CN110283851B - Target MYO9B related to malignant pleural effusion and application thereof - Google Patents
Target MYO9B related to malignant pleural effusion and application thereof Download PDFInfo
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Abstract
The invention discloses a target MYO9B related to malignant pleural effusion and application thereof. The invention also discloses any one application of the following 1) to 5): 1) the application of a substance inhibiting the activity of MYO9B protein in the preparation of a product for treating or assisting in treating malignant pleural effusion; 2) the application of a substance inhibiting the activity of MYO9B protein in preparing a product for reducing or decreasing the volume of pleural effusion of a patient with malignant pleural effusion; 3) the application of a substance for inhibiting or silencing MYO9B gene expression in the preparation of a product for treating or assisting in treating malignant pleural effusion; 4) the application of a substance inhibiting or silencing MYO9B gene expression in preparing a product for reducing or decreasing the volume of pleural effusion of a patient with malignant pleural effusion; 5) the MYO9B protein and related signal pathways thereof are used as targets for developing or designing products for treating or assisting in treating malignant pleural effusion.
Description
Technical Field
The invention belongs to the technical field of animal genetic engineering, and particularly relates to a target MYO9B related to malignant pleural effusion and application thereof.
Background
Tumor cell invasion into the pleura can lead to Malignant Pleural Effusion (MPE). In China, the incidence rate of lung cancer is the first of all cancers, the mortality rate is the highest, the median survival time of a patient with lung cancer accompanied by MPE is obviously shortened compared with that of a patient without MPE, and the life quality is worse. Therefore, the research on the pathogenesis of MPE, early discovery and diagnosis of MPE and the corresponding prevention and treatment measures are significant for improving the life quality of patients, but the pathogenesis of MPE is not very clear at present. The interaction between tumor and host is critical for MPE formation, tumors in the pleura are the driving force for MPE formation, and various immune cells of the host play an important role in the development of MPE.
MYO9B is a multidomain, single head motor signaling molecule in the immune system for a variety of tissues such as lymph nodes, thymus, spleen, and a variety of immune cells such as dendritic cells, macrophages, and CD4+High expression in T cells. In 2010, Hanley et al edited MYO9B gene of mouse embryonic stem cell by cre-loxP system to obtain MYO9B knockout mouse. Although the Cre/loxP system is well established in theory, there are still deficiencies and limitations in practical applications: (1) conditional gene knockout by Cre/loxP technology firstly constructs a targeting vector, amplifies a homology arm with the length of about 4-5kb, and simultaneously needs BAC cloning and gene recombination technology. The loxP fragment is targeted into the genome of the cell, and the probability of homologous recombination is extremely low in this step, and the loxP fragment is required to be introducedThe successful targeted cells can be identified only by complicated screening work such as long-fragment PCR and Northern hybridization. Therefore, this step requires much time and effort, and is a rate-limiting step for conditional gene knockout using the Cre/loxP system. (2) Cre enzyme has certain potential toxicity after being expressed in mammals as an exogenous recombinase, and can cause the problems of abnormal cell proliferation, DNA mismatching, chromosome deletion and the like. (3) The Cre/loxP system has the cutting efficiency of about 70 percent, a small amount of knocked-out genes in target organs or tissues are not knocked out, and the target genes which are not knocked out can be expressed in a trace amount in specific organs or tissues.
Disclosure of Invention
A first object of the present invention is to provide any one of the following applications 1) to 5):
1) the application of a substance inhibiting the activity of MYO9B protein in the preparation of a product for treating or assisting in treating malignant pleural effusion;
2) the application of a substance inhibiting the activity of MYO9B protein in preparing a product for reducing or decreasing the volume of pleural effusion of a patient with malignant pleural effusion;
3) the application of a substance for inhibiting or silencing MYO9B gene expression in the preparation of a product for treating or assisting in treating malignant pleural effusion;
4) the application of a substance inhibiting or silencing MYO9B gene expression in preparing a product for reducing or decreasing the volume of pleural effusion of a patient with malignant pleural effusion;
5) the MYO9B protein and related signal pathways thereof are used as targets for developing or designing products for treating or assisting in treating malignant pleural effusion.
In the application, the substance for inhibiting the activity of the MYO9B protein can be a protein, polypeptide or small molecule compound for inhibiting the synthesis of the MYO9B protein, promoting the degradation of the MYO9B protein or inhibiting the function of the MYO9B protein;
the substance for inhibiting or silencing MYO9B gene expression can be a substance for interfering MYO9B gene expression or a substance for knocking MYO9B gene out or a substance for mutating MYO9B gene.
Further, the MYO9B gene knockout substance can be a CRISPR/Cas9 system.
