first time, and is positively correlated with the survival rate. The SNORA8 lentivirus can significantly improve the expression level of SNORA8 of glioma, and the over-expression of SNORA8 inhibits the proliferation and colony formation of glioma cells, so that the SNORA8 lentivirus can be used as an effective medicament for preventing and/or treating glioma. The invention provides a more favorable means for diagnosis and prognosis evaluation analysis of glioma, which has important significance for research and treatment of glioma. Meanwhile, the method also lays an experimental foundation for developing high-efficiency drugs related to glioma treatment and provides a new visual field, so that the method has good practical application value.

Description

Molecular biomarker for diagnosis and treatment of glioma and application thereof

Technical Field

The invention belongs to the technical field of crude drug medicine and molecular biology, and particularly relates to a molecular biomarker for diagnosing and treating glioma and application thereof.

Background

The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.

Brain gliomas are the most common primary tumors of the central nervous system, accounting for 32% of all primary brain tumors, with more than half being the most malignant WHO grade IV Glioblastoma multiforme (GBM). Studies have shown that approximately 88% of GBM patients have less than 1 year of overall survival after diagnosis, and 5-year survival < 5%. The major causes of poor prognosis in patients with GBM are the malignant phenotypes and characteristics of tumor cells, such as hyperproliferation, invasive growth, intratumoral genetic heterogeneity, and microvascular hyperplasia. Currently, the international standard for GBM treatment is the administration of Temozolomide (TMZ) concurrent chemotherapy after surgical resection, combined with the application of radiation therapy, but the overall therapeutic effect is not ideal. Due to the infiltration characteristic of glioma cells to peripheral brain tissues and the poor permeability of the blood brain barrier to chemotherapeutic drugs, even if the current advanced microsurgical technique is utilized, the tumor is still difficult to completely resect, and the high resistance and tolerance of glioma cells to therapeutic intervention measures are easily caused. Therefore, finding a novel therapeutic approach that effectively inhibits the malignant biological properties of gliomas, thereby prolonging the life span of patients and improving the quality of life of patients, is an important challenge facing the field of neuro-tumor therapy.

Non-coding RNAs are a class of molecules that do not participate in coding proteins but function via RNA forms. In recent years, the method has become a hot research in the field of prevention and treatment of various diseases, particularly tumors. Non-coding RNAs such as miRNA, long non-coding RNA and circular RNA are all reported to be abnormally expressed in various tumors and participate in the generation and development of the tumors. The snorRNA (nucleolar RNA) is also a non-coding RNA, the length is 60-30nt, and since early researches find that the snorRNA is mainly positioned in nucleolus, is related to processing modification of rRNA and has single function, the snorRNA does not attract attention in a later period of time. However, more and more sequencing data show that snornas exhibit a general tendency to express aberrantly in tumors, and there are some studies showing that snornas play a key role in the progression of disease.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides a molecular biomarker for diagnosing and treating glioma and application thereof. The invention discovers that the expression quantity of SNORA8 is related to the prognosis of glioma for the first time, the malignant growth and invasion capacity of glioma cells can be controlled by regulating the expression of SNORA8, and the over-expression of SNORA8 can effectively inhibit the malignant progression of glioma and influence the downstream PI3K/AKT signal pathway. Therefore, SNORA8 can be used as a molecular biomarker for diagnosing and treating glioma, namely, can be used as a prognostic index and a potential therapeutic target of glioma, thereby completing the invention.

Specifically, the invention relates to the following technical scheme:

in a first aspect of the invention, there is provided the use of a substance for detecting SNORA8 in the preparation of any one or more of:

(a1) glioma diagnostic or auxiliary diagnostic products;

(a2) glioma prognosis evaluation or auxiliary prognosis evaluation products.

According to the invention, the expression level of SNORA8 is abnormally reduced in glioma expression and is related to tumor molecular subtype and grade, and SNORA8 expression is obviously related to prognosis, wherein in patients with glioma as a whole, LGG and GBM, high expression of SNORA8 indicates good prognosis, and conversely, low expression of SNORA8 indicates poor prognosis of glioma patients. Therefore, SNORA8 can be used as a new glioma prognostic marker and provides a basis for the evaluation of the postoperative survival period of a patient.

In a second aspect of the invention, there is provided the use of a substance for increasing the expression level of SNORA8 in the manufacture of a product.

