CN112048558A

CN112048558A - OLFML2A gene and application of protein as glioma drug target

Info

Publication number: CN112048558A
Application number: CN202010936199.4A
Authority: CN
Inventors: 张祎年; 李国强; 马世泽; 潘亚文; 段磊; 余跃; 马晓东; 许彦龙
Original assignee: Lanzhou University
Current assignee: Lanzhou University
Priority date: 2020-09-08
Filing date: 2020-09-08
Publication date: 2020-12-08
Anticipated expiration: 2040-09-08
Also published as: CN112048558B

Abstract

The invention relates to an OLFML2A gene and protein as an application of glioma drug targets, wherein OLFML2A is used as a new cancer suppressor gene, the gene and the protein can be used as glioma drug targets, OLFML 2A-based therapeutic drugs can effectively reduce the malignancy degree of glioma, slow down the growth of tumor bodies and inhibit the formation of tumors, the chemotherapy effect and prognosis of patients are predicted through the detection of the expression level of OLFML2A, and the drugs using OLFML2A as the targets can be used as a new means for targeted therapy of glioma. The invention provides a new method for the research and development of glioma targeted therapeutic drugs, lays a foundation for glioma targeted drug therapy, and has great application and popularization values.

Description

OLFML2A gene and application of protein as glioma drug target

Technical Field

The invention belongs to the technical field of gene medicines, and particularly relates to an OLFML2A gene and application of protein as glioma medicine targets.

Background

Glioma is the most common malignant primary tumor of the central nervous system, and the survival period of the glioma is less than 20 months even after the glioma is completely resected after operation and actively treated by postoperative radiotherapy, chemotherapy and the like. Heterogeneity of tumor cells is a major cause of clinical difficulty and poor prognosis, and also results in distinct clinical prognosis for patients with the same clinical grade and molecular typing, and the same treatment regimen. Therefore, finding a specific molecular target determining the high heterogeneity of glioma cells, defining the molecular mechanism of malignant proliferation and apoptosis imbalance of tumor cells, and further developing targeted therapeutic drugs is the key point in the research field of glioma.

The biological characteristics of glioma depend on the internal gene change, and the reliable tumor marking gene is searched and the characteristics of glioma, such as proliferation, invasiveness, differentiation and the like, are judged, which is an important prerequisite for glioma diagnosis, prognosis evaluation and determination of effective treatment measures. Therefore, under the condition that the effect of the existing treatment scheme is poor, research and development of a specific gene target of glioma are urgently needed, and further, a foundation is sought for gene diagnosis and targeted small molecule drug therapy of glioma, so that the method becomes necessary and urgent.

Disclosure of Invention

In order to solve the problems in the prior art, the invention provides an application of OLFML2A gene and protein as glioma drug targets. The inventor of the present application finds that the expression of the OLFML2A gene is increased in glioma in research, and finds that the expression of the gene is related to the prognosis of glioma, which suggests that OLFML2A can be used as a novel glioma regulatory factor. The discovery of the gene not only can provide a new theoretical basis for the pathogenesis of glioma, but also can provide a new way for the development of glioma drugs. The OLFML2A gene is used as a target spot to further screen novel glioma treatment drugs, and meanwhile, OLFML2A can also be used as a detection index to evaluate the drug curative effect of glioma.

The technical scheme adopted by the invention is as follows:

the OLFML2A gene is used as a glioma treatment/prognosis judgment target.

The application of the therapeutic target comprises the application of an OLFML2A inhibitor as an anti-glioma active ingredient or the application of an OLFML2A inhibitor in the preparation of anti-glioma drugs.

The application of the prognosis judgment target point comprises the application of applying the OLFML2A content detection reagent to a brain glioma prognosis judgment reagent or the application of preparing a prognosis judgment kit.

The OLFML2A gene is highly expressed in glioma cells.

The glioma cell is U251 or U87.