Further, two sgrnas, named sgRNA1 and sgRNA2, are included in the CRISPR/Cas9 system;
the target sequence recognized by the sgRNA1 is a DNA molecule shown as a sequence 1 in a sequence table;
the target sequence recognized by the sgRNA2 is a DNA molecule shown as a sequence 2 in a sequence table.
In a specific embodiment of the invention, the sgRNA1 is an RNA molecule shown as sequence 3 in a sequence table;
the sgRNA2 is an RNA molecule shown in a sequence 4 in a sequence table.
It is another object of the present invention to provide a product.
The active ingredients of the product provided by the invention are the substances for inhibiting the activity of MYO9B protein or the substances for inhibiting or silencing the expression of MYO9B gene;
the product has the function of treating or assisting in treating malignant pleural effusion or reducing the pleural effusion volume of malignant pleural effusion patients.
It is still another object of the present invention to provide a method for preparing a transgenic mouse.
The preparation method of the transgenic mouse provided by the invention comprises the following steps: and editing the MYO9B gene in the mouse genome based on a CRISPR/Cas9 system, and further losing the function of the MYO9B gene in the mouse genome to obtain the transgenic mouse.
In the method, two sgrnas, namely sgRNA1 and sgRNA2, are included in the CRISPR/Cas9 system;
the target sequence recognized by the sgRNA1 is a DNA molecule shown as a sequence 1 in a sequence table;
the target sequence recognized by the sgRNA2 is a DNA molecule shown as a sequence 2 in a sequence table;
further, the sgRNA1 is an RNA molecule shown as a sequence 3 in a sequence table;
the sgRNA2 is an RNA molecule shown in a sequence 4 in a sequence table.
The above method includes the step of using the sgRNA1, the sgRNA2, and Cas9 mRNA;
further, the sgRNA1, the sgRNA2, and the Cas9mRNA are in a mass ratio of (1-2): (1-2): 2;
further, the sgRNA1, the sgRNA2, and the Cas9mRNA are in a mass ratio of 1:1: 2.
In a specific embodiment of the present invention, the method comprises the steps of:
(1) electrotransfer liquid containing the sgRNA1, the sgRNA2 and the Cas9mRNA is electrotransferred into a mouse fertilized egg to obtain an electrotransferred fertilized egg;
(2) culturing the fertilized eggs after electrotransformation, selecting embryos which develop to a 2-cell stage, implanting the embryos into a surrogate mouse, and obtaining an F0 surrogate mouse after the embryos are completely developed;
(3) identifying F0-generation mice with MYO9B gene mutation (heterozygous) from the F0-generation mice;
(4) crossing the F0 mouse (hybrid) with wild mouse (C57BL/6 mouse) to obtain F1 mouse (hybrid);
(5) crossing the female and male in the F1 generation mouse (heterozygous) to obtain an F2 generation mouse (homozygous);
(6) the F2 generation mice (homozygous) were serially bred to F4 generation to give F4 generation mice (homozygous).
Compared with wild type C57BL/6 mice, the F4 generation mice (homozygous) obtained by the invention have deletion of the DNA fragment with the size of 16bp shown at 17697-17712 and the DNA fragment with the size of 7bp shown at 17866-17872 of MYO9B genes on two homologous chromosomes.
It is still another object of the present invention to provide a product for preparing the above transgenic mouse.
The product for preparing the transgenic mouse comprises the sgRNA1, the sgRNA2 and the Cas9 mRNA.
The following applications M1) or M2) or M3) also belong to the scope of protection of the present invention:
m1) the transgenic mouse prepared by the method is applied to a mouse model for researching the occurrence and development mechanism and/or prognosis of malignant pleural effusion;
m2) the application of the transgenic mouse prepared by the method or the product in preparing a mouse model for researching the occurrence and development mechanism and/or prognosis of malignant pleural effusion;
m3) the application of the transgenic mouse prepared by the method or the product in preparing and/or screening the medicine for treating or assisting in treating malignant pleural effusion.
The MYO9B gene in the mouse genome is edited by using a CRISPR/Cas9 system for the first time to obtain a MYO9B gene knockout mouse, and the function of MYO9B is further researched. The malignant pleural effusion model experiment result shows that: the MYO9B gene participates in immune response of malignant pleural effusion, and compared with a wild mouse, the pleural effusion quantity of the malignant pleural effusion mouse subjected to MYO9B gene knockout is obviously reduced.
Drawings
FIG. 1 is 01#Mouse mutational status. 01#The mice are heterozygotes containing mutation, and the mutation is as follows: 16-7 (deletion of 16bp + deletion of 7 bp).