The research result of the invention shows that SNORA8 inhibits the malignant phenotype of glioma cells by regulating cell growth and stem cell balling capacity. Therefore, the expression of SNORA8 can be activated to inhibit the proliferation and malignant invasion of glioma cells, thereby realizing the inhibition effect on tumors. At the same time, SNORA8 over-expression can inhibit glioma cell AKT signal pathway, therefore, the product can function as any one or more of the following:

(b1) inhibiting glioma cell proliferation;

(b2) inhibiting malignant invasion of glioma cells;

(b3) inhibiting glioma growth;

(b4) inhibiting glioma metastasis;

(b5) treating glioma;

(b6) inhibiting the AKT signaling pathway.

In a third aspect of the invention, there is provided the use of an agent for reducing the expression level of SNORA8 in the manufacture of a product.

The function of the product is any one or more of the following:

(c1) promoting glioma cell proliferation;

(c2) promoting malignant invasion of glioma cells;

(c3) promoting glioma growth;

(c4) promoting glioma metastasis;

(c5) preparing a glioma animal model;

(c6) facilitating the AKT signaling pathway.

In a fourth aspect of the invention, there is provided a product whose active ingredients comprise a substance for increasing the expression level of SNORA 8.

The function of the product is any one or more of the following:

(b1) inhibiting glioma cell proliferation;

(b2) inhibiting malignant invasion of glioma cells;

(b3) inhibiting glioma growth;

(b4) inhibiting glioma metastasis;

(b5) treating glioma;

(b6) inhibiting the AKT signaling pathway.

In a fifth aspect of the invention, there is provided a product whose active ingredients comprise a substance for reducing the expression level of SNORA 8.

The function of the product is any one or more of the following:

(c1) promoting glioma cell proliferation;

(c2) promoting malignant invasion of glioma cells;

(c3) promoting glioma growth;

(c4) promoting glioma metastasis;

(c5) preparing a glioma animal model;

(c6) facilitating the AKT signaling pathway.

In a sixth aspect of the invention there is provided the use of an agent for increasing the expression level of SNORA8 in any one or more of:

(b1) inhibiting glioma cell proliferation;

(b2) inhibiting malignant invasion of glioma cells;

(b3) inhibiting glioma growth;

(b4) inhibiting glioma metastasis;

(b5) treating glioma;

(b6) inhibiting the AKT signaling pathway.

In a seventh aspect of the invention there is provided the use of an agent for reducing the expression level of SNORA8 in any one or more of:

(c1) promoting glioma cell proliferation;

(c2) promoting malignant invasion of glioma cells;

(c3) promoting glioma growth;

(c4) promoting glioma metastasis;

(c5) preparing a glioma animal model;

(c6) facilitating the AKT signaling pathway.

The beneficial technical effects of one or more technical schemes are as follows:

the technical scheme firstly proves that the expression of SNORA8 is reduced along with the increase of the malignancy degree of glioma, and is positively correlated with the survival rate. The SNORA8 lentivirus can significantly improve the expression level of SNORA8 of glioma, and the over-expression of SNORA8 inhibits the proliferation and colony formation of glioma cells, so that the SNORA8 lentivirus can be used as an effective medicament for preventing and/or treating glioma.

The technical scheme provides a more favorable means for diagnosis and prognosis evaluation analysis of glioma, and has important significance for research and treatment of glioma. Meanwhile, the method also lays an experimental foundation for developing high-efficiency drugs related to glioma treatment and provides a new visual field, so that the method has good practical application value.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.

FIG. 1 shows the expression level of SNORA8 in glioma tissues in the present example, wherein A is the differential expression of SNORA8 in the TCGA database in normal brain tissues and Glioblastoma, B is the differential expression of SNORA8 in the TCGA database in five molecular pathological types of glioma, and C is the differential expression of SNORA8 in 6 normal brain tissues, 21 Low Grade Gliomas (LGG) and 9 Glioblastomas (GBM) by real-time fluorescence quantitative PCR method. P < 0.05; p < 0.01; p < 0.001.

Fig. 2 is a graph of the prognostic significance of SNORA8 in gliomas in an example of the present invention, where a and B are graphs of the prognosis of SNORA8 in all patients with gliomas, LGG and GBM, low grade gliomas, analyzed in the TCGA (n 667) and CGGA (n 325) databases, respectively.