A small interfering RNA for targeted inhibition of OLFML2A gene, wherein the nucleotide sequence of the small interfering RNA is at least one group of the following sequences:

siRNA-1 sequence: 5'-TCTATGTCACCAACTACTA-3', as shown in SEQ ID NO. 1;

siRNA-2 sequence: 5'-GCCAAACAAACATTCACTA-3', as shown in SEQ ID NO. 2.

An OLFML2A gene interfering nucleic acid construct, comprising a gene fragment of the OLFML2A gene small interfering RNA, capable of expressing the OLFML2A gene small interfering RNA.

The OLFML2A gene interference nucleic acid construct is OLFML2A gene interference lentivirus vector.

OLFML2A gene interference lentivirus is prepared by virus packaging of OLFML2A gene interference lentivirus vector under the assistance of lentivirus packaging plasmid and cell line.

A medicament for treating glioma, comprising a therapeutically effective amount of said OLFML2A gene small interfering RNA or said OLFML2A gene interfering lentivirus, and a pharmaceutically acceptable carrier, diluent or excipient.

The present inventors found through extensive studies that OLFML2A gene is a novel protooncogene according to the following: the expression of OLFML2A gene is obviously different between glioma and normal brain tissue and in glioma tissue of different grades, the expression level of OLFML2A has a significant influence on the survival time of the patient, and the higher the expression level of OLFML2A, the shorter the survival time of the patient. OLFML2A expression was sequentially increased from WHO grade I to WHO grade IV glioma as detected by immunohistochemical staining of OLFML2A on collected surgical specimens from glioma patients and on a portion of normal brain tissue.

The invention has the beneficial effects that:

the invention provides a new cancer suppressor gene OLFML2A and application of the gene and protein as glioma drug targets, a treatment drug based on OLFML2A can effectively reduce the malignant degree of glioma, slow down the growth of tumor body and suppress the formation of tumor, the chemotherapy effect and prognosis of a patient are predicted by detecting the expression level of OLFML2A, and the drug taking OLFML2A as the targets can be used as a new means for targeted therapy of glioma. The invention provides a new method for the research and development of glioma targeted therapeutic drugs, lays a foundation for glioma targeted drug therapy, and has great application and popularization values.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.

FIG. 1A is a graph comparing the expression levels of OLFML2A in normal brain tissue and in cancer tissue from a patient with glioma;

FIG. 1B is a graph comparing the expression levels of OLFML2A in cancer tissues from patients with different grade gliomas;

FIG. 1C is a Kaplan-Meier survival curve;

FIG. 1D is an immunohistochemistry of different grade tumors in clinical patient specimens;

FIGS. 2A and 2B are diagrams illustrating the proliferation potency of Celigo in detecting cells in different glioma cell lines, respectively;

FIGS. 2C and 2D are MTT observations of the effect of knockdown OLFML2A on glioma cell line proliferation in different glioma cell lines, respectively;

FIGS. 2E and 2F show the effect of FACS detection on apoptosis of knockdown OLFML2A in different glioma cell lines;

FIGS. 3A and 3B show the effect of knocking down the neoplasia of OLFML2A mice;

FIG. 3C and FIG. 3D are the comparison of tumor body weight and tumor body growth rate, respectively.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the examples given herein without any inventive step, are within the scope of the present invention.

The various reagents and the like referred to in the following examples are commercially available products known to those skilled in the art.

Example 1

The embodiment provides an OLFML2A gene for a glioma treatment/prognosis judgment target, wherein the nucleotide sequence of the OLFML2A gene is shown as SEQ ID No. 3;

the embodiment also provides a small interfering RNA for targeted inhibition of OLFML2A gene, wherein the nucleotide sequence of the RNA is as follows:

siRNA-1 sequence: 5'-TCTATGTCACCAACTACTA-3', as shown in SEQ ID NO. 1.

