Use of ARMC7 inhibitors in the preparation of drugs for treating melanoma

Abstract

The present invention discloses the use of an ARMC7 inhibitor in the preparation of a drug for treating melanoma. The present invention discovers for the first time that ARMC7 can be used for the early diagnosis, prognosis prediction and effective treatment of melanoma, has good diagnostic efficacy for the diagnosis and prognosis prediction of melanoma, and has good therapeutic effect for the treatment of melanoma. The present invention provides a new diagnostic and therapeutic target for the diagnosis and treatment of melanoma, and has important clinical significance.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular, relates to the use of an ARMC7 inhibitor in preparing a drug for treating melanoma. Background Art

[0002] Melanoma is a malignant tumor caused by the malignant transformation of melanocytes. It usually occurs in the skin, and rarely in non-skin areas such as the mouth, intestines, and eyes. Melanoma is the fifth most common cancer in the world. There are obvious racial differences in the incidence of melanoma, with the highest incidence in Caucasians and relatively low incidence in Asians and blacks. The incidence of melanoma in my country is 0.49 / 100,000, which is relatively low compared with high-incidence areas such as Europe and the United States. However, due to the high malignancy of melanoma, it is easy to metastasize to blood and lymph nodes, and the clinical prognosis is poor. In addition, there are relatively few analytical studies on melanoma in China. Therefore, there is still an urgent need to study the pathogenesis and treatment of melanoma, so as to provide new targets for the diagnosis and treatment of melanoma.

[0003] Melanoma is a malignant tumor with hidden onset, early metastasis, difficult early diagnosis and poor prognosis. The global incidence of melanoma is on the rise, and the mortality rate has decreased compared with the past due to the expansion of treatment methods. The treatment of melanoma is currently mainly surgical, supplemented by other treatments (targeted therapy, immunotherapy, cell therapy, etc.). Although targeted therapy and immunotherapy for melanoma have achieved remarkable results in recent years, secondary drug resistance and toxic side effects have affected the long-term efficacy of these treatments. Therefore, exploring new targets for melanoma treatment and finding factors related to the prognosis of the disease are of great significance for improving the prognosis of melanoma patients, prolonging their survival time and increasing their survival rate. Summary of the invention

[0004] In view of this, in order to overcome the above-mentioned technical problems currently faced in the art, the object of the present invention is to provide use of an ARMC7 inhibitor in the preparation of a drug for treating melanoma.

[0005] The present invention adopts the following technical solutions to achieve the above-mentioned invention objectives:

[0006] The first aspect of the present invention provides the use of an agent that inhibits the expression of ARMC7 and / or an agent that promotes the expression of ARMC12 in the preparation of a medicament for treating and / or preventing melanoma.

[0007] Further, the reagents for inhibiting ARMC7 expression include shRNA for ARMC7, siRNA for ARMC7, reagents for inhibiting ARMC7 transcriptional activity, reagents for inhibiting ARMC7 mRNA transcriptional levels, reagents for promoting ARMC7 mRNA degradation, reagents for inhibiting ARMC7 mRNA translation, and / or reagents for specifically recognizing guide nucleic acids of ARMC7 and shearing them to reduce the expression level of ARMC7;

[0008] Optionally, the sequence of the siRNA targeting ARMC7 is shown in SEQ ID NO: 1-2.

[0009] Furthermore, the reagents that promote ARMC12 expression include vectors that overexpress ARMC12, active peptides that promote ARMC12 expression, nanoparticles carrying ARMC12, viral vectors carrying ARMC12, PEG-modified proteins that encapsulate ARMC12, proteins that promote ARMC12 expression, oligonucleotides that promote ARMC12 expression and / or small molecule compounds that promote ARMC12 expression.

[0010] In the present invention, the Gene ID of ARMC7 in NCBI (https: / / www.ncbi.nlm.nih.gov / ) is 79637, and the Gene ID of ARMC12 in NCBI (https: / / www.ncbi.nlm.nih.gov / ) is 221481.