FIG. 3 is a series of charts demonstrating that SNORA8 specific overexpression reduces the proliferation and invasion function of glioma cells in an example of the present invention; wherein, A is real-time fluorescent quantitative PCR analysis, and shows that the relative RNA level of SNORA8 is obviously increased after cells are transfected by lentivirus lenti-SNORA8, and U6 is used as a control; b is a CCK-8 detection OD 450-based LN229 and GBM # P3 cell growth curve chart; c is a graph showing the reduction in clonality after transfection of lenti-SNORA8 into cells; d is a diagram of the reduction of the spheroidicity of glioma after lenti-SNORA8 transfection cells; e is a graph showing the reduction of glioma invasion capacity after lenti-SNORA8 transfection of cells; f is a real-time fluorescent quantitative PCR analysis showing that the relative RNA level of SNORA8 was significantly reduced after cells were transfected with lentiviral sh-SNORA8, with U6 as a control; g is a graph of the increase in clonogenic capacity following sh-SNORA8 transfection of cells. P < 0.05; p < 0.01; p < 0.001.

FIG. 4 is a graph showing by Western blot the inhibition of the level of the AKT signaling pathway in glioma cells following specific overexpression of SNORA8 in the examples of the invention.

FIG. 5 is an in vivo experiment showing that over-expression of SNORA8 inhibits tumor growth in an example of the invention. Wherein, A is the tumor growth condition of BALB/C nude mice in situ xenograft glioma evaluated by adopting a bioluminescence imaging (BLI) system, B is a statistical graph of fluorescence intensity of mice at the 20 th day, the control group of mice has high fluorescence intensity compared with the mice over-expressed by SNORA8, namely the tumors are larger, and C is the control group of mice has shorter survival time compared with the SNORA8 over-expressed group.

Detailed Description

It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise. The experimental procedures, if specific conditions are not indicated in the following detailed description, are generally in accordance with conventional procedures and conditions of molecular biology within the skill of the art, which are fully explained in the literature. See, e.g., Sambrook et al, "molecular cloning: the techniques and conditions described in the laboratory Manual, or according to the manufacturer's recommendations.

The present invention is further illustrated by reference to specific examples, which are intended to be illustrative only and not limiting. If the experimental conditions not specified in the examples are specified, they are generally according to the conventional conditions, or according to the conditions recommended by the sales companies; materials, reagents and the like used in examples were commercially available unless otherwise specified.

The skilled artisan will appreciate that the term "expression level" refers to the amount of a gene product present in vivo or in a sample at a particular time point. The expression level can be measured/quantified/detected, for example, by protein or mRNA expressed by the gene. The expression level can be quantified, for example, as follows: normalizing the amount of the gene product of interest present in the sample with the total amount (total protein or mRNA) of the same type of gene product in the same sample or reference sample (e.g., a sample obtained from the same individual at the same time or a fraction of the same size (weight, volume) of the same sample), or determining the amount of the gene product of interest/defined sample size (weight, volume, etc.). The expression level can be measured or detected by any method known in the art, such as a method for direct detection and quantification of a gene product of interest (e.g., mass spectrometry), or a method for indirect detection and measurement of a gene product of interest that generally works by binding the gene product of interest to one or more different molecules or detection devices (e.g., primers, probes, antibodies, protein scaffolds) specific for the gene product of interest. Also known to the skilled person is the determination of the level of gene copy, which also includes the determination of the absence or presence of one or more fragments (e.g. by nucleic acid probes or primers, such as quantitative PCR, Multiplex ligation-dependent probe amplification (MLPA) PCR).

The terms "indicator" and "marker" are used interchangeably herein and refer to a sign or signal of a condition or to monitor a condition. Such "disorder" refers to a biological state of a cell, tissue or organ, or to a health and/or disease state of an individual. The indicator may be the presence or absence of molecules including, but not limited to, peptides, proteins, and nucleic acids, or may be a change in the level or pattern of expression of such molecules in a cell, or tissue, organ, or individual. The indicator can be a sign of the occurrence, development or presence of a disease in an individual or of further progression of such a disease. The indicator may also be a sign of the risk of developing a disease in the individual.

The term "up-regulation", "increase" or "increase" of the level of an indicator means that the level of such indicator is reduced in a sample compared to a reference or reference sample.

The term "down-regulation", "reduction" or "decrease" of the level of an indicator refers to a reduction of the level of such indicator in a sample compared to a reference or reference sample.