The embodiment also provides a preparation method of the siRNA, which comprises the following steps:

(1) according to the design principle of RNA interference sequence, a plurality of 19-21ntRNA interference target sequences are designed by taking OLFML2A gene as a template. After evaluation and determination, siRNA sequences are selected as interference targets. And designing shRNA interference sequences according to the selected target sequences, and adding appropriate restriction enzyme cutting sites at two ends to complete vector construction. In addition, a TTTTT termination signal is added to the 3 '-end of the plus strand, and a termination signal complementary sequence is added to the 5' -end of the minus strand. After the design is completed, a single-stranded DNA oligo is synthesized. The synthesized single-stranded DNA oligo dry powder was dissolved in annealing buffer (final concentration 20. mu.M) and water-washed at 90 ℃ for 15 min. After naturally cooling to room temperature, a double strand with a cohesive end was formed. A20. mu.l reaction system was prepared according to Fermentas T4 DNA Ligase instructions and the double-stranded DNA oligo was ligated to the linearized vector. And transforming the connecting product into an escherichia coli competent cell, carrying out PCR identification on a positive clone, transferring the bacterial liquid with the correct sequencing into 150ml of LB liquid culture medium containing Amp antibiotics, and carrying out shake culture on a shaking table at 37 ℃ for overnight. Extracting plasmids according to the EndoFree Maxi Plasmid Kit instruction, and packaging the plasmids qualified by quality inspection with lentivirus.

(2) The slow virus packaging comprises plasmid transfection and slow virus harvesting, and the specific operations are as follows:

24h before transfection, 293T cells in the logarithmic growth phase were trypsinized and cell density was adjusted to about 5X 10 in medium containing 10% serum⁶Cells/15 ml, reseeded in 10cm cell culture dishes at 37 ℃ with 5% CO₂Culturing in an incubator. The cell can be used for transfection after 24 hours when the cell density reaches 70% -80%; replacing the medium with a serum-free medium 2h before transfection; adding each prepared DNA solution (20 μ g of GV vector plasmid, 15 μ g of pHelper1.0 vector plasmid, and 10 μ g of pHelper2.0 vector plasmid) into a sterilized centrifuge tube, mixing with corresponding volume of Gecky transfection reagent, adjusting the total volume to 1ml, and incubating at room temperature for 15 min; the mixed solution is slowly dripped into 293T cell culture solution, mixed evenly and treated at 37 ℃ with 5% CO₂Culturing in a cell culture box; culturing for 6h, and discardingRemoving the culture medium containing the transfection mixture, adding 10ml of PBS (phosphate buffer solution) for washing once, slightly shaking the culture dish to wash the residual transfection mixture, and then pouring and discarding; slowly adding 10% serum-containing cell culture medium 20ml, and heating at 37 deg.C with 5% CO₂Continuously culturing for 48-72h in the incubator;

(3) then, lentivirus concentration and purification are carried out, and the specific operation is as follows:

collecting 293T cell supernatant 48h after transfection (which can be counted as 0h after transfection) according to cell states; centrifuging at 4000g for 10min at 4 deg.C to remove cell debris; filtering the supernatant with a 0.45 μm filter in a 40ml ultracentrifuge tube; respectively balancing samples, putting ultracentrifuge tubes with virus supernatant into a Beckman ultracentrifuge one by one, setting the centrifugation parameters to be 25000rpm, setting the centrifugation time to be 2h, and controlling the centrifugation temperature to be 4 ℃; after centrifugation is finished, removing supernatant, removing liquid remained on the tube wall as much as possible, adding virus preservation solution (which can be replaced by PBS or cell culture medium), and lightly and repeatedly blowing and resuspending; after fully dissolving, centrifuging at high speed 10000rpm for 5min, and taking supernatant for quality detection.

Example 2

The present embodiment provides an OLFML2A gene and a small interfering RNA targeted for inhibiting OLFML2A gene for glioma therapy/prognosis target, which are different from those in embodiment 1 only in that: the nucleotide sequence of the RNA is as follows:

siRNA-2 sequence: 5'-GCCAAACAAACATTCACTA-3', as shown in SEQ ID NO. 2.

