CN117092337A - Application of LINC01315 coding small peptide - Google Patents

Abstract

The invention belongs to the technical field of biological medicines, and discloses application of LINC01315 coded small peptide. According to the invention, through over-expression or knocking down of LINC01315 coded small peptide in a mouse body, the tumor growth is slow when knocking down, and the tumor growth is fast when over-expression; meanwhile, the neutralizing antibody targeting LINC01315 coding small peptide is injected into a mouse body, so that the growth of tumors can be effectively inhibited, and the LINC01315 coding small peptide is related to eye tumors and can be used as a target point of an eye tumor diagnosis drug or an eye tumor treatment drug.

Description

Use of small peptides encoded by LINC01315

Technical field

The invention belongs to the field of biomedicine technology, and specifically relates to the use of small peptides encoded by LINC01315.

Background technique

Noncoding RNA (ncRNA) plays a variety of regulatory roles in cell migration, proliferation, development and immunity. With the continuous updating of sequencing technology, researchers have discovered that there are open reading frames (ORFs) that can encode small peptides in ncRNAs that do not have coding functions. Long noncoding RNA (LncRNA) is an ncRNA with a length of more than 200 nucleotides. It is a type of functional RNA molecule that has been identified in recent years as having strong potential to encode small peptides. It has been reported that small peptides encoded by LncRNA can be used as markers for colorectal cancer, triple-negative breast cancer, liver cancer, cervical cancer and other cancers, for diagnosis and treatment, or as cancer markers to prepare anti-tumor drugs.

Uveal melanoma (UM) is the most common primary intraocular malignant tumor in adults. Its incidence rate ranks first among intraocular tumors abroad, and second only to retinoblastoma in China. Combination treatment of early-stage, relatively small melanoma, including patch radiation therapy, laser and surgery, can not only prolong life, but also preserve the eyeball and vision. However, once metastasis occurs (to the liver), the median survival time is about 18 months, and the mortality rate is extremely high. Due to limited treatment methods, it seriously affects the quality of life of patients and imposes a serious burden on the economy and society.

With the successful launch of tumor immunomodulatory drugs targeting the linear antigen gp100, the treatment of uveal melanoma has achieved revolutionary success. However, due to the large number of immunosuppressive cells infiltrating in tumor tissues, the clinical response rate to tumor immunotherapy in patients with different types of tumors is still at a very low level. There are many reasons that limit the clinical response rate of tumor immunotherapy, including the number of killer T cells infiltrating in the tumor tissue, the presence of a large number of immunosuppressive factors such as TGFβ, IDO, and adenosine in the tumor microenvironment, and various immunosuppressive factors including Treg, MDSC, etc. Inhibit cell infiltration in tumor tissue.

Therefore, by identifying and developing monoclonal targeted therapeutic antibodies that can specifically target new targets and eliminate cancer cells in tumor tissues, it is expected to improve the tumor microenvironment, increase the clinical response rate of tumor patients to therapeutic drugs, and further extend the Survival goals for cancer patients.

The U.S. Food and Drug Administration (FDA) has approved only a novel T-cell receptor (TCR) bispecific immune antibody (tebentafusp) therapy for the treatment of HLA-A*02:01-positive metastatic uveal melanoma (mUM ) adult patients. Although tebentafusp monotherapy has demonstrated a clinically and statistically significant survival advantage: significantly extending overall survival (OS) and reducing the risk of death by 49%, it is hampered by its need to recruit native circulating T cells. There are limitations, and the curative effect may be different for people with different autoimmune abilities.

Therefore, it is desirable to generate soluble novel mechanism-of-action antibodies that can act specifically on UM, which antibodies are expected to have the highest affinity for their targets and thus treat UM more effectively and efficiently.

Contents of the invention

In view of the above-mentioned shortcomings of the prior art, the purpose of the present invention is to provide the use of small peptides encoded by LINC01315, in order to solve the problems existing in the prior art.

In order to achieve the above object, the present invention specifically adopts the following technical solutions.

The first aspect of the present invention protects the use of the small peptide encoded by LINC01315 as a target in the preparation or screening of ocular tumor diagnostic drugs or therapeutic drugs.

The second aspect of the present invention protects the use of inhibitors of small peptides encoded by LINC01315 in the preparation of drugs for the prevention and/or treatment of ocular tumors.

In certain embodiments, the nucleotide sequence of LINC01315 encoding a small peptide includes the sequence shown in SEQ ID No. 1.

In certain embodiments, the inhibitor refers to a substance that can inhibit the transcription or translation of the gene encoding the small peptide LINC01315, or that can inhibit the expression or activity of the small peptide encoded by LINC01315.

Preferably, the inhibitor is selected from nucleic acid molecules, small molecule chemicals, antibody drugs, polypeptides, proteins, interfering nucleic acid constructs, interfering lentiviruses, and adeno-associated viruses.

More preferably, the nucleic acid molecule is selected from one or more of siRNA, dsRNA, miRNA and shRNA.

More preferably, the inhibitor is a monoclonal antibody against a small peptide encoded by LINC01315.

In certain embodiments, the ocular tumor is selected from one or more of uveal melanoma, retinoblastoma, and conjunctival melanoma.

The third aspect of the present invention protects an shRNA encoding a small peptide of LINC01315, and the target sequence of the shRNA includes the sequence shown in SEQ ID No. 19.

The fourth aspect of the present invention protects a monoclonal antibody against a small peptide encoded by LINC01315, including a light chain and a heavy chain. The variable region of the heavy chain includes HCDR1, HCDR2 and HCDR3, and the variable region of the light chain includes LCDR1, LCDR2 and LCDR3,

The sequence of HCDR1 includes the amino acid sequence shown in SEQ ID No. 2;

The sequence of HCDR2 includes the amino acid sequence shown in SEQ ID No. 3;

The sequence of HCDR3 includes the amino acid sequence LPDY;

The sequence of LCDR1 includes the amino acid sequence shown in SEQ ID No. 4;

The sequence of LCDR2 includes the amino acid sequence shown in SEQ ID No. 5;

The sequence of LCDR3 includes the amino acid sequence shown in SEQ ID No. 6.

In certain embodiments, the amino acid sequence of the variable region of the heavy chain includes the sequence shown in SEQ ID No. 7.

In certain embodiments, the amino acid sequence of the variable region of the light chain includes the sequence shown in SEQ ID No. 8.

The fifth aspect of the present invention protects biological materials related to shRNA as described above or monoclonal antibodies as described above, and the biological materials include one or more of the following:

1) Nucleotide encoding the shRNA as described above or the monoclonal antibody as described above;

2) A recombinant expression vector containing the nucleotide described in 1);

3) Bioengineering bacteria containing the nucleotide described in 1), or bioengineering bacteria containing the recombinant expression vector described in 2).

The sixth aspect of the present invention protects the use of shRNA as described above or monoclonal antibody as described above or biological material as described above in the preparation of medicaments for preventing and/or treating ocular tumors.

The seventh aspect of the present invention protects a drug for preventing and/or treating ocular tumors, which drug includes an inhibitor of a small peptide encoded by LINC01315, and pharmaceutically acceptable excipients.