The inventor screens snorA members specifically expressed in glioma by gene sequencing technology in the early stage, wherein SNORA8 is used as a candidate gene, the difference is the largest, the research reports of SNORA8 are few, and no report is found in glioma, which suggests that the SNORA is possibly used as a new molecular biomarker for diagnosing and treating glioma, and the specific function of the SNORA is in urgent need of research.

In view of the above, in one embodiment of the present invention, there is provided the use of a substance for detecting SNORA8 in the preparation of any one or more of the following:

(a1) glioma diagnostic or auxiliary diagnostic products;

(a2) glioma prognosis evaluation or auxiliary prognosis evaluation products.

According to the invention, the expression level of SNORA8 is abnormally reduced in glioma expression and is related to tumor molecular subtype and grade, and SNORA8 expression is obviously related to prognosis, wherein in patients with glioma as a whole, LGG and GBM, high expression of SNORA8 indicates good prognosis, and conversely, low expression of SNORA8 indicates poor prognosis of glioma patients. Therefore, SNORA8 can be used as a new glioma prognostic marker and provides a basis for the evaluation of the postoperative survival period of a patient.

Therefore, in the application (a1), the glioma diagnosis or the auxiliary diagnosis comprises the diagnosis analysis of molecular subtype and grade of the glioma.

In the use (a2), the prognosis of glioma comprises predicting the overall survival of the patient after surgery for glioma.

In yet another embodiment of the invention, the means for detecting SNORA8 comprises amplification primers for SNORA8, including SNORA8-F and SNORA 8-R.

In still another embodiment of the present invention, the SNORA8-F is any one of the following single-stranded DNA of F1) to F4):

f1) single-stranded DNA shown in a sequence 1 in a sequence table;

f2) single-stranded DNA obtained by adding one or several nucleotides to the 5 '-end and/or 3' -end of f 1);

f3) a single-stranded DNA having 85% or more identity to the single-stranded DNA defined by f1) or f 2);

f4) a single-stranded DNA which hybridizes with the single-stranded DNA defined by f1) or f2) under stringent conditions;

in still another embodiment of the present invention, the SNORA8-R is any one of the following single-stranded DNA of R1) to R4):

r1) single-stranded DNA shown in sequence 2 in the sequence table;

r2) single-stranded DNA obtained by adding one or more nucleotides to the 5 '-end and/or the 3' -end of r 1);

r3) has 85% or more identity with the single-stranded DNA defined by r1) or r 2);

r4) under stringent conditions with the single-stranded DNA defined in r1) or r 2).

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" includes a nucleotide sequence having 85% or more, or 90% or more, or 95% or more identity to the nucleotide sequence shown in SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, or SEQ ID No. 4 of the present invention. Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to assess the identity between related sequences.

The stringent conditions are hybridization and washing of the membrane 2 times, 5min each, at 68 ℃ in a solution of 2 XSSC, 0.1% SDS, and 2 times, 15min each, at 68 ℃ in a solution of 0.5 XSSC, 0.1% SDS; alternatively, hybridization was carried out at 65 ℃ in a solution of 0.1 XSSPE (or 0.1 XSSC), 0.1% SDS, and the membrane was washed.

In another embodiment of the present invention, the identity of 85% or more may be 85%, 90% or 95% or more.

In one embodiment of the invention, the product comprises a test kit.

In one embodiment of the invention, there is provided the use of an agent for increasing the expression level of SNORA8 in the manufacture of a product.

The research result of the invention shows that SNORA8 inhibits the malignant phenotype of glioma cells by regulating cell growth and stem cell balling capacity. Therefore, the expression of SNORA8 can be activated to inhibit the proliferation and malignant invasion of glioma cells, thereby realizing the inhibition effect on tumors. At the same time, SNORA8 over-expression can inhibit glioma cell AKT signal pathway, therefore, the product can function as any one or more of the following:

(b1) inhibiting glioma cell proliferation;

(b2) inhibiting malignant invasion of glioma cells;

(b3) inhibiting glioma growth;

(b4) inhibiting glioma metastasis;

(b5) treating glioma;

(b6) inhibiting the AKT signaling pathway.

The product may be a medicament.

In one embodiment of the invention, the substance that increases the expression level of SNORA8 comprises a substance that upregulates the expression and/or promotes the activity of SNORA8 using gene-specific Mimics-based technology; such as short hairpin RNA (shRNA) that artificially synthesizes SNORA8 or a promoter or lentivirus that up-regulates expression of SNORA 8; compound accelerators are also included.