Example 3

the embodiment also provides a small interfering RNA for targeted inhibition of OLFML2A gene, wherein the nucleotide sequences of the RNA are siRNA-1 sequence and siRNA-2 sequence, and specifically comprises the following steps:

siRNA-1 sequence: 5'-TCTATGTCACCAACTACTA-3', as shown in SEQ ID NO. 1;

siRNA-2 sequence: 5'-GCCAAACAAACATTCACTA-3', as shown in SEQ ID NO. 2.

Examples of the experiments

First, detecting OLFML2A gene in glioma population tissue sample, and analyzing the relationship between the gene and prognosis

Tissue level:

(1)RT-PCR

tissue samples to be ground were placed in 1.5ml lep tubes containing 1ml of lysis solution. Grinding the tissue by using a superfine homogenizer; centrifuging at 4 deg.C and 5000rpm for 3min, and removing precipitate; adding 200 μ L chloroform into each tube, standing at room temperature for 10 min; centrifuging at 12800rpm at 4 deg.C for 15 min; absorbing the upper layer liquid, transferring to a new 1.5mLEP tube, adding equal volume of precooled isopropanol, mixing uniformly, and standing for 10min at 4 ℃. Centrifuging at 12800rpm for 12min at 4 deg.C, and removing supernatant; 1mL of 75% ethanol (freshly made with DEPC water) was added and the precipitate was washed. Centrifuging at 11800rpm for 5min at 4 deg.C, discarding supernatant, and drying at room temperature. When the RNA precipitate is basically transparent, adding RNase-free water until the RNA precipitate is completely dissolved, and analyzing and determining the concentration and the quality of the extracted RNA by using a Nanodrop2000/2000C spectrophotometer; RNA reverse transcription, and placing the obtained RT product-cDNA at-20 ℃ for storage for later use; Real-timePCR detection and melting curve making; and (6) analyzing the data.

(2) Tissue immunohistochemistry and survival assays

After conventional deparaffinization of the tissue sections, gradient ethanol hydration was performed: placing the slices in xylene I15 min-xylene II 15 min-absolute ethyl alcohol I5 min-absolute ethyl alcohol II 5 min-85% alcohol 5 min-75% alcohol 5 min-distilled water washing. The tissue sections were placed in a repair box filled with EDTA antigen repair buffer (pH 8.0) for antigen repair in a microwave oven. Stopping fire for 8min and 7 min. After natural cooling, washing with PBS buffer solution, adding rabbit anti-OLFML 2A antibody diluted at a ratio of 1:200, incubating overnight in a refrigerator at 4 deg.C, incubating with anti-mouse secondary antibody labeled with horseradish peroxidase at room temperature for 45min, DAB developing, and sealing with neutral gum. Expression of NQ01 mRNA in TCGA database glioma samples was analyzed on-line using GEPIA (http:// GEPIA. cancerpku. cn) and Kaplan-Meier survival curves were plotted.

As shown in fig. 1A, the expression levels of OLFML2A in Normal brain tissue and cancer tissue of glioma patients were compared, wherein the ratio of Normal: normal brain tissue; a Cancer: glioma tissue. It can be seen that the expression of OLFML2A gene in tumor tissue was significantly increased in patients with glioma.

Fig. 1B is a graph comparing the expression levels of OLFML2A in cancer tissues of patients with different grade glioma, wherein LGG: low grade glioma; GBM: high grade glioma. Different grade tumor immunohistochemistry in clinical patients is shown in fig. 1D, and the arrow indicates OLFML2A positive cells, it can be seen that the higher the WHO tumor grade, the higher the expression level of OLFML2A gene.

FIG. 1C shows the Kaplan-Meier survival curve, and it can be seen that the expression level of OLFML2A has a significant effect on the survival time of patients, and the prognosis of patients with higher expression level of OLFML2A is worse. "high expression" in FIG. 1C indicates that the OLFML2A gene was expressed more highly in the patient sample, and "low expression" indicates that the OLFML2A gene was expressed less in the patient sample, including those with lost visits and those with no deaths at the follow-up endpoint.