[0011] In some embodiments, the reagents that inhibit ARMC7 expression include but are not limited to: reagents that inhibit the expression of the gene encoding the ARMC7 protein or reduce the expression level of the gene, and reduce the activity of the ARMC7 protein, for example, reagents that inhibit the expression of the ARMC7 gene or reduce its expression level, these reagents include but are not limited to: reagents that inhibit the transcriptional activity of the ARMC7 gene, reagents that inhibit the transcriptional level of ARMC7 mRNA, reagents that promote the degradation of ARMC7 mRNA, siRNA against the ARMC7 gene, shRNA against the ARMC7 gene, reagents that inhibit the translation of ARMC7 mRNA, reagents that specifically recognize the guide nucleic acid of the ARMC7 gene and cut it to reduce its expression level, dsRNA against the ARMC7 gene, microRNA against the ARMC7 gene, and antisense nucleic acid against the ARMC7 gene.

[0012] In some embodiments, the ARMC7 gene can be knocked out by administering a targeting vector to inhibit or reduce ARMC7 gene expression. In other embodiments, the agent that reduces ARMC7 protein activity can be, for example, a specific antibody targeting ARMC7 or a small molecule compound that inhibits ARMC7 protein activity.

[0013] In some embodiments, the activity of the ARMC7 protein can also be reduced by introducing mutations into the ARMC7 protein. In other embodiments, a mutation that causes the corresponding activity to be weakened or lost is introduced into the functional domain of the ARMC7 protein. The mutation can be the insertion, deletion or substitution of one or several or even more (e.g., more than 10, more than 20, more than 30) amino acids. By administering an agent that acts on the ARMC7 gene, a mutation that causes the relevant biological activity to be weakened or lost can be present in the functional domain of the ARMC7 protein encoded by it. Such agents can change the sequence of the ARMC7 gene, resulting in the presence of corresponding mutations in the ARMC7 protein encoded by it, thereby having weakened activity or loss of activity. For example, the wild-type ARMC7 gene can be replaced by a mutant ARMC7 gene by homologous recombination technology, resulting in the expression of a weakly active or inactive ARMC7 protein.

[0014] In some embodiments, the agent that promotes ARMC12 expression refers to any substance that can promote ARMC12 expression, including but not limited to: natural purified substances that can promote ARMC12 expression, modified natural purified substances, semi-synthetic substances, chemically synthesized substances and / or any combination thereof. Any substance that can promote ARMC12 expression is within the scope of protection of the present invention.

[0015] In some embodiments, the vector in the vector overexpressing ARMC12 includes a DNA plasmid vector, a lentiviral vector, a retroviral vector, a poxvirus vector, a herpes simplex virus vector, an adenovirus vector, an adeno-associated virus vector, a liposome bound to a DNA plasmid, a molecular couple bound to a DNA plasmid, and / or a polymer bound to a DNA plasmid. In a specific embodiment of the present invention, the vector is not particularly limited.

[0016] In the present invention, the shRNA refers to short hairpin RNA, which includes two short inverted repeat sequences. The shRNA cloned into the shRNA expression vector includes two short inverted repeat sequences separated by a stem-loop sequence in the middle, forming a hairpin structure, which is controlled by the polⅢ promoter. Subsequently, 5-6 Ts are connected as the transcription terminator of RNA polymerase Ⅲ. shRNA can be stably integrated into the genome of the cell, allowing long-term gene knockout.

[0017] In the present invention, the siRNA refers to small interfering ribonucleic acid, i.e. a relatively short-length double-stranded nucleic acid or optionally a longer precursor thereof. In some embodiments, the length of the available siRNA in the present invention is preferably a length of about 20-50 bp. However, there is no particular restriction on the length of the available siRNA in the present invention. For example, the siRNA may initially be present in the cell in a precursor form, and the final or processed form of the siRNA that performs and exerts gene silencing activity when or after the precursor form is delivered to the target cell is substantially different. For example, the precursor form of the siRNA may include a precursor sequence element, which may be processed, degraded, changed or cut when or after delivery, to produce an siRNA that mediates gene silencing activity in the cell.

[0018] In the present invention, the dsRNA refers to a double-stranded ribonucleic acid, an RNA molecule formed by the renaturation of two complementary chains, which can be cleaved by the Dicer enzyme to form siRNA. dsRNA inhibits gene expression through RNA interference (RNAi), and dsRNA does not need to have 100% homology with the target gene sequence, as long as it can inhibit the expression of the target gene.