In one embodiment of the invention, there is provided the use of an agent for reducing the expression level of SNORA8 in the manufacture of a product.

The function of the product is any one or more of the following:

(c1) promoting glioma cell proliferation;

(c2) promoting malignant invasion of glioma cells;

(c3) promoting glioma growth;

(c4) promoting glioma metastasis;

(c5) preparing a glioma animal model;

(c6) facilitating the AKT signaling pathway.

Among the substances that reduce the expression level of SNORA8 are RNA interference molecules or antisense oligonucleotides directed against SNORA8, small molecule inhibitors, sirnas, and substances that effect lentiviral infection or gene knock-out.

The product may be a medicament.

In one embodiment of the invention, a product is provided wherein the active ingredient comprises a substance for increasing the expression level of SNORA 8.

The function of the product is any one or more of the following:

(b1) inhibiting glioma cell proliferation;

(b2) inhibiting malignant invasion of glioma cells;

(b3) inhibiting glioma growth;

(b4) inhibiting glioma metastasis;

(b5) treating glioma;

(b6) inhibiting the AKT signaling pathway.

The product may be a medicament.

In one embodiment of the invention, a product is provided wherein the active ingredient comprises a substance for reducing the expression level of SNORA 8.

The function of the product is any one or more of the following:

(c1) promoting glioma cell proliferation;

(c2) promoting malignant invasion of glioma cells;

(c3) promoting glioma growth;

(c4) promoting glioma metastasis;

(c5) preparing a glioma animal model;

(c6) facilitating the AKT signaling pathway.

The product may be a medicament.

In one embodiment of the invention, there is provided the use of an agent for increasing the expression level of SNORA8 in any one or more of:

(b1) inhibiting glioma cell proliferation;

(b2) inhibiting malignant invasion of glioma cells;

(b3) inhibiting glioma growth;

(b4) inhibiting glioma metastasis;

(b5) treating glioma;

(b6) inhibiting the AKT signaling pathway.

In one embodiment of the invention, there is provided the use of an agent for reducing the expression level of SNORA8 in any one or more of:

(c1) promoting glioma cell proliferation;

(c2) promoting malignant invasion of glioma cells;

(c3) promoting glioma growth;

(c4) promoting glioma metastasis;

(c5) preparing a glioma animal model;

(c6) facilitating the AKT signaling pathway.

According to the present invention, the concept of "treatment" means any suitable measure for the treatment of a glioma-related disease, either for the prophylactic treatment of such an manifested disease or manifested symptoms, or to avoid the recurrence of such a disease, for example after the end of a treatment period or the treatment of symptoms of an already established disease, or the prevention or inhibition or reduction of the occurrence of such a disease or symptoms with prior intervention.

According to the invention, the above-mentioned medicament also comprises at least one pharmaceutically inactive ingredient.

The pharmaceutically inactive ingredients may be carriers, excipients, diluents and the like which are generally used in pharmacy. Further, the composition can be prepared into oral preparations such as powder, granule, tablet, capsule, suspension, emulsion, syrup, and spray, external preparations, suppositories, and sterile injectable solutions according to a conventional method.

Such pharmaceutically inactive ingredients, which may include carriers, excipients and diluents, are well known in the art and can be determined by one of ordinary skill in the art to meet clinical criteria.

In still another embodiment of the present invention, the carrier, excipient and diluent include, but are not limited to, lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, and the like.

In yet another embodiment of the present invention, the medicament of the present invention may be administered into the body by known means. For example, by intravenous systemic delivery or local injection into the tissue of interest. Optionally via intravenous, transdermal, intranasal, mucosal or other delivery methods. Such administration may be via a single dose or multiple doses. It will be understood by those skilled in the art that the actual dosage to be administered in the present invention may vary greatly depending on a variety of factors, such as the target cell, the type of organism or tissue thereof, the general condition of the subject to be treated, the route of administration, the mode of administration, and the like.

In still another embodiment of the present invention, the subject to which the medicament is administered may be a human or non-human mammal, such as a mouse, rat, guinea pig, rabbit, dog, monkey, orangutan, or the like.