The eighth aspect of the present invention protects and detects a method for screening drugs for the prevention and/or treatment of ocular tumors. The method includes: taking the small peptide encoded by LINC01315 as a drug target and looking for the expression of the small peptide encoded by LINC01315. and/or functional substances as drug candidates.

In certain embodiments, the method includes: applying the candidate drug to the cells in vitro, and detecting the content of the small peptide encoded by LINC01315 in the cells after co-culture.

Compared with the prior art, the present invention has the following beneficial effects:

1) The small peptide encoded by LINC01315 in this application can be abundantly expressed in uveal melanoma cells and is an effective specific target.

2) LINC01315 encodes a small peptide that can promote the occurrence and development of uveal melanoma, a member of the key YAP signaling pathway, and is effective against most uveal melanomas.

3) Mouse tumor-bearing experiments have demonstrated that the use of neutralizing antibodies to neutralize the small peptide encoded by LINC01315 has a significant inhibitory effect on tumor growth, making it an ideal candidate for therapeutic applications.

Description of the drawings

Figure 1A shows a structural analysis diagram of LINC01315 proteins from different species in Example 1 of the present application.

Figure 1B shows a schematic diagram of the ORF-4xHA and ORFmut-4xHA fusion proteins constructed in Example 1 of the present application.

Figure 1C shows the protein electrophoresis patterns of ORF-4xHA and ORFmut-4xHA in Example 1 of the present application.

Figure 1D shows a schematic diagram of constructing GFPwt, GFPmut, ORF-GFPmut and ORFmut-GFPmut fusion proteins in Example 1 of the present application.

Figure 1E shows the immunofluorescence results of GFPwt, GFPmut, ORF-GFPmut and ORFmut-GFPmut in Example 1 of the present application.

FIG. 1F shows laser confocal microscopy images of GFPwt, GFPmut, ORF-GFPmut and ORFmut-GFPmut in Example 1 of the present application.

Figure 2 shows a diagram of mice overexpressing ORF or inhibiting ORF in Example 3 of the present application.

Figure 3 shows the results of tumor growth after overexpressing ORF or inhibiting ORF in Example 3 of the present application.

Figure 4 shows the effect of monoclonal neutralizing antibodies targeting ORF in treating tumor growth in Example 4 of the present application.

Detailed ways

This application analyzed LINC01315 from different species and found that it contains an open reading frame ORF (LINC01315-ORF) with coding potential. This coding small peptide is highly conserved in primates, so it is predicted to be an effective specific Sexually targeted eye tumors, and then through electrophoresis, immunofluorescence, and western blot detection, it was found that the encoded small peptide can be expressed. It was found by overexpressing or knocking down LINC01315-ORF in mice, and knocking down LINC01315-ORF. Tumors grow slowly, but overexpression of LINC01315-ORF causes tumors to grow rapidly; in addition, by injecting neutralizing antibodies targeting LINC01315-ORF into mice, it was found that it can effectively inhibit tumor growth. Studies have proven that LINC01315-ORF is related to ocular tumors and can be used as a target for ocular tumor diagnostic drugs or ocular tumor therapeutic drugs. On this basis, the present invention was completed.

The first aspect of the present invention protects the use of the small peptide encoded by LINC01315 as a target in the preparation or screening of ocular tumor diagnostic drugs or ocular tumor treatment drugs.

In the use of the present invention, the nucleotide sequence encoding the small peptide of LINC01315 includes the sequence shown in SEQ ID No. 1.

The drug uses the small peptide encoded by LINC01315 as the target of the drug.

The drug can inhibit or block the expression and/or function of the small peptide encoded by LINC01315.

The second aspect of the present invention protects the use of inhibitors of small peptides encoded by LINC01315 in the preparation of drugs for the prevention and/or treatment of ocular tumors.

In the use of the present invention, the nucleotide sequence encoding the small peptide of LINC01315 includes the sequence shown in SEQ ID No. 1. In the present invention, the LINC01315-encoded small peptide may be the encoded small peptide or LINC01315-ORF or ORF.

The LINC01315-ORF sequence is shown below:

ATGGTGCAGGCGGGACCCTCGAGCTGCAGCATCTCCGGTGACCCGGGGTTGCCGAGGAGGTGGAGACCAGCACAGGTGGTCCGGCCCGGGCGCCTCCGAATCCGGGGGTGGTCAAGACGGATCCCCAAGGCTGAGGTCGGCAGTCCCGGGGACTCGCAGCTGTTGAGCCTGTGGAGACGCGGCCCCGTGACCGAGGCACCCTTCAGCAACCCGGGGGCAGCGTTTTCCCCCTACCGGAAATCTGATGGGCTTATGACAT CATGGCTGGCTGCTGAGCGA(SEQ ID NO.1)

In the use of the present invention, the ocular tumor is selected from one or more of uveal melanoma, retinoblastoma and conjunctival melanoma. Preferably, it is uveal melanoma (UM).

In the use of the present invention, the drug uses the small peptide encoded by LINC01315 as the drug target.

In the use of the present invention, the drug can inhibit or block the expression and/or function of the small peptide encoded by LINC01315.

In the use of the present invention, the drug is a pharmaceutical preparation made of an inhibitor of the small peptide encoded by LINC01315 and pharmaceutical excipients.

In the use of the present invention, the inhibitor is a single active ingredient or a combination of several ingredients. In a specific embodiment, the use of the inhibitor in the preparation of drugs for the prevention and/or treatment of ocular tumors specifically refers to: using the inhibitor as the main active ingredient of the drug for the preparation of drugs for the prevention and/or treatment of ocular tumors. Cancer drugs.

In the use of the present invention, the inhibitor refers to a substance that can inhibit the transcription or translation of the gene encoding the small peptide LINC01315, or a substance that can inhibit the expression or activity of the small peptide encoded by LINC01315. For example, the substances include but are not limited to: nucleic acid molecules, small molecule chemicals, antibody drugs, polypeptides, proteins, interfering nucleic acid constructs, interfering lentiviruses, and adeno-associated viruses. The nucleic acid molecules include but are not limited to: antisense oligonucleotides, double-stranded RNA (dsRNA), ribozymes, substances for knocking out or knocking down the expression of small peptides encoded by LINC01315, and small peptides prepared by endoribonuclease III. Interfering RNA or short hairpin RNA (shRNA).

Preferably, the shRNA includes a sense strand fragment and an antisense strand fragment, and a stem-loop structure connecting the sense strand fragment and the antisense strand fragment, the sequences of the sense strand fragment and the antisense strand fragment are complementary, and The target sequence contained in the sense strand fragment and the antisense strand fragment is consistent with the sequence of LINC01315 encoding small peptide gene. The shRNA can be processed into small interfering RNA (siRNA) to specifically silence the expression of the LINC01315-encoding small peptide gene. The target sequence of the shRNA includes the sequence shown in SEQ ID No. 19, and the coding sequence of the shRNA includes the base sequence shown in SEQ ID NO. 17 or SEQ ID NO. 18.

The bold underlined part is the target

The bold underlined part is the target

More preferably, the coding sequence of the stem-loop structure of the shRNA can be selected from any of the following: UUCAAGAGA, AUG, CCC, UUCG, CCACC, CTCGAG, AAGCUU and CCACACC.