[0019] In the present invention, the microRNA refers to microribonucleic acid (miRNA), which is a non-coding RNA of about 22 nt in length and widely exists in various organisms from viruses to humans. The main function of mature miRNA is to negatively regulate the post-transcriptional level of genes, and participate in many life processes such as cell proliferation, apoptosis, immunity, neuroendocrine and stem cell differentiation by causing the degradation of its target mRNA or interruption of the translation process.

[0020] In the present invention, the antisense nucleic acid refers to a nucleic acid containing a sequence complementary to that encoding ARMC7. Antisense nucleic acids can be composed of DNA, RNA or both. Antisense nucleic acids may contain non-complementary bases as long as they can specifically hybridize under stringent conditions. When the antisense nucleic acid is introduced into a cell, it binds to the target polynucleotide and inhibits transcription, RNA processing, or stability. In addition to antisense polynucleotides, antisense nucleic acids also include polynucleotide mimics, which contain a modified main chain, and 3' and 5' end portions. Such antisense nucleic acids can be appropriately designed based on the sequence information of ARMC7 and prepared using methods known to those skilled in the art.

[0021] A second aspect of the present invention provides a pharmaceutical composition for treating and / or preventing melanoma.

[0022] Furthermore, the pharmaceutical composition comprises the agent for inhibiting ARMC7 expression and / or the agent for promoting ARMC12 expression as described in the first aspect of the present invention.

[0023] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient.

[0024] In some embodiments, the pharmaceutical composition may further comprise a second active ingredient, which is any drug or agent that can be used to treat and / or prevent melanoma, and / or assist in the treatment and / or assistance in the prevention of melanoma.

[0025] Further, the pharmaceutically acceptable carriers and / or excipients are described in detail in Remington's Pharmaceutical Sciences (19th ed 1995). These substances are used to help the stability of the formulation or to help improve its activity or biological effectiveness as needed. The pharmaceutically acceptable carriers and / or excipients include, but are not limited to: buffers, such as neutral buffered saline, phosphate buffered saline, etc.; carbohydrates, such as glucose, mannose, sucrose or dextran, mannitol; proteins; polypeptides or amino acids, such as glycine; antioxidants; chelating agents, such as EDTA or glutathione; adjuvants (such as aluminum hydroxide); preservatives; and any other pharmaceutically or physiologically acceptable carriers, diluents or excipients reported in any prior art that can be used in pharmaceutical compositions.

[0026] A third aspect of the present invention provides a pharmaceutical preparation for treating and / or preventing melanoma.

[0027] Furthermore, the pharmaceutical preparation comprises the pharmaceutical composition described in the second aspect of the present invention.

[0028] Furthermore, the dosage form of the pharmaceutical preparation includes solution, lyophilized powder, tablet, capsule, granule, suspension, controlled release agent, nanocrystal, microemulsion and / or solid dispersion.

[0029] In some embodiments, the administration route of the pharmaceutical composition or pharmaceutical preparation of the present invention is not particularly limited, as long as it can exert the desired therapeutic effect or preventive effect, and the administration route of the pharmaceutical composition or pharmaceutical preparation includes but is not limited to: topical, through the skin, intravenous, intraperitoneal, intraocular, intraarterial, intrapulmonary, oral, intravesicular, intramuscular, intratracheal, subcutaneous, inhalation, through the pleura, through the mucosa, skin, gastrointestinal, intraarticular, intraventricular, rectal, vaginal, intracranial, intraurethral, ​​intrahepatic. In some cases, it can be administered systemically, and in some cases, it can be administered locally.

[0030] In some embodiments, the suitable dosage of the pharmaceutical composition or pharmaceutical preparation described in the present invention can be prescribed in a variety of ways according to factors such as formulation method, administration mode, patient's age, weight, gender, morbidity, diet, administration time, administration route, excretion rate and reaction sensitivity. Generally, a skilled physician can easily determine the prescription and the dosage that is effective for the desired treatment or prevention.

[0031] In the present invention, the treatment and / or prevention refers to slowing down, interrupting, blocking, alleviating, stopping, reducing, or reversing the progression or severity of existing symptoms, symptoms, conditions, or diseases (e.g., melanoma). The desired therapeutic effects include, but are not limited to: preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and alleviating or improving prognosis.