The invention is further illustrated by the following examples, which are not to be construed as limiting the invention thereto. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

Examples

Materials and methods

1. Tissue sample and database

This example includes glioma samples and normal brain tissue from neurosurgery at the university of Shandong, Qilu Hospital. The study protocol was approved by the hospital institutional review board and was approved by each subject with written informed consent. Patients were treated routinely with 6 normal brain tissues, 21 WHO grade II-III, 9 grade IV (GBM) tissue specimens. Glioma specimens were validated and classified by two experienced clinical pathologists according to WHO tumor classification. Clinical information and expression data from public databases glioma specimens were obtained from the TCGA and CGGA databases.

2. Cell culture and reagents

Human glioblastoma cells were purchased from the cell bank of the Chinese academy of sciences. All cells were cultured using DMEM medium containing 10% fetal bovine serum and incubated at 37 ℃ in a cell culture chamber containing 5% carbon dioxide.

Overexpression and knockdown of SNORA8

lenti-SNORA8 and lenti-Control lentivirus which are constructed and synthesized by adopting lentivirus vectors and are used for overexpression; and sh-SNORA8 lentivirus for knockout, sequence: 5'-GCTTGTTAAACCTGGTATTTC-3' (Genephrma, Shanghai, SEQ ID NO.3) glioma cells were infected according to standard protocols. Subsequent experiments were performed after infection by puromycin screening.

4. Real-time quantitative PCR

RNA was extracted from glioma cells using Trizol reagent (Invitrogen, Life Technologies). And reverse transcription is performed. The primers for SNORA8 were forward primers: 5'-TGCACTCAGCAGTTTACACCT-3' (SEQ ID NO. 1); reverse primer: 5'-CCAAGAAAACCAACGATGCCA-3' (SEQ ID NO. 2).

5.Western blot

Harvested cells were lysed in RIPA cell lysis buffer with heat denaturation. Protein lysates (20 μ g) were analyzed and proteins were transferred to polyvinylidene difluoride membranes (PVDF). Hatching of the primary antibodies p-AKT (S473), AKT, p-S6K (T421/S424), S6K, GAPDH (CST). Specific proteins were detected by enhanced chemiluminescence (ECL, Millipore, Bredford, USA).

6. Cell proliferation potency assay

Glioma cells were taken in 96-well cell culture dishes at a density of 3000 cells/well. Cell proliferation was analyzed 24, 48, 72, 96h after transfection using cell counting Kit-8 (CCK-8). Add 10. mu.L of CCK-8 solvent to each well and incubate for 1h in a cell incubator. Optical density was then measured at 450nm using Ensight (PerkinElmer) and analyzed to plot cell proliferation curves.

7. Cell colony formation assay

Cells were seeded into 6-well plates at a density of 1000 cells/well. DMEM containing 10% fetal bovine serum was replaced every three days. After 15 days, methanol was fixed, stained with crystal violet for 15 minutes and photographed. Each experiment was repeated 3 times.

8. Cell balling experiment

Cells were seeded into 12-well plates at a density of 1000 cells/well and cultured using Neurobasal stem cell broth. After 10 days, the number of the balls is detected by a microscope and is photographed for statistics. Each experiment was repeated 3 times.

9. Cell invasion ability detection experiment

Glioma spheronization cells were incubated in a sphere forming matrix for 72 hours, spheroids >2mm in diameter were implanted in 96-well plates, and invasion gel was added. Glioma spheroids were photographed every 24 hours with a microscope. The ellipsoid at 0h served as the reference point for measuring the area of invasion of the invading cells.

10. In vivo experiments in mice

Establishment of intracranial glioma, GBM # P3 fluorescent cells (1 × 10⁶) Lenti-SNORA8 or Lenti-Control virus was transfected and then implanted stereotactically into mouse brain. The growth condition of intracranial tumors is detected on days 4 and 20 by using a bioluminescence imaging technology. Kaplan-Meier survival curves were used to describe survival time and body weight.

11. Statistical analysis

ANOVA or t-test was applied using GraphPad Prism 7 software. All experiments were repeated 3 times and the mean ± standard error was taken. The Kaplan-Meier survival curves were analyzed using the log-rank test. Chi-square test and fisher's exact analysis were used to determine the relationship between GALNT2 expression and clinical pathology. P <0.05 is statistically significant for the differences.