Second, siRNA sequence knockdown expression of OLFML2A gene

(1) Lentiviral transfection of cells of interest

The cell density of U87 and U251 was adjusted to 1X 10⁵each/mL, the cell suspension was inoculated into 6-well plates, 2mL was added to each well, the cells were cultured until 20% confluence, the medium was changed and 25-fold dilution with transfection enhancing reagent (Polybrene) Polybrene was added to each well, 40 μ l was added to each well, and 10 μ l was calculated according to MOI ═ 5⁶The virus was added to 6 well plates per well in a final volume of 1ml per well. And after transfection for 16h, changing the liquid of the cells in the culture plate, continuously culturing for 72h, and observing the GFP expression under a fluorescence microscope to verify the transfection efficiency. The wells were then aspirated of medium, and 2ml of fresh growth medium containing puromycin (2. mu.g/ml) was added to each well and the relevant experiments were performed after 24h of incubation. Multiplicity of viral Infection (MOI) — (viral titer × viral volume)/cell number.

(2) Celigo imaging and enumeration

After pancreatin digestion of U87 and U251 cells of each experimental group in logarithmic growth phase, the complete culture medium was resuspended into cell suspension, counted, and 96-well plate was spreadPlates, 1500 cells/well, 3 replicates per group, 100. mu.l/well culture system, 5% CO at 37 ℃₂Culturing in an incubator; starting the next day after the plate is paved, detecting and reading the plate once by Celigo every day, and continuously detecting and reading the plate for 3-5 days; adjusting parameters, accurately calculating the number of cells with green fluorescence in the scanning pore plate every time, performing statistical drawing on the data, and drawing a cell proliferation curve for 5 days.

(3) MTT assay

The cell density of plated cells (2000 cells/well) of each experimental group of U87 and U251 in the logarithmic growth phase is determined according to the cell growth speed, the number of plated cells is determined according to the experimental design (if 5 days of detection are carried out, 5 96-well plates are plated), and the cell density of each experimental group is adjusted to be uniform. Starting the day after plating, 20. mu.l of 5mg/ml MTT was added to the wells 4h before termination of the culture without changing the medium. After 4h, the culture was completely aspirated, and 100. mu.l of DMSO was added to dissolve formazan particles. Oscillating for 2-5min with oscillator, and detecting OD value with enzyme labeling instrument 490/570 nm. And (6) carrying out data statistical analysis.

(4) FACS apoptosis assay

Washing cells twice by 1 × Wash Buffer, and centrifuging for 5min at 500 rpm; removing the supernatant, leaving 50 μ l of the lower layer liquid, adding the antibody, and incubating for 30min at 4 ℃ in a dark place; mixing the liquid evenly, and centrifuging for 5min at 1000 rpm; washing twice by the Wash Buffer, and resuspending the cells by the Wash Buffer; moving into a flow tube for flow cytometry detection.

The expression of the target genes OLFML2A of glioma cell strains U87 and U251 is observed by knocking down OLFML2A through small interfering ribonucleic acid (siRNA); wherein: FIGS. 2A and 2B show the proliferation potency of Celigo cells, and FIGS. 2C and 2D show the proliferation rate of glioma cell lines observed in MTT assay, from which: the knocking-down of OLFML2A in glioma cell lines U87 and U251 can obviously inhibit the growth of cells.

Fig. 2E, 2F show the effect of FACS detection of knockdown of OLFML2A on apoptosis. It can be seen that the knockdown of OLFML2A in glioma cell lines U87, U251 promotes apoptosis.

In FIGS. 2A-F, shCtrl represents the control group, shOLFML2A-1 represents the sequence siRNA1 knockdown group, and shOLFML2A-1 represents the sequence siRNA2 knockdown group.