[0032] In the present invention, the effective amount refers to a therapeutically effective amount. The effective amount used herein refers to the amount of a pharmaceutical composition or pharmaceutical preparation sufficient to treat a specified disorder, condition or disease (e.g., to improve, alleviate, weaken and / or delay one or more of its symptoms). In some embodiments, the effective amount is an amount sufficient to delay the development of a disease (e.g., melanoma). In some embodiments, the effective amount is an amount sufficient to prevent or delay the recurrence of a disease. The effective dose can be administered in one or more administrations. The pharmaceutically effective amount depends on a variety of factors, including but not limited to: characteristic factors of the subject (subject) being treated (such as height, weight, sex, age and medication history), the severity of the disease, etc.

[0033] In the present invention, the subject refers to any animal, including humans and non-human animals. The non-human animals include all vertebrates, for example, mammals, such as non-human primates (especially higher primates), sheep, dogs, rodents (such as mice or rats), guinea pigs, goats, pigs, cats, rabbits, cattle, and any livestock or pets; and non-mammals, such as chickens, amphibians, reptiles, etc. In a specific embodiment of the present invention, the subject is preferably a human.

[0034] A fourth aspect of the present invention provides any of the following applications:

[0035] (1) Use of reagents for detecting ARMC7 and / or ARMC12 expression levels in the preparation of melanoma diagnosis and / or prognosis prediction products;

[0036] (2) Use of reagents for detecting ARMC7 and / or ARMC12 expression levels in the preparation of melanoma diagnosis and / or prognosis prediction reagents;

[0037] (3) Use of the methylation sites cg00850747, cg02186444, cg10590861, cg25749101, cg23179678 and / or cg19875472 of the ARMC7 gene in the preparation of melanoma diagnosis and / or prognosis prediction products;

[0038] (4) Use of the methylation sites cg00850747, cg02186444, cg10590861, cg25749101, cg23179678 and / or cg19875472 of the ARMC7 gene in the preparation of melanoma diagnosis and / or prognosis prediction reagents;

[0039] (5) Use of reagents for detecting ARMC7 methylation levels in the preparation of melanoma diagnosis and / or prognosis prediction products;

[0040] (6) Use of reagents for detecting ARMC7 methylation levels in the preparation of melanoma diagnosis and / or prognosis prediction reagents;

[0041] (7) Use of ARMC7 and / or ARMC12 in screening candidate drugs for treating and / or preventing melanoma.

[0042] Further, the reagent for detecting the expression level of ARMC7 and / or ARMC12 includes primers for specific amplification of ARMC7 and / or ARMC12, and / or probes for specific recognition of ARMC7 and / or ARMC12;

[0043] Optionally, the sequences of primers for specific amplification of ARMC7 are shown in SEQ ID NO:3-SEQ ID NO:4;

[0044] Optionally, the sequences of primers for specifically amplifying ARMC12 are shown in SEQ ID NO:9-SEQ ID NO:10.

[0045] A fifth aspect of the present invention provides any of the following products:

[0046] (1) A diagnostic product for melanoma diagnosis and / or prognosis prediction, the diagnostic product comprising the reagent for detecting the expression level of ARMC7 and / or ARMC12, the reagent for detecting the methylation site of the ARMC7 gene and / or the reagent for detecting the methylation level of ARMC7 as described in the fourth aspect of the present invention;

[0047] (2) A diagnostic reagent for melanoma diagnosis and / or prognosis prediction, the diagnostic reagent comprising the reagent for detecting the expression level of ARMC7 and / or ARMC12, the reagent for detecting the methylation site of the ARMC7 gene and / or the reagent for detecting the methylation level of ARMC7 as described in the fourth aspect of the present invention.

[0048] In some embodiments, the diagnostic product includes a detection kit, a detection chip and / or a detection test strip. The diagnostic product is to diagnose melanoma by detecting the expression level of ARMC7 and / or ARMC12 in a test sample derived from a subject, detecting whether the methylation site of the ARMC7 gene exists in the test sample derived from the subject, and / or detecting the methylation level of ARMC7 in the test sample derived from the subject.

[0049] In some embodiments, the kit is an RT-PCR kit, which may further comprise elements necessary for reverse transcription polymerase chain reaction. The RT-PCR kit comprises a pair of primers specific for ARMC7 and / or ARMC12. The primers are nucleotides having nucleic acid sequences specific for the ARMC7 and / or ARMC12, and may be about 7 to 50 bp in length, more particularly about 10-39 bp in length.