Second, experimental results

Expression levels of SNORA8 are down-regulated in gliomas and correlated with tumor molecular subtype, grade

Differences in gene expression levels of SNORA8 in GBM and normal brain tissues were first analyzed using the TCGA database. The results show a significant reduction in the expression level of SNORA8 in GBMs compared to normal brain tissue (shown in figure 1A). The expression difference of SNORA8 expression level in different glioma molecule subtypes is further analyzed in detail, and the expression of SNORA8 in LGG-IDHwt subgroup with higher malignancy degree is lower in low-grade glioma LGG; in GBM, SNORA8 also had lower expression levels in the more malignant IDHwt group (shown in fig. 1B). Further detection of SNORA8 expression level in clinical specimens using real-time quantitative PCR, similar results were observed, with SNORA8 highly expressed in normal brain tissue and significantly reduced expression in gliomas, with the lowest expression level in GBM (shown in fig. 1C). The above results indicate an important role for SNORA8 in glioma malignancy grading. Further, applicants analyzed the correlation of the expression level of SNORA8 with multiple clinicopathological risk indicators of glioma in TCGA, and the results showed that molecular genetic features such as IDH mutation, MGMT promoter methylation, and 1p/19q co-deletion were significantly associated with high expression of SNORA8 (as shown in table 1).

TABLE 1 relationship between SNORA8 expression and clinical pathological characteristics in glioma patients

High expression of SNORA8 as a marker for good prognosis in patients with glioma

The prognostic value of SNORA8 expression in Overall Survival (OS) of glioma patients was tested using the Kaplan-Meier survival curve. In the TCGA and CGGA databases, there was a significant correlation between SNORA8 expression and prognosis, with high expression of SNORA8 predicted good prognosis in patients with global glioma, LGG, and GBM. Therefore, SNORA8 can be used as a new glioma prognostic marker and provides a basis for the evaluation of the postoperative survival period of a patient.

3. Verifying over-expression efficiency of SNORA8 in glioma cells and up-regulating inhibition effect of SNORA8 on proliferation and invasion of glioma cells

Based on the abnormal down-regulation of expression of SNORA8 in glioma, suggesting that it may exert an anti-oncogene function, in order to evaluate the role of SNORA8 in the malignant biological properties of glioma, lentivirus lenti-SNORA8 was constructed to up-regulate the expression of SNORA8 in glioma cell lines LN229 and GBM # P3, GBM # BG 7. Real-time quantitative PCR analysis confirmed that expression levels of SNORA8 were significantly elevated after lenti-SNORA8 infection compared to NC group (fig. 3A). These results further demonstrate the effectiveness of lenti-SNORA8 in up-regulating SNORA8 in glioma cell in vitro functional assays. The effect of SNORA8 on cell proliferation was determined using CCK-8, colony formation, and cell spheronization experiments. The CCK-8 results show that the glioma cells of the SNORA8 overexpression group have obviously lower proliferation after 4 days than the control group; at the same time, the colony forming ability and the balling ability of the glioma cells of the SNORA8 overexpression group are inhibited. In the invasiveness test experiment, the glioma cells of the SNORA8 overexpression group invaded peripheral matrigel at 72 hours at a significantly lower distance than the control group (FIG. 3E). On the other hand, lentiviral sh-SNORA8 was constructed to knock down the expression of SNORA8 in glioma cell line LN 229. Real-time quantitative PCR analysis confirmed that expression levels of SNORA8 were significantly reduced after infection with sh-SNORA8 compared to NC group (fig. 3F). Further colony formation experiments showed an enhanced colony forming ability of glioma cells of the SNORA8 knockout group (FIG. 3G). These results indicate that upregulation of the SNORA8 gene can inhibit the malignant growth and invasiveness of glioma cells.

Over-expression of SNORA8 inhibits glioma cell AKT signaling pathway

The AKT signaling pathway and downstream targets are key signaling pathways that regulate malignant growth of gliomas, with hyperactivated states present in a variety of tumors including gliomas. The applicant detects AKT signal activity of glioma cells after overexpression of SNORA8 by using Western method, and the result shows that the phosphorylation activity of p-AKT (S473) is remarkably inhibited by SNORA8 overexpression, and the phosphorylation activity of p-S6K (T421/S424) which is a downstream molecule of AKT is also remarkably reduced. These data indicate that overexpression of SNORA8 inhibits the glioma cell AKT signaling pathway.