The interference nucleic acid construct contains a gene fragment encoding the shRNA described above and can express the shRNA. The interference nucleic acid construct can load the shRNA fragment into a known lentivirus vector. After the interference lentivirus is packaged into infectious virus particles, it infects HEK-293T and then transcribes the shRNA of the present invention. Through steps such as enzyme digestion and processing, it is used to specifically silence the expression of LINC01315 encoding small peptide genes. The known lentiviral vector may be selected from pLKO.1-TRC.

The interference lentivirus refers to the interference nucleic acid construct being packaged by viruses with the assistance of lentivirus packaging plasmids and cell lines. The interfering lentivirus can infect tumor cells and produce small interfering RNA targeting the small peptide gene encoding LINC01315, thereby inhibiting the proliferation of tumor cells. The interfering lentivirus can be used to prepare drugs for preventing or treating eye tumors.

The antibody drug is a monoclonal antibody against a small peptide encoded by LINC01315. Including a light chain and a heavy chain, the variable region of the heavy chain includes HCDR1, HCDR2 and HCDR3, and the variable region of the light chain includes LCDR1, LCDR2 and LCDR3,

The sequence of HCDR1 includes the amino acid sequence shown in SEQ ID No. 2;

The sequence of HCDR2 includes the amino acid sequence shown in SEQ ID No. 3;

The sequence of HCDR3 includes the amino acid sequence LPDY;

The sequence of LCDR1 includes the amino acid sequence shown in SEQ ID No. 4;

The sequence of LCDR2 includes the amino acid sequence shown in SEQ ID No. 5;

The sequence of LCDR3 includes the amino acid sequence shown in SEQ ID No. 6.

The third aspect of the present invention protects an shRNA encoding a small peptide of LINC01315, and the target sequence of the shRNA includes the sequence shown in SEQ ID No. 19.

AGGCGTGAAGTCAGGAGAATT( SEQ ID NO.19 )

The fourth aspect of the present invention protects an anti-LINC01315 encoded small peptide-binding monoclonal antibody, including a light chain and a heavy chain. The variable region of the heavy chain includes HCDR1, HCDR2 and HCDR3. The variable region of the light chain Including LCDR1, LCDR2 and LCDR3,

The sequence of HCDR1 includes the amino acid sequence shown in SEQ ID No. 2;

The sequence of HCDR2 includes the amino acid sequence shown in SEQ ID No. 3;

The sequence of HCDR3 includes the amino acid sequence LPDY;

The sequence of LCDR1 includes the amino acid sequence shown in SEQ ID No. 4;

The sequence of LCDR2 includes the amino acid sequence shown in SEQ ID No. 5;

The sequence of LCDR3 includes the amino acid sequence shown in SEQ ID No. 6.

Table 1

sequence serial number HCDR1 SDYAWN SEQ ID No.2 HCDR2 YISYSGSTSYNPSLKS SEQ ID No.3 HCDR3 LPDY LCDR1 KASQDVITAVA SEQ ID No.4 LCDR2 SASYRYT SEQ ID No.5 LCDR3 QQHYSIPYT SEQ ID No.6

In certain embodiments, the variable region of the heavy chain further includes the framework regions FR1-FR4 of the sequence SEQ ID No. 9-SEQ ID No. 12, and the variable region of the light chain further includes the sequence SEQ ID No. 9-SEQ ID No. 12. ID No. 13 - Framework regions FR1 to FR4 of SEQ ID No. 16.

Table 2

In certain embodiments, the amino acid sequence of the variable region of the heavy chain includes the sequence shown in SEQ ID No. 7.

In certain embodiments, the amino acid sequence of the variable region of the light chain includes the sequence shown in SEQ ID No. 8.

Variable region of heavy chain:

MRVLILLWLFTAFPGILSDVQLQESGPGLVKPSQSLSLTCTVTGYSITSDYAWNWIRQF PGNKLEWMGYISYSGSTSYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYYCAILPDYW GQGTTLTVSS(SEQ IDNo.7)

Variable region of light chain:

MESQIQVFVFVFLWLSGVDGDIVMTQSHKFMSTSVGDRVSITCKASQDVITAVAWYQ QKPGQSPKLLIYSASYRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYYCQQHYSIPYTFG GGTKLEIK(SEQ ID No.8)

The preparation method of the anti-LINC01315-encoded small peptide-binding monoclonal antibody provided by the invention includes the following steps: cultivating a monoclonal antibody expression plasmid or a monoclonal antibody-encoding gene under conditions suitable for expressing the monoclonal antibody. Host cells are used to express the monoclonal antibody, and the monoclonal antibody is purified and isolated.

After obtaining the nucleic acid sequence encoding the monoclonal antibody of the present invention, the antibody of interest can be prepared and produced according to the following method. For example, a vector containing a nucleic acid encoding a target monoclonal antibody is directly introduced into a host cell, and the cells are cultured under appropriate conditions, thereby inducing the expression of the encoded monoclonal antibody. The expression vectors and host cells used in the present invention are existing technologies and can be directly obtained through commercial channels. The 10% FBS 1640 culture medium used in culture is also a variety of conventional mammalian cell culture media. Those skilled in the art can Select the appropriate 1640 medium based on experience and culture it under conditions suitable for the growth of host cells. After the host cells grow to an appropriate cell density, the selected promoter is induced using an appropriate method (such as temperature shift or chemical induction), and the cells are cultured for a further period of time. The recombinant polypeptide in the above method can be expressed within the cell, on the cell membrane, or secreted outside the cell. Once the monoclonal antibody of the present invention is obtained, its physical, chemical and other properties can be used to isolate and purify the monoclonal antibody through various separation methods. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitating agents (salting out method), centrifugation, osmotic sterilization, ultratreatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.

The fifth aspect of the present invention protects biological materials related to shRNA as described above or monoclonal antibodies as described above, and the biological materials include one or more of the following:

1) Nucleotides encoding shRNA as described above or monoclonal antibodies as described above;

2) A recombinant expression vector containing the nucleotide described in 1);

3) Bioengineering bacteria containing the nucleotide described in 1), or bioengineering bacteria containing the recombinant expression vector described in 2).

The recombinant expression vector can usually be constructed by inserting the nucleotide into a suitable vector, and those skilled in the art can select a suitable expression vector. For example, the types of vectors may include, but are not limited to, plasmids, phagemids, phage derivatives, animal viruses, cosmids, and the like. For another example, it is any one of a lentiviral vector, a retroviral vector, or an adeno-associated virus vector. Bioengineered bacteria contain the recombinant expression vector as described above or the polynucleotide as described above is integrated into the genome, so that the monoclonal antibody can be expressed.

The sixth aspect of the present invention protects the use of shRNA as described above or monoclonal antibody as described above or biological material as described above in the preparation of medicaments for preventing and/or treating ocular tumors.

The seventh aspect of the present invention protects a drug for preventing and/or treating ocular tumors, which drug includes an inhibitor of a small peptide encoded by LINC01315, and pharmaceutically acceptable excipients.

Among the drugs described in this application, the inhibitor is used alone or in combination with other drugs. That is, the inhibitor can be the only active ingredient or one of the active ingredients of the drug.