Thirdly, constructing an OLFML2A-siRNA stable knock-down glioma-bearing mouse model, and observing the tumor growth of the tumor-bearing mouse

Selecting cells in logarithmic growth phase, digesting, re-suspending, counting, and adjusting concentration to 2 × 10⁷One per ml. 200 μ l of the resuspended cells were inoculated subcutaneously into 10 nude mice per group. Tumor volumes were measured every 2 days, nude mice were sacrificed on day 30 and data analyzed.

Fig. 3A and 3B show the effect of knocking down the tumor formation of OLFML2A mice (photographs of mice and tumor bodies), fig. 3C shows the comparison of tumor body weights, fig. 3D shows the comparison of tumor body growth rates, in the figures, "NC" represents a control group, and "KD" represents an OLFML2A knock-down group, which indicates that siRNA can reduce the malignancy of glioma by inhibiting the expression of OLFML2A gene, thereby achieving drug therapy of glioma.

The invention points out that OLFML2A plays an extremely important role in the process of glioma development and progression, particularly in the process of malignant glioma evolution, and provides a new pathogenesis theoretical basis and a therapeutic target for glioma.

The above description is only for the specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention, and all the changes or substitutions should be covered within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims.

SEQUENCE LISTING

<110> Lanzhou university

<120> OLFML2A gene and application of protein as glioma drug target

<130> 2010

<160> 3

<170> PatentIn version 3.3

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tctatgtcac caactacta 19

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gccaaacaaa cattcacta 19

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catatcctag aggttgggtg accaaccctg tcccagtttg cctgggactt gagggttttt 60

ccaggatgca gcactttcag tgctaaaact aggaatgttc caggcactct gggatgagtt 120

ggtcaccttg cctaggtgcc taagaacctc tcccctgccc acgtactaag accaatgagt 180

aagagagaca aggctgggaa ggacacagct agaggaaaag gcaaggacat cagcaagtat 240

ggcagtgtgc agaaaagctt tgcagacaga ggcctcccaa aacctcccaa ggagaagctg 300

cttcaggtgg agaagctgag aaaggagagc ggcaagggca gtttcctcca gcccacagcc 360

aagccccgcg ccctggccca gcagcaggct gtgatccggg gcttcaccta ctacaaggca 420

ggcaagcagg aggtgaccga ggcggtggca gacaacaccc tccagggcac ttcctggctg 480

gagcaactgc cgcccaaggt ggagggcagg tccaactccg cagagcccaa ctccgcagag 540

caggatgagg ctgagcccag gtcctccgag cgagtggacc tggcttctgg cacccccact 600

tcaatccctg ccaccaccac caccgccacc accaccccaa cccccaccac cagtctcctg 660

cccaccgagc caccttcagg tccagaagtc tccagccaag gcagagaggc gagctgtgag 720

ggcaccctcc gggctgtgga cccccctgtg aggcaccaca gctatgggcg ccacgaggga 780

gcctggatga aggaccctgc agctcgagac gacaggatct atgtcaccaa ctactactat 840

ggaaacagcc tggtggagtt ccgcaacctg gaaaacttca agcaaggccg ctggagtaac 900

atgtacaagc taccctacaa ctggatcggc acaggccacg tggtgtacca gggcgccttc 960

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gcctcctggg cgctgctgcc cgacgtggta tatgaggaca ccacaccttg gaagtggcgc 1080

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gtggacgacc gcgatgaggc ccagcccgag gtgatcgtcc tgagtcgctt ggaccccggc 1200