FIG. 2 shows a schematic representation of the formula 02#Mouse mutational status. 02#The mice are heterozygotes containing mutation, and the mutation is as follows: 16-1 (deletion of 16bp + deletion of 1 bp).
FIG. 3 is a comparison of pleural effusion volume of MYO9B knockout mice and wild-type mice.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.
The product name of Opti-MEM in the following examples is GibcoTMOpti-MEMTMIs a Thermo product.
The product name of Cas9mRNA in the following examples is GeneArtTMCRISPR nucleic mRNA, product of Invitrogen, lot number 00556295.
Both the C57BL/6 mouse and the CD-1 mouse in the examples described below are products of Experimental animals technology, Inc. of Wei Tony Hua, Beijing.
Example 1 construction method of MYO9B Gene editing mouse model
One, sgRNA and Cas9mRNA preparation
1. Design of target sequences
According to the MYO9B gene sequence (the Genbank number of the mRNA sequence of the MYO9B gene is NM-001142322.1, and the sequence is shown as a sequence 5 in a sequence table, wherein the DNA sequence shown as the 171 st-6557 nd site of the sequence 5 is a CDS sequence for encoding mouse MYO9B protein), the designed target sequences are as follows:
1 #: TGGCTCGAAGCACTACGTGC (SEQ ID NO: 1);
2 #: AGGCTGGCAGCTCGGGCCGT (SEQ ID NO: 2).
2. Preparation of sgRNA
(1) Carrying out PCR amplification by using an eSPAs 9(1.1) plasmid (adddge) as a template and adopting an invitro-sgMyo9b-1#. S primer and a hU6.R primer to obtain a PCR product, namely an in vitro transcription template 1;
the eSPAs 9(1.1) plasmid (adddge) is used as a template, and invitro-sgMyo9b-2#. S and hU6.R primers are adopted to carry out PCR amplification to obtain a PCR product, namely the in vitro transcription template 2.
The primer sequences are as follows:
invitro-sgMyo9b-1#.S：TAATACGACTCACTATAGGGTGGCTCGAAGCACTACGTGCGTTTTAGAGCTAGAAATAG；
invitro-sgMyo9b-2#.S：TAATACGACTCACTATAGGGAGGCTGGCAGCTCGGGCCGTGTTTTAGAGCTAGAAATAG；
hU6.R：AAAAGCACCGACTCGGTGCC。
(2) and (3) carrying out agarose gel electrophoresis on the PCR product, then recovering an agarose gel DNA recovery kit, and then carrying out in-vitro transcription by using a T7 in-vitro transcription kit (purchased from Ambion company, with the product number of AM1334) by using the recovered in-vitro transcription template 1 and the recovered in-vitro transcription template 2 as templates to obtain sgRNA1 and sgRNA2 respectively.
The sgRNA1 sequence is as follows: UGGCUCGAAGCACUACGUGCGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU (sequence 3)
The sgRNA2 sequence is as follows: AGGCUGGCAGCUCGGGCCGUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 4).
(3) And (3) carrying out agarose gel electrophoresis on the transcription product, then recovering the RNA purification kit, dissolving the RNA purification kit with DEPC (diethyl phthalate) water to obtain a sgRNA1 solution and a sgRNA2 solution, and finally storing the sgRNA1 solution and the sgRNA2 solution at the temperature of-80 ℃ for later use.
3. Preparation of electrotransfer mixture
The Opti-MEM, the sgRNA1 solution, the sgRNA2 solution and the Cas9mRNA solution were mixed well to obtain an electrotransfer mixture (total volume 50. mu.L), and the final concentrations of the sgRNA1, the sgRNA2 and the Cas9mRNA in the electrotransfer mixture were 200 ng/. mu.L, 200 ng/. mu.L and 400 ng/. mu.L, respectively.
Second, superovulation
1 male C57BL/6 mouse at 8 weeks of age and 3 female C57BL/6 mouse at 8 weeks of age. Female mice in day one 14: pregnant mare serum gonadotropin PMSG (5U/mouse) was intraperitoneally injected at 00 days 14: 00 intraperitoneal injection of human chorionic gonadotropin hCG (5U/mouse), followed by immediate caging of 3 female mice and 1 male mouse, day four 8: 00-9: 00, checking vaginal suppository of the female mouse, and selecting the female mouse of which the vaginal suppository is checked.
Thirdly, collecting and culturing fertilized eggs
Euthanasia female mouse with vaginal embolus detected in the second step, aseptically collecting oviduct, placing into a 35mm petri dish (drop culture medium prepared from M2 culture solution and hyaluronidase solution, pre-warming at 37 deg.C), tearing the ampulla of oviduct under stereomicroscope with forceps, releasing zygote group surrounded by cumulus cells, placing into hyaluronidase drop for several minutes, collecting fertilized egg, transferring into M2 drop culture medium, selecting fertilized egg with normal shape, and culturing at 37 deg.C and 5% CO2Culturing under the condition for later use.