In the medicines described in this application, the pharmaceutically acceptable excipients mean that they will not produce adverse, allergic or other adverse reactions when the medicine is properly administered to animals or humans. The pharmaceutically acceptable excipients should be compatible with the inhibitor, that is, capable of being blended therewith without substantially reducing the effectiveness of the inhibitor under normal circumstances. The pharmaceutically acceptable excipients are selected from one or more of carriers, diluents, adhesives, lubricants and wetting agents. Specific examples of some substances that may serve as pharmaceutically acceptable carriers, diluents, binders, lubricants and wetting agents are sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium methylcellulose, ethylcellulose and methylcellulose; tragacanth powder; malt; gelatin; talc; solid lubricants, such as stearic acid and magnesium stearate; calcium sulfate ; Vegetable oils, such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and cocoa butter; Polyols, such as propylene glycol, glycerin, sorbitol, mannitol and polyethylene glycol; Alginic acid; Emulsifiers, such as Tween; Moisturizing agents Wetting agents, such as sodium lauryl sulfate; colorants; flavoring agents; tableting agents, stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic saline solutions; and phosphate buffers, etc. These substances are used as needed to aid the stability of the formulation or to help enhance the activity or its bioavailability or to produce an acceptable mouthfeel or odor in the case of oral administration.

Among the medicines described in this application, the medicine is one or more of solutions, injections, sprays, nasal drops, aerosols, powder sprays, tablets, capsules and granules. The above-mentioned pharmaceutical compositions in various dosage forms can be prepared according to conventional methods in the pharmaceutical field. Injections are preferred.

Among the drugs described in this application, the medicine can be introduced into the body through injection, spraying, nasal instillation, eye instillation, penetration, absorption, physical or chemical mediated methods, such as muscle, intradermal, subcutaneous, vein, mucosal tissue; or Mixed or wrapped with other substances and then introduced into the body. Preferably, administration is by injection. The pharmaceutical composition can also be used in combination with other treatment methods, including surgery, radiotherapy, chemotherapy, and targeted therapy.

The eighth aspect of the present invention protects a method for screening drugs for the prevention and/or treatment of ocular tumors. The method includes: taking the small peptide encoded by LINC01315 as a drug target, and searching for drugs that can inhibit or block the expression of the small peptide encoded by LINC01315 and /or functional substances as drug candidates.

In the method of the present invention, the method includes: applying the candidate drug to the cells in vitro, and detecting the content of the small peptide encoded by LINC01315 in the cells after co-culture. The cells may be from mammals.

Experimenters can determine whether the drug is of therapeutic significance by detecting the content of the small peptide encoded by LINC01315 after co-culture. Generally speaking, drugs that can reduce the content of small peptides encoded by LINC01315 by 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% respectively compared with the control group can be judged as Therapeutic drugs.

Furthermore, if the drug can reduce the small peptide encoded by LINC01315 in cells by at least 50%, it is determined to be a drug of therapeutic significance.

The present invention further provides a method for treating or preventing ocular tumors, which includes administering an effective amount of an inhibitor of the small peptide encoded by LINC01315 to a subject in need thereof (eg, a mammal). The method may also be in vitro or non-therapeutic.

The subject or individual for therapeutic or preventive treatment is preferably a mammal, such as but not limited to humans, primates, livestock (such as sheep, cattle, horses, donkeys, pigs), pets (such as dogs, cats), laboratory experiments Animals (such as mice, rabbits, rats, guinea pigs, hamsters) or captured wild animals (such as foxes, deer). The subject is preferably a primate. The subject is most preferably a human. The subject may be a patient with an ocular tumor or an individual in whom prevention of an ocular tumor is desired.

The small peptide inhibitor or drug encoded by LINC01315 can be administered to the subject before, during or after receiving anti-cancer treatment.

Through genome pathogenic analysis in the current technology, more than 95% of uveal melanoma patients carry gain-of-function mutations in GNAQ/11. Previous studies on the mechanism have found that GNAQ or GNA11 mutations can abnormally activate the YAP signaling pathway and promote tumors. For cell growth, about 83% of GNAQ or GNA11 mutations can activate YAP and induce the proliferation of cancer cells. Knocking down or inhibiting YAP activity can alleviate the malignant phenotype of tumors. Therefore, excessive activation of the YAP signaling pathway is the cause of uveal melanoma.

This application found that the ORF can bind GNAQ or GNA11 and import it into the nucleus, and the interaction with the mutant results in more nuclear import. After entering the nucleus, mutant GNAQ or GNA11 can inhibit the transcription of PRP4K and mediate the interaction between ORF and PRP4K, thereby regulating the phosphorylation of YAP in the nucleus and promoting the growth of tumor cells. Using uveal melanoma as a model, the present invention confirms the regulatory effect of ORF on the YAP signaling pathway, provides a new target for the development of inhibitors that interfere with the YAP signaling pathway, and illustrates the great potential of ORF as a new drug.

In addition, the present invention designs a specific monoclonal antibody that is effective against the small peptide encoded by LINC01315 and uses intratumoral injection to endogenously block the regulation of the YAP signaling pathway by ORF, and ultimately can specifically target and eliminate tumor tissue. cancer cell.

Taken together, the present invention clarifies the new pathogenic mechanism of uveal melanoma, and provides a new theoretical basis for disease diagnosis and prognosis judgment; in terms of economic benefits, ORF has great pharmaceutical value in treating diseases caused by excessive activation of YAP signaling. , forming a broad market prospect for the development of new drugs.

The implementation of the present invention is described below with specific embodiments. Those familiar with this technology can easily understand other advantages and effects of the present invention from the content disclosed in this specification.

Before further describing the specific embodiments of the present invention, it should be understood that the protection scope of the present invention is not limited to the following specific specific embodiments; it should also be understood that the terms used in the embodiments of the present invention are for describing specific specific embodiments, It is not intended to limit the scope of the present invention. Test methods without specifying specific conditions in the following examples usually follow conventional conditions or conditions recommended by each manufacturer.

When the examples give numerical ranges, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints can be selected. 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. In addition to the specific methods, equipment, and materials used in the embodiments, those skilled in the art can also use methods, equipment, and materials described in the embodiments of the present invention based on their understanding of the prior art and the description of the present invention. Any methods, equipment and materials similar or equivalent to those in the prior art may be used to implement the present invention.

In the following examples of this application, the reagent information used is: cloning point mutation kit (Cat#C214, Vazyme), homologous recombination kit (Cat#C115, Vazyme), Western and IP lysis solution (Cat#P0013, Beyotime), Lipofectamine2000 transfection reagent (Cat#11668030, ThermoFisher), recombinant plasmid DNA: pCS2+ (http://www.rzpd.de/info/vectors/pCS2plus_pic.shtml), competent cells DH5α (Cat#CB101, TIANGEN )

Cell culture reagents: PBS (Cat#70011069) and Opti-MEM (Cat#31985070) were purchased from Gibco.