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cagccccctc agcccagccc accccacccg ctgtccggcc acattccaaa cctctaccgt 2160

cacctagctg ctgagcagaa accgcacccc gagagaaaat cccatcctct gttccaaggc 2220

ccctgtctgc tctatgctca tttttatttt ctcttattct tcatcagtgc cgtcatttgt 2280

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caaacccaca acctttacac actcagggat acctccgggt ctgccatgaa taagacccta 2520

ggcccaagtc tggtgtggtg ccaggatggc acagtttccc tcttccttgc cagccctgac 2580

ctggtcactg ggcaggctgg cccagcagcc tgggggctgc agaagacatg gtgtgagtag 2640

ttgggtccag gggaggcatc aggccttctt ctggttgcag gagagaacca gagggtggga 2700

acggggaggg aagaactgag ggtctgcaga ctggactttt cctggctcga cccaggactt 2760

gggttgagga tgcacggggg ccaccttgcc cggggccact ggtggctccc cagagcctca 2820

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gaggctcaga aaagtttaga gagcagctca gtgtcacact gggagctggg cacagccgat 3000

ctccttccag aagggttctg tctgtatcct ttattctccg cacatggagg ctgccctacc 3060

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cccttgctgc cttcattctg tcccaatgcc agctccctgg atgtctgtat gtttgaatac 3180