Fourth, electrotransfer and embryo transplantation in oviduct
Adding 45 mu L of the electrotransfer mixed solution prepared in the step one into an CUY505P5 electrode cup, adding the selected fertilized eggs, measuring the resistance to enable the resistance to be 0.48-0.52 kOmega, and carrying out electrotransfer, wherein the specific parameters are as follows: poring Pulse: 225V, Length 1ms, interval 50ms, No.4, D.Rate 10%, Polarity +; transfer Pulse: 20V, Length 50ms, interval 50ms, No.5, D.Rate 10%, Polarity +/-. Fertilized egg after electrotransformationTransferred to KSOM medium and 5% CO at 37 deg.C2Culturing overnight under the condition, and selecting embryos which develop to 2-cell stage for implanting into surrogate mice.
The embryo transplantation comprises the following specific steps: a CD-1 female mouse pseudopregnant for 0.5d (vaginal embolus is detected in the morning of the day) is anesthetized, a 1cm incision is made on the back, the ovary and the oviduct are exposed, an embryo cultured to a 2-cell stage after microinjection is implanted into the ampulla of the oviduct under a stereomicroscope, the ovary and the oviduct are placed back to the abdominal cavity and then sutured, the left and right oviducts are implanted, and 20 embryos at the 2-cell stage are implanted into each mouse.
Fifth, strain establishment
1. Identification of Positive F0 Generation mice
1) CD-1 mice born approximately 20 days after transplantation, giving F0 generation mice. After birth, the F0 mouse was tailed at 3-4 weeks of age, and the obtained tissue was used for extraction of genomic DNA, while the wild-type C57BL/6 mouse was used as a control.
2) And (3) performing PCR amplification by using the extracted genome DNA as a template and adopting an upstream primer and a downstream primer to obtain a PCR product, and sequencing the PCR product. The primer sequences are as follows:
My09b.dec.S：TCTGGTATGAACTGCCTTG；
My09b.dec.A：TAGTAGCCATCCTCCTGAG。
the sequencing result shows that: positive F0 mouse 01 compared to wild-type C57BL/6 mouse#The 16bp DNA fragment at position 17697-17712 (corresponding to position 189-204 of the mRNA sequence) and the 7bp DNA fragment at position 17866-17872 (corresponding to position 358-364 of the mRNA sequence) of the MYO9B gene on one of the two homologous chromosomes were deleted, while the other chromosome was not mutated (FIG. 1).
Positive F0 generation of mice 02 compared to wild type C57BL/6 mice#The 16bp DNA fragment at position 17695-17710 (corresponding to position 187-202 of the mRNA sequence) and the base G at position 17868 (corresponding to position 360 of the mRNA sequence) of the MYO9B gene on one of the two homologous chromosomes are deleted, and the other chromosome is not mutated (FIG. 2).
2. Establishment of strains
Positive F0 mouse generation 01#And (5) establishing a system. The method comprises the following specific steps: positive F0 mouse generation 01#Hybridizing with wild C57BL/6 mouse to obtain F1 mouse, and identifying and screening the gene type of tailed mouse and positive F0 mouse 01 at 3-4 weeks#Mice with consistent genotypes are obtained, and F1 generation heterozygous mice 01 are obtained#(ii) a F1 generation hybrid mouse 01#Hybridizing to obtain F2 mouse, and identifying the genotype of the scissored tail at 3-4 weeks to screen out F2 homozygous mouse 01#F2 generation homozygous mouse 01#The female and male can be used as breeding mice for mating, and the breeding is continuously crossed and propagated to F4 generation, so as to obtain F4 generation homozygous mouse 01#And carrying out genotype identification on the gene. Genotype identification primers my09b.
F4 generation homozygous mouse 01, compared to wild type C57BL/6 mouse#The 16bp DNA fragment shown in 17697-17712 and the 7bp DNA fragment shown in 17866-17872 of the MYO9B genes on the two homologous chromosomes were deleted.
Functional verification of MYO9B gene knockout mice
For wild type C57BL/6 mouse and MYO9B gene knockout mouse (F4 generation homozygous mouse 01)#) Malignant pleural effusion model experiments are performed.