Antibodies: Anti-GFP(Cat#AE012,ABclonal), Anti-HA(Cat#30701ES20,Yeasen), Anti-GAPDH(Cat#30201ES20,Yeasen), Anti-Flag(Cat#14793S,CST), Anti-mouse IgG ,HRP-linked Antibody(Cat#7076,CST)、Anti-rabbit IgG,HRP-linked Antibody(Cat#7074,CST)

Example 1

In this example, the small peptide encoded by LINC01315 is obtained, and the protein expression is verified through experiments, including the following steps:

1.1. Gene structure analysis

The online databases LncRNAdisease2.0 (http://www.rnanut.net/lncRNAdisease/) and NCBI (https://www.ncbi.nlm.nih.gov/) were used to analyze lncRNAs related to ocular tumor diseases, and then used Coding ability prediction database (http://regrna2.mbc.nctu.edu.tw/) analysis locked long non-coding chain LINC01315.

Using POSTAR3 (http://postar.ncrnalab.org) and GWIPSviz (https://gwips.ucc.ie/index.html) analysis, it was found that there is a region with high coding potential in LINC01315, the open reading frame LINC01315-ORF of LINC01315. The nucleotide sequence is shown in SEQ ID No: 1.

At the same time, the LINC01315 protein structure analysis of humans (homo sapiens), gorilla (gorilla), lizard (Piliocolobustephrosceles) and papaya (Papio Anubiscollagen alpha-2(IX) chain-like) was analyzed. The results are shown in Figure 1A.

As can be seen from Figure 1A, LINC01315 contains an open reading frame ORF with coding potential, which is highly conserved in primates. Therefore, this sequence is predicted to be an effective specific target.

1.2. Construction of eukaryotic expression vector pCS2-ORF-GFP/4xHA

1) LINC01315-ORF sequence amplification

Design the primers ORF-F and ORF-R to amplify the LINC01315-ORF sequence online through https://crm.vazyme.com/cetool/multipoint.html, use the LINC01315-ORF sequence as a template, perform a PCR amplification reaction, and obtain PCR product.

The sequences of primers ORF-F and ORF-R are as follows:

ORF-F: CGTTTAAACGGGCCCTCTAGAATGGTGCAGGCGGGACCC (SEQ ID NO.20)

ORF-R:CTCGTCGCTCTCAATTCTAGATCGCTCAGCAGCCAGCCA(SEQ ID NO.21)

The PCR amplification system is shown in Table 1.

Table 1

The PCR reaction procedures are shown in Table 2.

Table 2

step Temperature(℃) time Predenaturation 98 30s transsexual 98 15s annealing 60 15s extend 72 2min 30 cycles last extension 72 5min final stage 4

The amplified product was double digested with XbaI and EcoRI to obtain the PCR product.

2) Enzyme digestion vector

According to the specificity of the pCS2-GFP/4xHA ((http://www.rzpd.de/info/vectors/pCS2plus_pic.shtml) backbone enzyme cleavage site, XbaI and EcoRI were selected for double enzyme digestion. The enzyme digestion method was the same as above to obtain the enzyme. Cut the skeleton.

3) Purification

The PCR product in step 1) and the enzyme digestion skeleton in step 2) are purified to obtain the purified PCR product and the purified P enzyme digestion skeleton respectively.

4)Connect

Recombine the purified enzyme digestion backbone and purified PCR product in step 3) with a homologous recombination kit (Cat#C115, Vazyme) to construct a recombinant DNA plasmid.

5) Screen positive clone strains and sequence them

Transform the recombinant DNA plasmid obtained in step 4) into the DH5α (Cat#CB101, TIANGEN) strain, plate it on an LB plate (containing 50 mg/L cananamycin), and culture it overnight at 37°C.

PCR was performed on the overnight colonies to identify positive clones. The positive clones were shaken, and the plasmids were extracted and verified to be correct by sequencing. The plasmids were extracted, DNA was quantified, and frozen at -20°C. The correct recombinant DNA plasmid is labeled pCS2-ORF-GFP/4xHA (abbreviated as ORF-HA).

At the same time, the ORFmut-F primer was used to mutate the pCS2-ORF-GFP/4xHA plasmid. Clone point mutation kit (Cat#C214, Vazyme) was used for mutation.

ORFmut-F:TGCGAGAAAAGCCTTGTTTAAACTTGGTGCAGGCGGGACCC(SEQ ID NO.22)

The plasmid after ORFmut mutation is pCS2-ORFmut-GFP/4xHA (abbreviated as ORFmut-4xHA).

At the same time, pCS2-GFP/4xHA was used as a control group (Vehicle).

pCS2-GFP/4xHA, labeled GFPwt.

pCS2-GFP/4xHA was mutated into GFPmut using a cloning point mutation kit (Cat#C214, Vazyme).

pCS2-ORF-GFP (ORF-HA) was mutated into ORF-GFPmut using a cloning point mutation kit (Cat#C214, Vazyme).

ORF-GFPmut was mutated using a cloning point mutation kit (Cat#C214, Vazyme).

The electropherogram results are shown in Figure 1C.

As can be seen from Figure 1, all ORFs are expressed.

1.3. Expression and purification of expression vector

1) Plate HEK293T cells in a 6-well plate at 10 ⁵ cells per well, repeat 3 wells for each group, and culture overnight in a 37°C 5% CO ₂ incubator. When the cell confluence reaches more than 80%, follow Table 3 and Table 4 to prepare the system. pCS2-ORF-GFP/4xHA, pCS2-ORFmut-GFP/4xHA and pCS2-GFP/4xHA were transfected into HEK293T cells using Lipofectamine2000 transfection reagent (Cat#11668030, ThermoFisher).

table 3

Element Dosage pCS2-ORF-GFP/4xHA or pCS2-ORFmut-GFP/4xHA or pCS2-GFP/4xHA 2μg Opti-MEM 50μL

Table 4

Element Dosage Lipofectamine2000 5μL Opti-MEM 50μL

Mix the systems in Table 3 and Table 4 and let stand at room temperature for more than 5 minutes. Add evenly to the HEK293T cell culture supernatant, shake gently, and continue culturing in a constant-temperature cell incubator for 6-8 hours. Then replace with fresh medium and continue culturing for 24 hours. Hour.

1.4. Activity detection

Collect 10 ⁷ cells obtained in step 1.3, add 1 mL IP lysis buffer (Cat#P0013, Beyotime), pipette evenly, place on ice for 40 minutes, vortex every 10 minutes for 15 seconds, after full lysis, centrifuge at 12000 rpm for 3 minutes at 4°C, and transfer to Pour into a clean 1.5mL centrifuge tube, add 5×SDS Loading buffer, mix by pipetting, boil at 100°C for 10 minutes, mix lightly, cool to room temperature, and then use 4-20% Bis-Tris acrylamide gels to precast the gel. (Cat#M00657, Genscript) and Tris-MOPS-SDS Running Buffer (Cat#AWB0076a, Abiowell) electrophoresis buffer for protein separation. After protein gel electrophoresis is completed, Western blot processing is performed. Among them, the composition of 5×SDSLoading buffer is: 250mM Tir-HCL (pH=6.8), 10w/v% SDS, 0.5w/v% BPB, 50w/v% glycerol and 5w/v% β-mercaptoethanol.