cagttgccat gttaggaagg tcagctgcac agccaagagt gtaagagtgt aaagaaatgc 3240

cttttttttt ttttttgagt tggagttttg ctcttgtcgc ccaggctgga gtgcagtggc 3300

gcgatctcgg ctcactgcaa cctctgcccc ctgggttcaa gcgattctcc tgcctcagcc 3360

tccctagtag ctgggactac agcacccacc accacaccca gctaattttt gtatttttag 3420

tagagacagg ggtttcacca cgttggccag gctggtctcg aactcctgac cttaggtgat 3480

ccgcccgcct cagcctccca aagtgttggg attacaggca tgagccactg cgcctggcca 3540

gcaaatgctt tttgtgcaga atacacttct ttcaggcatt gtcaggtgct gttttgttta 3600

agctctaact cacccctgga atacagggga atgatgacaa ccagcccagc caggcctgac 3660

tcatcatggt cacatccagc ccccaccccc ggccaactaa ccactgcagg ctcctcttcc 3720

agactcacca gggggcctcg aggccccggc atctcccttg gccctgggtg tgggttttac 3780

aagactgtgt ctttcatgac atcatagccc aaccatgtga gaagaaggag aaggcccccc 3840

tttcttcatt aatctgaaaa aaaggaaagt gagaataggc tgatttttaa aagttaaggg 3900

gcaagcagca ttgcattctg ggggaacgat cctggccaca gccgccaaac aaacattcac 3960

taggcctctt ctgttttcat acccttgtaa gtgggttatg tggtgggtat ggtcagtttt 4020

ttcttttttc ttttcttttc ttttttttga gacagagttt cgcttttgtt gcccgggctg 4080

gaatgcaatg gcgcgattca gctcactgca atctccgcct cccgggttca agtgattctc 4140

ctgccttagc ctcctgaaaa gctgggatta cagggccctg ccaccaagcc cagctaattg 4200

tatttttagt agagacagga tttcaccatg ttggccaggc cagtctcaaa ctcctgacct 4260

caggtgatcc acctgcctca gcctcccaga ctgttgggat tacaggcatg agccaccacg 4320

cctggccagt ttcttcattt tacatatggt cacattggcg cctagaacag ttaggtcgct 4380

cgtcacatag gcagttaagt ggagaaccag gtttcaaaat caggtaagaa aaccatcatc 4440

attaactgag caccagctgt gctaagcctg ccacgggcgt atccttgcag cctcacaaca 4500

gtgggaggtc tgtatcctga atgtcctcat tttacagatg aggacattga ggagaagaga 4560

cttacccagg ctcacacagc agctcagcct gttccaggcg ctggtcagtg cgtgttcttt 4620

gccaccagcc tgtcactcca gtggcagctc cagaaacgga ggctgttgct tttatcccta 4680

aactgcatcc acagagaagc cccaagaagg aggttggggc cagctcataa aaagcctgaa 4740

tgccaagcca aggagtggat gcctccagtc atatttagaa caaagtcaag tataaattta 4800

cagagaaaaa attctaagac agttggatgt tgtcctgttg gtgaggaagg gaaaggtttt 4860

tcttgtaggg aactggaacc agcccacaac tgcacacttg tgagctgtca tggaaacctg 4920

atccccaaca gcttttgagg ttgtttgttt gtttgtttgt ttacctgtct tgggctttgt 4980

tgcttttggc aaaaggtact tcaaacaagg gagggcctgg actgaggggg accaggtctt 5040

cttgctgacc tcgtctacaa aggcaaagga aggcaaagga agctgtctcg ggtgtttctg 5100

aacaacgtga ctcatgaggg gctttggcta cctcttgcgt tccccctaga gatgtccagg 5160

ccttacattt aatcggcttt ctctgcggtg gggtagagaa tggagctccc gccttgcggg 5220

cagtgctaaa ggtggagctg ggggattttc ctgggaatga tttgagggct cttgaaagcc 5280

catgtgttcc aaagcgtctt taactctggg atagcattgg aagccgctgt catgacagga 5340

catggcactg gatggctggc agagagccct ggctgggagt tagggagccc tgggttggaa 5400

tccagcccca cctcttttat gccacaggtt tggtcaagtt ctctcccgct cagggtaggg 5460

ctgtgaactc cctcttacag ctaagaacat gcagcttagt gaggacaaga cccttctaga 5520

gctttacccc taatcccccc ccaggagccc cgaggccggc attattcctc cccattacag 5580

gtgatgagcc tcaaattcag agagcttaag caacctgctc agggtcacgt ctccaacagg 5640

cagtagagtc aaggtataaa ccaggtctgt ttttgtacca gagtcccaga ctaactgttg 5700

gtaggaatct tgtaaccagt catgttttct tccttgtttt ggccgctggg aagctcaaag 5760

tcaaattcga gacccttttt tttccaattg tgctgagtct cctactagac tcgcttcatt 5820

ctagctttct gcttttacct ttaccctaat ctttttattt ttatgctatt gtactttatt 5880

tttgtaagtt gctgagatat ctgttttgca acaagatggg ctatatctaa ataaagacat 5940

gatcaaaggt ttgatttaaa a 5961

Claims

The application of OLFML2A gene as a glioma treatment/prognosis judgment target.
2. The use according to claim 1, wherein the use of the therapeutic target comprises the use of an OLFML2A inhibitor as an anti-glioma active ingredient, or the use of an OLFML2A inhibitor for the manufacture of an anti-glioma medicament.
3. The use of claim 1, wherein the use of the prognosis target comprises the use of an OLFML2A content detection reagent in a brain glioma prognosis reagent or the use of a prognosis kit.
4. The use according to claim 1, wherein the OLFML2A gene is highly expressed in glioma cells.
5. The use of claim 4, wherein the glioma cell is U251 or U87.
6. A small interfering RNA for targeted inhibition of OLFML2A gene, wherein the nucleotide sequence of the small interfering RNA is at least one group of the following sequences:

siRNA-1 sequence: 5'-TCTATGTCACCAACTACTA-3', as shown in SEQ ID NO. 1;

siRNA-2 sequence: 5'-GCCAAACAAACATTCACTA-3', as shown in SEQ ID NO. 2.
7. An OLFML2A gene interfering nucleic acid construct, comprising a gene fragment of the OLFML2A gene small interfering RNA of claim 6, capable of expressing said OLFML2A gene small interfering RNA.
8. The OLFML2A gene interfering nucleic acid construct of claim 7, wherein the OLFML2A gene interfering nucleic acid construct is an OLFML2A gene interfering lentiviral vector.
9. OLFML2A gene interfering lentivirus, wherein the OLFML2A gene interfering lentivirus vector of claim 7 or 8 is packaged into a virus with the help of a lentivirus packaging plasmid and a cell line.
10. A medicament for treating glioma, comprising a therapeutically effective amount of the OLFML2A gene small interfering RNA of claim 6 or the OLFML2A gene interfering lentivirus of claim 9, and a pharmaceutically acceptable carrier, diluent or excipient.