1. Construction of malignant pleural effusion mouse model
Based on wild type C57BL/6 mouse and MYO9B gene knockout mouse (F4 generation homozygous mouse 01)#) According to the literature "Stathopoulos GT et al, J Natl Cancer Inst, 2008; stathopoulos GT et al, Am J Respir Crit Care Med, 2010; the method in Wu XZ et al, Am J Respir Cell Mol Biol, 2017' constructs a malignant pleural effusion mouse model, and respectively obtains a wild type MPE mouse model and an MYO9B gene knockout MPE mouse model. The method comprises the following specific steps: after the mouse enters a deep anesthesia state, the limbs of the mouse are fixed by a sterilization adhesive tape to be in a supine position, the hair on the right front chest wall of the mouse is cut off, and then the skin on the right front chest wall of the mouse is disinfected by iodophor and 75% alcohol in sequence. The skin is cut with a small opening with the length of about 5mm at the position of about 1cm at 45 degrees on the right upper part of the mouse xiphoid process,the subcutaneous fascia and muscle were then separated blunt layer by layer until the ribs were exposed. Inoculation of tumor cells into the pleural cavity of mice: mu.l of mouse Lewis lung carcinoma cell suspension (1.5X 10) was extracted under direct visualization with a 100. mu.l range microsyringe5PBS buffer), and injecting the tumor cell suspension into the pleural cavity of the mouse through the intercostal space. The muscle and epidermal layers were sutured in this order with nylon thread, and the wound was then disinfected with 75% alcohol. Finally, the mice were waited for to wake up and returned to their cages.
2. Pleural effusion quantity determination for malignant pleural effusion mouse model
After 14 days of mouse model establishment, the mice were anesthetized and sacrificed, fixed in supine position with adhesive tape, and then the thoracico-abdominal area was disinfected with 75% alcohol, a "T" incision was made in the midline of the abdomen, the liver was opened to visualize the pleural cavity, and there was a bloody pleural effusion (fig. 3). A1 mL range syringe was used, with a minimum division of 0.01 mL. The syringe is held by hand, the needle punctures the septum, carefully absorbs the pleural effusion, empties the bubbles in the syringe, and records the pleural effusion volume according to the reading shown by the syringe.
The results are shown in FIG. 3. The results show that: the volume of pleural fluid in the wild type MPE mouse model (n 10) was 0.61 ± 0.01mL (mean ± SEM), and the volume of pleural fluid in the MYO9B knockout MPE mouse model (n 10) was 0.26 ± 0.05mL (mean ± SEM). Compared with wild type MPE mice, the MYO9B gene knockout MPE mouse model has obviously less pleural effusion volume, and the difference has statistical significance (P < 0.0001). The MYO9B gene deletion can significantly reduce the pleural effusion volume of malignant pleural effusion mice.
Sequence listing
<110> Beijing Chaoyang Hospital affiliated to capital medical university
<120> malignant pleural effusion related target MYO9B and application thereof
<160>5
<170>PatentIn version 3.5
<210>1
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>1
tggctcgaag cactacgtgc 20
<210>2
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>2
aggctggcag ctcgggccgt 20
<210>3
<211>20
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>3
uggcucgaag cacuacgugc guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210>4
<211>100
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>4
aggcuggcag cucgggccgu guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210>5
<211>100