Using the classic "sandwich" wet transfer method, the separated proteins were transferred to a 0.22 μm PVDF membrane at 300 mA for 150 min. The PVDF membrane was blocked with 5% skim milk at room temperature for 1 hour, then transferred to milk diluted with the primary antibody, shaken at 4°C overnight, and then washed three times with TBST buffer (8gNaCl, 20mM Tris-HCl, 0.1% Tween20, pH8.0) , then incubate the diluted secondary antibody solution at room temperature for 2 hours, wash the membrane with TBST buffer and treat it with ECL developer, and finally collect the chemiluminescence signal. The image results were analyzed using Fiji software. The results are shown in Figure 2.

As can be seen from Figure 1E, fluorescence signals can be observed in pCS2-GFP/4xHA (GFPwt); after mutating the fluorescent protein in pCS2-GFP/4xHA (GFPmut), no fluorescence signal is observed; in pCS2-ORF-GFP/4xHA ( After the ORF in (ORF-HA) is mutated (ORF-GFPmut), a fluorescent signal can be observed; after further mutating the ORF in (ORF-GFPmut) (ORFmut-mut), no fluorescent signal is observed, indicating that the ORF can Translation, expression.

At the same time, the obtained protein was electrophoresed, and the results are shown in Figure 1F.

As can be seen from Figure 1F, the ORF-GFPmut protein is expressed in large quantities.

Schematic diagrams of fusion proteins are shown in Figure 1B and Figure 1D.

As can be seen from Figures 1C, 1E and 1F, ORF-4xHA can be stably expressed in eukaryotes.

Example 2

In this example, preparing a monoclonal neutralizing antibody targeting ORF specifically includes the following steps:

2.1. Animal immunity

2.1. Immunization: Rapid immunization protocol, using target protein to immunize 3 Balb/c mice. The sequence of the target protein is MHHHHHHVQAGPSSCSISGDPGLPRRWRPAQVVRPGRLRIRGWSRRIPKAEVGSPGDSQLL SLWRRGPVTEAPFSNPGAAFSPYRKSDGLMTSWLAAER (SEQ ID NO. 23)

2.1.2. Blood collection and testing: 7 days after immunization, use the indirect ELISA method to detect the serum of immunized animals to determine the level of immune response.

After the immunization is completed, the immunized animals can reach the immune response level against the immunogen (OD value >1.0, titer reaches 1:8,000).

2.2. Cell fusion and screening

2.2.1. Cell fusion and plating: Select 2 mice for fusion, and use electrofusion method to perform one round of cell fusion. The average fusion efficiency is that approximately 2,500 spleen cells can be fused to produce one hybridoma cell, so the number of hybridoma cells is expected to reach approximately 2×10 ⁴ . All cells in each round of fusion will be plated into a 96-well plate, with a maximum of 30 96-well plates.

2.2.2. Preliminary screening: Use the indirect ELISA method to screen the supernatant of the fused cells and select the supernatant that is positive for the target protein.

2.2.3. Confirmation screening: For all positive clones obtained in the preliminary screening stage, use the indirect ELISA method to screen all positive mother clone cell supernatants, and those that are positive for the target protein and negative for His.

2.2.4. Clone expansion culture and cryopreservation: Transfer the positive mother clone cells to a 24-well plate for expansion culture, and collect 2 mL of supernatant from each expanded culture clone for indirect ELISA detection. Cryopreserve all specifically positive clonal cells to avoid clonal loss.

2.3. Subcloning, expanded culture and cryopreservation

2.3.1. Decision point: Select 5 clones for subcloning: 12D2, 3C4, 9E4, 14F3, 4B1, backup: 14G2, 15D1.

2.3.2. Subcloning: Use the limiting dilution method to subclone the positive mother clones to ensure that these positive mother clones are derived from a single mother clone cell. A maximum of 3 rounds of subcloning were performed. The general success rate of subcloning is 80%. Possible loss of positivity occurs during the subclonal stage. (If the obtained positive mother clone cannot meet the specificity requirements, the alternative mother clone will be used for re-subcloning).

2.3.3. Subclone screening: Use indirect ELISA for subclone screening.

2.3.4. Cryopreservation of monoclonal cells: Select 2 stable daughter clones for each mother clone for amplification and liquid nitrogen storage. Collect 5 mL of supernatant from each sub-clone before freezing in liquid nitrogen. Provide 2 tubes of cells per subclone.

2.4. Antibody production

2.4.1. Antibody production: Monoclonal antibody production, 10-15mg/strain.

2.4.2. Antibody purification: Use Protein A affinity chromatography to purify the antibody, and use dialysis to store the purified antibody in phosphate buffer saline (PBS).

2.4.3. Quality control (QC): Use polyacrylamide gel electrophoresis (SDS-PAGE) and indirect ELISA for QC detection, and measure the antibody concentration on NanoDrop2000.

2.5. Detection

The Elisa method was used to detect the reactivity of monoclonal antibodies 12D2, 3C4, 9E4, 14F3, and 4B1 with the antigen respectively. The antigen was DKT69 (small peptide antigen).

Screen the hybridoma culture fluid obtained in step 2.3 by enzyme-linked immunoassay (Elisa) to form a sample to be tested. Coat a 96-well enzyme plate (1 μg/ml, 100 μL/well) with Antigen A (DKT69) and Antigen B (His-tagged unrelated protein), and incubate at 4°C overnight; add PBS (phosphate buffer, pH 7.4), Block for 1 hour at 37°C; add the sample to be detected, and incubate at 37°C for 2 hours; then add the secondary antibody, incubate for 1 hour, and finally read the absorbance value OD450nm. The results are shown in Table 5. The secondary antibody was peroxidase-labeled goat anti-mouse IgG, Fcγ fragment.

table 5

After comprehensive consideration, the 14F3 clone was selected and sent to a third-party institution for sequencing.

The sequencing results are: the nucleotide sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID No. 7, wherein the heavy chain variable region includes the amino acid sequence HCDR1 and the amino acid sequence shown in SEQ ID No. 2 HCDR2 as shown in SEQ ID No. 3 and HCDR3 with the amino acid sequence LPDY; the nucleotide sequence of the light chain variable region is as shown in SEQ ID No. 8, wherein the light chain variable region includes the amino acid sequence as SEQ ID No. LCDR1 shown in .4, LCDR2 whose amino acid sequence is shown in SEQ ID No. 5, and LCDR3 whose amino acid sequence is shown in SEQ ID No. 6.

2.6. Recombinant expression of monoclonal antibodies

After the heavy chain cDNA and light chain cDNA of the monoclonal antibody are connected using a linker peptide (GGGGSGGGGSGGGGS), they are cloned into the pCMV vector to obtain the recombinant expression plasmid of the monoclonal antibody. The recombinant plasmid was transfected into HEK293F cells. The HEK293F cell culture supernatant was collected and tested after affinity purification. Monoclonal antibodies against the small peptide encoded by LINC01315 were obtained.

Example 3

In this example, experiments were performed by knocking out or overexpressing LINC01315-ORF in vivo. Includes the following:

3.1. Lentivirus knockout ORF

1) Sequence design and synthesis

The shRNA knockout sequences shRNA-LINC01315-F and shRNA-LINC01315-R targeting LINC01315 were designed through The GPP Web Portal Online Tool (https://portals.broadinstitute.org/gpp/public/). The sequence design results were submitted to Shanghai Sangon Biotechnology Co., Ltd. for synthesis. The target sequence of shRNA is AGGCGTGAAGTCAGGAGAATT (SEQ ID NO. 19).