[0050] In some embodiments, the RT-PCR kit may further include test tubes or suitable vessels, reaction buffers (different pH values ​​and magnesium concentrations), deoxynucleotides (dNTPs), enzymes (e.g., Taq polymerase and reverse transcriptase), deoxyribonuclease inhibitors, ribonuclease inhibitors, DEPC-water and / or sterile water.

[0051] In some embodiments, the kit is a DNA chip kit, which may further include elements necessary for operating the DNA chip. The DNA chip kit may include a substrate bound to a cDNA corresponding to ARMC7 and / or ARMC12 or an oligonucleotide equivalent to a fragment thereof, and reagents, agents and enzymes for constructing a fluorescently labeled probe. In addition, the substrate may include a control cDNA or an oligonucleotide equivalent to a fragment thereof.

[0052] In some embodiments, the diagnostic reagent further comprises a reagent for detecting the expression level of ARMC7 and / or ARMC12 by sequencing technology, nucleic acid hybridization technology and / or nucleic acid amplification technology. The diagnostic reagent diagnoses melanoma or predicts the prognosis of the subject by detecting the expression level of ARMC7 and / or ARMC12 in the sample to be tested.

[0053] A sixth aspect of the present invention provides any of the following methods:

[0054] (1) A method for screening candidate drugs for treating and / or preventing melanoma, the method comprising the following steps:

[0055] ① Treating the system expressing or containing ARMC7 and / or ARMC12 with the test substance;

[0056] ② Detecting the expression of ARMC7 and / or ARMC12 in the system;

[0057] ③ Select test substances that can inhibit ARMC7 expression and / or promote ARMC12 expression as candidate drugs;

[0058] (2) An in vitro method for inhibiting melanoma cell growth for non-therapeutic purposes, the method comprising the following steps: adding an effective amount of an agent for inhibiting ARMC7 expression and / or an agent for promoting ARMC12 expression as described in the first aspect of the present invention to a system in need thereof.

[0059] In some embodiments, the system in the method of screening candidate drugs includes a cell system, a subcellular system, a solution system, a tissue system, an organ system and / or an animal system.

[0060] In some embodiments, the substance to be tested is any substance that may have a therapeutic and / or preventive effect on melanoma, or any substance that may inhibit the expression level of ARMC7 described in the present invention and / or may promote the expression level of ARMC12 described in the present invention. In the present invention, the substance to be tested is not particularly limited.

[0061] In addition, the present invention also provides a method for treating and / or preventing melanoma, the method comprising the following steps: administering to a subject in need thereof a therapeutically and / or preventively effective amount of an agent for inhibiting ARMC7 expression, an agent for promoting ARMC12 expression, the pharmaceutical composition described in the second aspect of the present invention, and / or the pharmaceutical preparation described in the third aspect of the present invention.

[0062] In some embodiments, when the agent for inhibiting ARMC7 expression, the agent for promoting ARMC12 expression, the pharmaceutical composition described in the second aspect of the present invention, and / or the pharmaceutical preparation described in the third aspect of the present invention are administered, the agent can be administered systemically, or the agent can be administered directly to a specific part where cancer cells or precancerous cells are present. Therefore, administration can be accomplished in any manner that effectively delivers the agent to cancer cells or precancerous cells.

[0063] In some embodiments, the modes of administration include, but are not limited to, topical, transdermal, parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, oral, intranasal instillation, intracavitary or intravesical instillation, intraocular, intraarterial, intralesional, or by administration to mucous membranes such as the nose, throat, and bronchial tubes.

[0064] In addition, the present invention also provides a method for diagnosing and / or assisting in the diagnosis of melanoma, and / or predicting the prognosis of melanoma patients, the method comprising the following steps: detecting the expression level of ARMC7 and / or ARMC12 described in the first aspect of the present invention in a sample derived from a subject, detecting the methylation site of the ARMC7 gene in the sample derived from the subject, and / or detecting the methylation level of ARMC7 in the sample derived from the subject, and predicting and diagnosing the prognosis of the subject or whether the subject has melanoma based on the detection results.