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>5
ggaagtgaaa tcgcacggtg cagcggcggg cgaggctggt cgcttcgggg cggggcggcc 60
gggagcagcg ggcaggctgg ccggggaccg gtggcgcgca gcggccgagg tccagttaca 120
ggaggtgtgc acccgccggt gaggtatgct gaggccagga ggccggcagt atgagtgcgc 180
acgaggctgg cagctcgggc cgtcggcagc aggccaccta ccacctgcac atctacccgc 240
agctgtccag cgctgggagc cagacctcat gccgtgtgac cgccaccaag gacagcacaa 300
caagcgatgt catccaggat gtggtggcca gcctacacct ggatggctcg aagcactacg 360
tgctggtgga ggtgaaggag tcaggcgggg aggagtgggt gttggatgcc agcgactcac 420
ctgtacaccg agtgctactg tggcctcggc gagctcagga cgagcaccct caggaggatg 480
gctactactt cttgctgcag gaacgcaatg ctgatggcag tattcagtac ctgcccatac 540
agctgctggc tcagcctaca gctgcatgtc gcctggtaga gcgagggctg ttgccgaggc 600
ctcaagcaga ctttgatgac ctgtgcaacc tgccagagct gactgaagcc aacctgctgc 660
agaacctgaa gctgcgcttc atgcagcaaa agatctacac atacgcgggc agcatcctgg 720
tggccatcaa cccctttaag ttcctgccca tttacaaccc caagtatgtg aagatgtatg 780
agaaccaaca gctgggcaaa ctggagccac atgtgttcgc tctggccgat gtagcctact 840
acgccatgct gcgcaagcac gtgaaccagt gtatcgtcat ctctggtgag agtggctctg 900
gcaagacaca gagcaccaac ttcctcatcc actgcctcac agcgctcagc cagaagggct 960
atgccagtgg cgtcgaaagg accatcctgg gagcagggcc tgtgctggag gcttttggga 1020
atgccaagac agcccacaac aacaactcca gccgcttcgg gaagttcatc caagtcaact 1080
acctagaaaa tggcattgtg aggggagctg ttgtggaaaa ataccttctt gaaaagtctc 1140
gcctggtttc ccaggagaag gatgagcgga actaccatgt gttttattat ctgctgctgg 1200
gtgtcagtga ggaggagcgc ctggaatttc agctgaagca gcctcaagac tatttctacc 1260
tcaaccagca taacttgaat attgaagatg gagaagacct caaacatgac tttgaaaggc 1320
ttcagcaggc catggagatg gtgggcttcc tgcctgccac caagaagcag atcttctctg 1380
tcctctcagc catcctgtac cttggcaatg tcacctacaa gaagagagcc acaggccgag 1440
atgaaggcct ggaggtcggt cctccagagg tgttggacac cctctcccag ctcttaaagg 1500
taaagcggga gaccctggtg gaggtcttaa ccaagagaaa aacagttaca gtcaatgaca 1560
aactcatcct gccttacagc ctcagtgagg ccataactgc acgagactcc atggccaagt 1620
ccctatacag tgccctattt gactggattg tgctaaggat caaccacgcc ctcctcaaca 1680
agaaggacat ggaagaggct gtttcctgct tgtccattgg cgtcctggac atctttggat 1740
ttgaggactt cgaaaggaat agctttgagc agttttgcat caactatgcc aatgagcagt 1800
tgcagtacta cttcacccag cacatcttca agctggagca ggaggagtac cagggtgagg 1860
gcatctcgtg gcacaacatt gactacaccg acaacgtggg ctgtatccac ctcatcagca 1920
agaagcccac tggcctcttc tacctgctgg acgaggagag caacttccca catgccacaa 1980
gccacacttt gctggccaag ttcaagcagc agcatgagga caataagtac ttcctgggca 2040
caccagtcct ggagcccgcc ttcatcatcc agcacttcgc gggcagagtg aaataccaga 2100
tcaaggactt ccgggagaag aacatggact acatgcggcc tgacatcgtg gcactgctaa 2160
ggggcagtga cagctcctat gtgcgccagc tcattggcat ggacccggta gctgtgttcc 2220
gctgggctgt actacgagca gccatcaggg ccatggctgt gctgcgggag gctgggcgcc 2280
tgcgtgcaga gagagcggag aaggcagcag gtataagtag ccctgccact cgaagtcaca 2340
tggaagagct accaagagga gccagtaccc cttcagaaaa actgtaccgc gatttgcata 2400
accaaatcat caagagcctc aaaggactgc catggcaggg cgaggacccg aggaggcttc 2460
tccagtccct cagtctgttc cagaagcccc gcacctcctt cctgaagagt aaaggtatca 2520
aacaaaagca gatcattccc aagaacctgc tggactcgaa gtccctgagg ctcatcatca 2580
gcatgacact gcatgaccga actaccaagt cactgctgca cctgcacaaa aagaagaagc 2640
cacctagcat cagtgcacag ttccagacat ctcttaacaa gctgttggag gcactgggca 2700
aggccgagcc cttcttcatc cgctgcatcc gctccaatgc cgagaagaag gaactttgct 2760
ttgatgatga actggtgctg cagcaactgc gctacacagg catgctagag actgtgcgca 2820
tccgacgctc tggctacagc gccaagtaca ccttccagga cttcacggag cagttccagg 2880
tgttgctgcc caaggatgtc cagccctgta gggaggccat tgctgccctg ctggagaagc 2940
tgcaggtgga caggcagaac taccagattg gaaagacgaa ggtcttcctg aaggagacag 3000
agcgtcagac cctgcaggag aagctgcatg gtgaggtcct gcgtagaatc ctgcagctgc 3060