Table 6 annealing system is as follows

Element volume shRNA-LINC01315-F(20μM) 5μL shRNA-LINC01315-R(20μM) 5μL 10×NEB Buffer2 5μL ddH ₂ O Add to 50μL

The annealing procedure is as follows: 95℃×10min, (95℃～25℃)×(-1℃/min), 25℃×Hold on to obtain the annealing product shRNA.

shRNA-LINC01315-F:

CCGGAGGCGTGAAGTCAGGAGAATTCTCGAGAATTCTCCTGACTTCACGCCTTTTTTG(SEQ IDNO.17)

shRNA-LINC01315-R:

AATTCAAAAAAGGCGTGAAGTCAGGAGAATTCTCGAGAATTCTCCTGACTTC(SEQ ID NO.18)

2) Enzyme digestion vector

The vector used was pLKO.1-TRC (Addgene Company, Cat#10878). Choose AgeI and EcoRI for step-by-step digestion. Proceed as follows:

Table 7

Element Dosage pLKO.1-TRC 6μg AgeI 1μL 10×NEB Buffer1.1 5μL ddH ₂ O Add to 50μL

37°C water bath for 2 hours. Use a DNA purification kit (Cat#DP219, TIANGEN) to recover the enzyme digestion products. For specific steps, refer to the instructions. Finally, use 30 μL Elution Buffer to elute the DNA product. Perform a second enzyme digestion on the DNA eluate. The reaction system is shown in Table 8.

Table 8

Element Dosage DNA product 30μL EcoRI 1μL 10×NEB Buffer3.1 5μL ddH ₂ O Add to 50μL

After 2 hours in a 37°C water bath, perform electrophoresis on a 1% DNA gel, and then purify the DNA gel product. Finally, a spectrophotometer was used for DNA quantification to obtain double enzyme digestion products.

3) Enzyme connection

The primer annealing product of step 1) and the double enzyme digestion product of step 2) are enzymatically ligated and recombined. The connection reaction system is shown in Table 9.

Table 9

Element Dosage Annealing primer 2μL Double enzyme cutting vector 20ng T4 DNA ligase 2μL 10×DNA ligase buffer 2μL ddH ₂ O Add to 20μL

16℃×30min. Add 10 μL of the connection system to the melted HEK-293T competent cells (RRID: CVCL_1926), flick it evenly and place it on ice for 10 minutes. Heat shock at 42°C for 60 seconds and immediately bathe in ice for 2 minutes. Apply the mixed system evenly to the ampicillin-resistant cells. On the plate, select 4-10 positive clones and shake them for 3-5 hours after continuous culture at 37°C for 12-18 hours. Take a small amount of bacterial liquid and send it to Beijing Liuhe BGI for sequencing. The bacterial liquid clones with correct sequencing results will be expanded and cultured. Extract plasmid DNA and freeze at -20°C for later use. The positive clone is pLKO.1-shRNA.

4) Lentivirus production

Spread 5-8×10 ⁶ HEK-293T cells evenly in a 10cm cell culture dish treated with polylysine, and culture it overnight in a 37°C 5% CO ₂ incubator until the cell confluence reaches more than 85%. Change the culture medium to Opti-MEM, and then transfect according to the following system:

Table 10 System 1

Element Dosage pLKO.1-shRNA 6μg psPAX2 4.5μg pMD2.G 1.5μg Opti-MEM 600μL

Table 11 System 2

Element Dosage Lipofectamine2000 30μL Opti-MEM 600μL

Mix system 1 and system 2, let stand at room temperature for 5 minutes, add to the cell culture supernatant, and shake evenly. After continuous culture for 6-8 hours at 37°C and 5% _CO2 , change to fresh medium containing 10% FBS and 1% penicillin-streptomycin double antibody solution and continue to culture for 48 hours. Collect the cell culture supernatant and use a 0.22 μm filter to remove impurities. , then concentrate the supernatant with virus concentrate (Cat#631231, Clontech), and finally resuspend the pellet in 100 μL of PBS containing 0.1% BSA. The resulting solution is the virus suspension, and freeze it at -80°C for later use.

Plate the above-mentioned cells to be established in each well of a 6-well plate at 2-3 × 10 ⁵ cells, and culture them overnight in a 5% CO ₂ incubator at 37°C. When the cell density reaches 30-50%, add 5 μL of concentrated virus. Suspension and polybrene with a final concentration of 5 mg/mL was added to the cell culture supernatant, shaken evenly, and cultured continuously for 48 hours. Replace with fresh culture medium and culture for another 48 hours. Passage processing was performed depending on the cell culture density, and then screening drugs were added or flow cytometry was added. Cell sorting, the resulting cells are stably transfected cell lines, and ORF knockout cell lines are obtained.

3.2. Construction of overexpression ORF vector

Replace the vector used to construct the ORF knockdown plasmid pLKO.1-TRC (i.e. 2) in step 3.1) with PCDH-EF1-MCS-T2A-Puro (SBI Company, Cat#CD513B-1), and the others are the same. In step 3.1 of this example, an ORF overexpressing cell line is obtained.

Mel 920-ORF-NC negative control group and 92.1 control group were also established.

Mel 920-ORF-NC negative control group is a stably transfected strain with unrelated sequences.

The 92.1 control group is the PCDH-EF1-MCS-T2A-Puro empty-loaded stable transfection strain.

3.3. Immunofluorescence experiment

The ORF knockout cell line in step 3.1 and the ORF overexpression cell line in step 3.2 are evenly spread on the cell slide that has been treated with 0.1 mg/mL polylysine solution (PLL) and dried, at 10 ³ cells. Cultivate overnight in a 37°C 5% _CO2 constant temperature incubator. Wash twice with pre-cooled PBS, then fix the cells with 4% PFA at room temperature for 20 minutes. Wash the slides twice with PBS, and add 0.5% Triton X-100 to permeabilize the cells for 15 minutes at room temperature. , wash once with 3% BSA, and then block with 3% BSA for 1 hour at room temperature. Discard the supernatant, add diluted primary antibody, and incubate at 4°C overnight. After recovering the primary antibody, wash the slide with PBS 2 times, 5 minutes each time, and add Alexa-labeled secondary antibody was incubated at room temperature for 2 hours, washed twice with PBS, and nuclear stained with Hoechst 33342 diluted in PBS. The image results were obtained using a laser confocal microscope ZEISS LSM 880, and subsequent image processing was performed using Fiji software. The results are shown in Figure 2. Among them, Mel 920-ORF-KO is the knockout ORF group, and Mel 920-ORF-OE is the overexpression ORF group.

As can be seen from Figure 2, ORF is not expressed after endogenous knockout.

3.4. Investigation of overexpressed ORF and inhibited ORF in mice

Female nude mice aged 4-6 weeks were used as experimental subjects. The mice were randomly divided into 3 groups, with 12 mice in each group. The 3 groups were the control group (Scramble), the overexpression group (ORF) and the inhibition group ( sh-ORF). Except for the control group, the other 2 groups constructed uveal melanoma models, as follows:

1) Both groups of mice were subcutaneously injected with the stably transformed cell line obtained in step 3.3 from the root of the thigh, and each mouse was injected with 1×10 ⁷ cells. The control group (Scramble) was injected with Mel 92.1-wt tumor cells.