Effect of SNORA8 on growth status of intracranial tumors

The growth progress of glioma after intracranial glioma in nude mice is detected by using a bioluminescence imaging (BLI) system. The results showed that the fluorescence intensity was significantly reduced 20 days after the implantation of the lenti-SNORA8 group compared to the control group (FIGS. 5A, 5B). The total survival time of the control group was shorter than that of the lenti-SNORA8 group (P <0.05, FIG. 5C). These results indicate that overexpression of SNORA8 results in a decrease in the growth rate of glioma cells in vivo.

Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

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Claims (10)

1. Use of a substance for detecting SNORA8 in the preparation of any one or more of the following:

(a1) glioma diagnostic or auxiliary diagnostic products;

(a2) glioma prognosis evaluation or auxiliary prognosis evaluation products.

2. The use of claim 1, wherein in said use (a1), the diagnosis or the auxiliary diagnosis of glioma comprises a diagnostic analysis of the molecular subtype, grade, of glioma; or the like, or, alternatively,

in the use (a2), the prognosis of glioma comprises predicting the overall survival of the patient after surgery for glioma.

3. The use of claim 1, wherein the means for detecting SNORA8 comprises an amplification primer for SNORA 8;

preferably, the amplification primers include SNORA8-F and SNORA 8-R;

wherein the SNORA8-F is any one of the following single-stranded DNA from F1) to F4):

f1) single-stranded DNA shown in a sequence 1 in a sequence table;

f2) single-stranded DNA obtained by adding one or several nucleotides to the 5 '-end and/or 3' -end of f 1);

f3) a single-stranded DNA having 85% or more identity to the single-stranded DNA defined by f1) or f 2);

f4) a single-stranded DNA which hybridizes with the single-stranded DNA defined by f1) or f2) under stringent conditions;

the SNORA8-R is any one of the following single-stranded DNA of R1) to R4):

r1) single-stranded DNA shown in sequence 2 in the sequence table;

r2) single-stranded DNA obtained by adding one or more nucleotides to the 5 '-end and/or the 3' -end of r 1);

r3) has 85% or more identity with the single-stranded DNA defined by r1) or r 2);

r4) under stringent conditions with the single-stranded DNA defined in r1) or r 2).

4. Use of a substance for increasing the expression level of SNORA8 in the manufacture of a product, said product functioning as any one or more of:

(b1) inhibiting glioma cell proliferation;

(b2) inhibiting malignant invasion of glioma cells;

(b3) inhibiting glioma growth;

(b4) inhibiting glioma metastasis;

(b5) treating glioma;

(b6) inhibiting the AKT signaling pathway.

5. Use of an agent for reducing the expression level of SNORA8 in the manufacture of a product, said product functioning as any one or more of:

(c1) promoting glioma cell proliferation;

(c2) promoting malignant invasion of glioma cells;

(c3) promoting glioma growth;

(c4) promoting glioma metastasis;

(c5) preparing a glioma animal model;

(c6) facilitating the AKT signaling pathway.

6. A product characterized in that its active ingredients comprise substances for increasing the expression level of SNORA 8;

the function of the product is any one or more of the following:

(b1) inhibiting glioma cell proliferation;

(b2) inhibiting malignant invasion of glioma cells;

(b3) inhibiting glioma growth;

(b4) inhibiting glioma metastasis;

(b5) treating glioma;

(b6) inhibiting the AKT signaling pathway.

7. A product characterized in that its active ingredients comprise substances for reducing the expression level of SNORA 8;

the function of the product is any one or more of the following:

(c1) promoting glioma cell proliferation;

(c2) promoting malignant invasion of glioma cells;

(c3) promoting glioma growth;

(c4) promoting glioma metastasis;

(c5) preparing a glioma animal model;

(c6) facilitating the AKT signaling pathway.

8. Use of an agent that increases the expression level of SNORA8 in any one or more of:

(b1) inhibiting glioma cell proliferation;

(b2) inhibiting malignant invasion of glioma cells;

(b3) inhibiting glioma growth;

(b4) inhibiting glioma metastasis;

(b5) treating glioma;

(b6) inhibiting the AKT signaling pathway.

9. Use of an agent for reducing the expression level of SNORA8 in any one or more of:

(c1) promoting glioma cell proliferation;

(c2) promoting malignant invasion of glioma cells;

(c3) promoting glioma growth;

(c4) promoting glioma metastasis;

(c5) preparing a glioma animal model;

(c6) facilitating the AKT signaling pathway.

10. Use according to claim 4 or 5 and/or product according to claim 6 or 7, wherein the product is a medicament.

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