agagttggtt ccgtatggtg ctggaacgca agcactttgt acagatgaag catgctgcct 3120
tgaccatcca ggcctgctgg cggtcttacc gtgtgcgccg tgcactggaa aggacgcagg 3180
cagctgtgta cctgcaggct gcctggaggg gctacctgca gagacaggcc taccaccacc 3240
agaggcatag catcatccgc ctgcagagcc tctgccgtgg ccacctgcag cgcaggagct 3300
tcagccagat ggtgtcagag aagcagaagg cagagcaagc cagggaggca gcaggaggaa 3360
agctgtcaga gggtgagcct ggccccgtgg ccgctgggga gcagctatct gagcaccctg 3420
tggaagaccc tgagagcctg ggtgtggagg ctgaaacctg gatgaacaag tccccagacg 3480
gcatgtcacc taagaaggag acacccagcc cagagatgga gaccgcagcc caaaagacag 3540
tgccagctga aagtcatgag aaagtctcca gtagccgaga gaagcgagag tcacggcggc 3600
aacgagggtt ggagcatgtt gaacgacaga ataaacacat ccaatcctgc agggaggaga 3660
gcagcaccca ccgagaacct tccagaaggg caagcctgga aataggggaa agcttccctg 3720
agggcacaaa gggacccaga gaagatggac ttgaggcatg gactgagacc acagccccct 3780
ctagttcaaa gcaggcacag gttgtgggag acccacctgg gagtcccagt cccgtgcaga 3840
ggcccaccac cctggcccta gacagtaggg ttagcccaat gctccccagc agctccctgg 3900
aggttagccc agtgctcccc agcagctccc tggaatcccc caaagataag gacaaggatg 3960
agagcagcac caaggctcag gacaagcccg agagtcccag tggctccacc cagatccaac 4020
gataccaaca cccagacaca gagcggctgg ccactgctgt agagatatgg cgaggcaaga 4080
agcttgccag tgccgtgctg agccaatccc tggacctgag tgagaagcac cgggctacag 4140
gggcagccct gactcccaca gaggagaggc gcatctcttt ctccaccagt gacgtctcca 4200
agctgtcccc agtcaaggta cagacttcag ctgaaatcga tggggacttt agcagcaaga 4260
agccatccat ccataagaag aagtcaggag atccatccgc tggtcctgat gcaggcctgt 4320
ctccaggctc ccagggtgac tctaaatctg catttaagcg gctcttcctg cacaaagcca 4380
aggataagaa gcccagcctg gagggtgtag aggagacaga gagcaatgga gggcaggctg 4440
cacaggagac cccggccagg aagactctag acgtaccttc tagccagcag caccgccata 4500
ccacaggtga gaagccccta aaagggaaga agaaccgaaa tcgcaaagtt ggccagatca 4560
cagtgtccga gaagtggcga gagtcagtgt tccgtaagat cactaatgcc aatgagctca 4620
agtttctgga cgagttcctg ctcaacaagg tgaatgacct tcgctcacag aagacaccca 4680
tcgaaagctt gttcattgag gccactgagc gcttcaggag caacatcaag accatgtatt 4740
cggtgcctaa tgggaagatc catgtaggct acaaggacct gatggagaac taccagatcg 4800
ttgtcagtaa cctggctgcc gagcgtgggg agaaggacac caatctggtc ctcaatgtct 4860
tccagtcact gctggatgag ttcacccgca gctacaacaa gactgacttt gagcgggcca 4920
agcagagcaa agcccagaag aagaagcgga agcaggagcg tgctgtccag gaacacaatg 4980
gacatgtgtt tgccagctac caggtgaaca ttccacagtc atgtgagcag tgtctgtcct 5040
acatctggct catggacaag gctctactgt gcagtgtgtg caagatgacc tgccacaaga 5100
aatgcgtgca caagattcag agctattgct cctacactgg aaggaggaag agtgagctgg 5160
gtgccgaacc aggccacttc ggtgtgtgtg tagacagcct gaccagtgac aaggcctccg 5220
tgcccattgt gctggagaag cttctggaac acgtagagat gcatggcctg tacactgagg 5280
gcctttaccg caagtcagga gctgccaacc ggacacggga attacgccag gcactgcaga 5340
cagaccctgc tgcagttaag ctggaagact tccctatcca cgctatcacc ggggtcctga 5400
agcaatggct tcgtgagctg cctgagccac tcatgacttt tgcccagtat ggagatttcc 5460
tcagggctgt tgagcttcca gagaagcagg agcagctgtc tgccatctat gcagtcctgg 5520
accacctgcc agaagccaac cacacctccc tggagcgact catcttccac cttgtcaaag 5580
tggccctgct tgaagatgtg aaccgcatgt ctccgggagc tctagctatc atctttgcac 5640
cctgcctgct tcgctgccct gacaactccg accccctgac cagcatgaag gatgtactga 5700
agatcaccac gtgtgtggag atgctcatca aagaacagat gaggaagtac aagatgaaga 5760
tggaggaaat caaccacctg gaggctgctg agagcattgc attccgcagg ctctccctgt 5820
tgaggcagaa tgctccgtgg cctctcaaac tggggttttc atcaccctat gagggggtcc 5880
ggatcaaaag ccccaggacc ccagtggtcc aagacctgga gctgggggct ctccccgagg 5940
aggctgcagg tggcgacgag gaccgagaaa aggagattct catggagagg atccagtcca 6000
tcaaggaaga gaaggaggac atcacatatc gactgccgga gctggaccca cggggttctg 6060
atgaggagaa ccttgactca gagacatcgg ccagcactga gagcctgctg gaggagaggg 6120
gcgtgcgggg ggccgtggaa gggccccccg cacctgctct cccctgcccc atttcgccca 6180