Tumor volume=0.5×length×width×width

2) When the volume of the tumor tissue is about 100mm, measure and record the size of the tumor tissue every ^two days, that is, record the size of the tumor tissue on 6d, 9d, 12d, 15d, 18d, 21d and 24d. The results are shown in Figure 3.

Figure 3 shows the tumor volume curve and real photos of mice injected with a stably transduced cell line overexpressing ORF (ORF) and a stably transduced cell line inhibiting ORF (sh-ORF) in this example.

As can be seen from Figure 3, the tumor size in the overexpression group (ORF) was significantly larger than the control group (Scramble), while the tumor size in the inhibition group (sh-ORF) was significantly smaller than the control group, indicating that knocking out ORF can significantly inhibit tumors.

Example 4

In this example, the ORF-targeting monoclonal neutralizing antibody prepared in Example 2 was subjected to in vivo experiments. Includes the following:

Female nude mice aged 4-6 weeks were used as the experimental subjects. The mice were randomly divided into 2 groups. The two groups were the control group (vehicle) and the antibody group (anti-ORF), with 12 mice in each group.

1) Mice in the control group and antibody group were injected subcutaneously with Mel 92.1-wt tumor cells (Mel92.1, RRID: CVCL_8607) from the root of the thigh, and each mouse was injected with 1×10 ⁷ cells.

2) Start treatment when the volume of the tumor tissue is about ^100mm . Inject multiple points into the tumor. The control group is injected with PBS, and the treatment group is injected with anti-ORF antibody, 200 μg/animal, once every two days. Measure and record the size of the tumor tissue every 2 days, that is, on 1d, 2d, 3d, 4d, 5d and 6d. The results are shown in Figure 4.

As can be seen from Figure 4, the tumor volume in the antibody group (anti-ORF) was significantly smaller than that in the control group (vehicle), indicating that monoclonal neutralizing antibodies targeting ORF can inhibit tumor growth.

It can be seen from Example 3 and Example 4 that knocking out the small peptide encoded by LINC01315 (LINC01315-ORF) or using a monoclonal neutralizing antibody targeting the ORF can effectively inhibit the growth of tumors.

The above embodiments only illustrate the principles and effects of the present invention, but are not intended to limit the present invention. Anyone familiar with this technology can modify or change the above embodiments without departing from the spirit and scope of the invention. Therefore, all equivalent modifications or changes made by those with ordinary knowledge in the technical field without departing from the spirit and technical ideas disclosed in the present invention shall still be covered by the claims of the present invention.

Claims (12)

1. The application of LINC01315 coded small peptide as a target in preparing or screening eye tumor diagnosis drugs or therapeutic drugs.
2. Use of an inhibitor of linc01315 encoding a small peptide for the preparation of a medicament for the prevention and/or treatment of ocular tumors.
3. 3. The use according to claim 1 or 2, wherein the nucleotide sequence encoding the small peptide LINC01315 comprises the sequence set forth in SEQ ID No. 1;

   and/or the eye tumor is selected from one or more of uveal melanoma, retinoblastoma and conjunctival melanoma.
4. 4. The use according to claim 2, wherein the inhibitor is a substance capable of inhibiting the transcription or translation of the gene encoding the small peptide LINC01315 or of inhibiting the expression or activity of the small peptide LINC 01315;

   and/or the inhibitor is selected from the group consisting of a nucleic acid molecule, a small molecule chemical, an antibody drug, a polypeptide, a protein, an interfering nucleic acid construct, an interfering lentivirus, and an adeno-associated virus; preferably, the nucleic acid molecule is any one or more of the following: antisense oligonucleotides, double stranded RNAs, sirnas or shrnas.
5. 5. The use of claim 4, wherein the inhibitor is an shRNA, the target sequence of which comprises the sequence shown in SEQ ID No. 19;

   And/or the inhibitor is an antibody drug, which is a monoclonal antibody against LINC01315 encoding a small peptide;

   preferably, the monoclonal antibody comprises a light chain and a heavy chain, characterized in that the variable region of the heavy chain comprises HCDR1, HCDR2 and HCDR3, the variable region of the light chain comprises LCDR1, LCDR2 and LCDR3,

   the HCDR1 sequence comprises the amino acid sequence shown in SEQ ID No. 2;

   the HCDR2 sequence comprises the amino acid sequence shown in SEQ ID No. 3;

   the sequence of HCDR3 comprises the amino acid sequence LPDY;

   the sequence of LCDR1 comprises the amino acid sequence shown as SEQ ID No. 4;

   the sequence of LCDR2 contains the amino acid sequence shown in SEQ ID No. 5;

   the sequence of LCDR3 comprises the amino acid sequence shown in SEQ ID No. 6.
6. 6. A shRNA encoding a small peptide of LINC01315, wherein the shRNA has a target sequence comprising a sequence as set forth in SEQ id No. 19.
7. 7. A monoclonal antibody against LINC01315 coding small peptide, comprising a light chain and a heavy chain, characterized in that the variable region of the heavy chain comprises HCDR1, HCDR2 and HCDR3, the variable region of the light chain comprises LCDR1, LCDR2 and LCDR3,

   the HCDR1 sequence comprises the amino acid sequence shown in SEQ ID No. 2;

   the HCDR2 sequence comprises the amino acid sequence shown in SEQ ID No. 3;

   The sequence of HCDR3 comprises the amino acid sequence LPDY;

   the sequence of LCDR1 comprises the amino acid sequence shown as SEQ ID No. 4;

   the sequence of LCDR2 contains the amino acid sequence shown in SEQ ID No. 5;

   the sequence of LCDR3 comprises the amino acid sequence shown in SEQ ID No. 6.
8. 8. The monoclonal antibody of claim 7, wherein the amino acid sequence of the variable region of the heavy chain comprises the sequence set forth in seq id No. 7;

   and/or the amino acid sequence of the variable region of the light chain comprises a sequence as shown in SEQ ID No. 8.
9. 9. Biological material associated with shRNA according to claim 6 or monoclonal antibody according to claims 7-8, characterized in that the biological material comprises one or more of the following:

   1) Nucleotides encoding the shRNA of claim 6 or the monoclonal antibody of claims 7-8;

   2) A recombinant expression vector comprising 1) said nucleotide;

   3) The bioengineering bacteria containing 1) the nucleotide or the bioengineering bacteria containing 2) the recombinant expression vector.
10. 10. Use of shRNA according to claim 6 or monoclonal antibody according to claim 7 or 8 or biomaterial according to claim 8 for the preparation of a medicament for the treatment or screening of ocular tumors, or for the preparation of a medicament for the diagnosis of ocular tumors.
11. 11. A medicament for preventing and/or treating eye tumors, which is characterized by comprising an inhibitor of LINC01315 coded small peptide and pharmaceutically acceptable auxiliary materials.
12. 12. A method of screening for a drug for preventing and/or treating an ocular tumor, the method comprising: taking LINC01315 coded small peptide as a drug target spot, and searching a substance capable of inhibiting or blocking the expression and/or the function of the LINC01315 coded small peptide as a candidate drug.