ccctgagtcc cctccccgag gccgccgccc ctccacgagg aaggccgaca tccttcgtca 6240
cggtcagagt gaagacacct cggaggaccc ccatcatgcc catggccaat atcaagctcc 6300
ctccgggcct gcccttgcac ctgacaagct gggcacctgc tctccaggag gctgttgttc 6360
cagtgaagcg ccgagagcca cctgcccgca gacaggacca ggtacattcc gtatacatcg 6420
cccctggggc tgacctgcca tcacagagta cactgatagc cctggaccat gataccatac 6480
ttcctgggac caagcgcagg tattcggacc cccctaccta ctgcctgccc cccagctccg 6540
gccaggccaa tggctgagga ccatgactgg cagtctgcat ctcctaacat ccccgaactg 6600
gcatcccagc tgtggagctg gccttcactt tctgagaagg atctagaatg aaaagctccc 6660
aaagggatgc agtggccagc tctgtgtgtt gtggagactg ggagctgctg gccaggagcc 6720
atcagagccc caacctgcac agcagtggct cctttgtcct ttcagtaact gtttctcttt 6780
ttgtggttta cataactttt aagttcataa cagccttaat ggaggaccaa acttttgtat 6840
ttgtatgtct gaacttttat attaactctg cacccttgta acctggacat gggcagggca 6900
agcctgcaaa gtggacatgt gggctacaga tgactgctgc actcactgca tagtggtaga 6960
gagtgaactc acaggacgtc tgtccatctt gaatgcctcc cagcagtagt catctgccca 7020
gagaaccttc cagagattgc tggagctttc cacagctgaa ggcaggacag gagtgggaag 7080
tccctgagtg ccagggaagc ctgaatgtag cttacagctc tgcccactgt gcctgtgaaa 7140
tgcacggagc cagggacttg gaacctttag gaacaatcag tgcatccggt gacagcctgg 7200
gttctttaga ggctggctct cttctcaggc tctacccagt cctggagaca aggaagcccc 7260
acaggaggtg tgaaataaaa gtacttgaga agggtt 7296
Claims (3)
1. Any one of the following 1) to 4):
1) the application of a substance inhibiting the activity of MYO9B protein in the preparation of a product for treating or assisting in treating malignant pleural effusion;
2) the application of a substance inhibiting the activity of MYO9B protein in preparing a product for reducing or decreasing the volume of pleural effusion of a patient with malignant pleural effusion;
3) the application of a substance for inhibiting or silencing MYO9B gene expression in the preparation of a product for treating or assisting in treating malignant pleural effusion;
4) application of a substance for inhibiting or silencing MYO9B gene expression in preparing a product for reducing or decreasing volume of pleural effusion of a patient with malignant pleural effusion.
2. Use according to claim 1, characterized in that: the substance inhibiting the activity of the MYO9B protein is a protein, polypeptide or small molecule compound inhibiting the synthesis of the MYO9B protein, promoting the degradation of the MYO9B protein or inhibiting the function of the MYO9B protein;
or the substance for inhibiting or silencing MYO9B gene expression is a substance for interfering MYO9B gene expression or a substance for knocking MYO9B gene out or a substance for mutating MYO9B gene.
3. Use according to claim 2, characterized in that: the MYO9B gene knockout substance is a CRISPR/Cas9 system; the CRISPR/Cas9 system comprises two sgRNAs which are named sgRNA1 and sgRNA2 respectively; the target sequence recognized by the sgRNA1 is a DNA molecule shown as a sequence 1 in a sequence table; the target sequence recognized by the sgRNA2 is a DNA molecule shown as a sequence 2 in a sequence table; the sgRNA1 is an RNA molecule shown as a sequence 3 in a sequence table; the sgRNA2 is an RNA molecule shown in a sequence 4 in a sequence table.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2019104625419 | 2019-05-30 | ||
| CN201910462541.9A CN110055285A (en) | 2019-05-30 | 2019-05-30 | Target spot MYO9B relevant to malignant pleural effusion and its application |
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| Publication Number | Publication Date |
|---|---|
| CN110283851A CN110283851A (en) | 2019-09-27 |
| CN110283851B true CN110283851B (en) | 2021-12-28 |
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