[0065] In some embodiments, the sample derived from the subject can be obtained from the subject's blood and other fluid samples and tissue samples of biological origin, such as biopsy tissue samples or tissue cultures or cells derived therefrom. The source of the tissue sample can be solid tissue, such as fresh, frozen and / or preserved organ or tissue samples, biopsy tissues or aspirates; blood or any blood component; body fluid; cells from any time of individual pregnancy or development; or plasma. The sample includes biological samples that have been processed in any way after it is obtained, such as reagent treatment, stabilization, or enrichment for certain components (such as proteins or polynucleotides), or embedded in a semi-solid or solid matrix for sectioning purposes. The samples described in the present invention include, but are not limited to: blood, tissue, blood-derived cells, serum, plasma, lymph, synovial fluid, cell extracts and combinations thereof. In a preferred embodiment, the sample is selected from a tissue sample or a blood sample of the subject.

[0066] Compared with the prior art, the present invention has the following beneficial effects:

[0067] The present invention discovers for the first time that ARMC7 and ARMC12 can be used for early diagnosis, prognosis prediction and effective treatment of melanoma. They are two brand-new melanoma diagnostic and therapeutic targets, have good diagnostic efficacy for the diagnosis and prognosis prediction of melanoma, and have good therapeutic effect for the treatment of melanoma. The present invention provides a brand-new diagnostic method and prognosis prediction method for the diagnosis and prognosis of melanoma, provides a brand-new therapeutic target for the treatment of melanoma, and has important clinical significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] Figure 1 : The results of differential expression of ARMC7 in melanoma tissues in the TCGA-CKCM-GTEx dataset;

[0069] Figure 2 : The results of differential expression of ARMC7 in melanoma tissues in the GSE3189 dataset;

[0070] Figure 3 : Result diagram of differential expression of ARMC12 in melanoma tissues in TCGA-CKCM-GTEx dataset;

[0071] Figure 4 :The result graph corresponding to the diagnostic efficacy of ARMC7 for melanoma;

[0072] Figure 5 :The corresponding result graph of the diagnostic efficacy of ARMC12 for melanoma;

[0073] Figure 6 :The corresponding result diagram of ARMC7 for prognosis prediction of melanoma;

[0074] Figure 7 :The corresponding result diagram of ARMC12 for prognosis prediction of melanoma;

[0075] Figure 8 : The result graphs corresponding to the matrix score, immune score and ESTIMATE score between the ARMC7 high expression and ARMC7 low expression groups were evaluated using the estimation algorithm;

[0076] Fig. 9 : The corresponding result diagram of the DNA methylation level of ARMC7 gene in CMM in the TCGA database;

[0077] Fig.10 :Analysis results of the correlation between ARMC7 methylation and prognosis of melanoma patients;

[0078] Fig.11 : Result graphs corresponding to the infiltration of multiple immune cells in the ARMC12 high and low expression groups;

[0079] Fig.12 : The corresponding result graph of knocking down ARMC7 inhibiting tumor cell growth in A375 cell line;

[0080] Fig.13 : The corresponding result graph shows that knocking down ARMC12 promotes tumor cell growth in the A375 cell line. DETAILED DESCRIPTION

[0081] The present invention is further described below in conjunction with specific embodiments, which are only used to explain the present invention and cannot be understood as limiting the present invention. It can be understood by those skilled in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and purpose of the present invention, and the scope of the present invention is defined by the claims and their equivalents.

[0082] The reagents and raw materials used in the present invention are easily obtained by those of ordinary skill in the art, and can be obtained from commercial sources unless otherwise specified. The experimental methods for which specific conditions are not specified in the present invention are usually tested under conventional conditions or under conditions recommended by the manufacturer. In particular, the following examples are only used to illustrate the present invention and should not limit the scope of the present invention in any way. It should be noted that the experimental conditions and results described in the following examples are only used to illustrate the present invention, and should not and will not limit the present invention described in detail in the claims.

[0083] Example 1 Application of ARMC7 and ARMC12 in diagnosis and prognosis prediction of melanoma

[0084] 1. Data acquisition

[0085] In this example, human gene expression profile data of cutaneous malignant melanoma (CMM) were extracted from TCGA (https: / / portal.gdc.cancer.gov / ) and GTEx databases. The CMM data included mRNA expression levels and patient clinical information of 469 melanoma tissues, 1 adjacent tissue, and 812 normal skin tissues.

[0086] In addition, this example downloaded the mRNA expression data of the GSE3189 dataset from the GEO (GEO, http: / / www.ncbi.nlm.nih.gov / geo / ) database, and compared the expression differences of ARMC7 between CMM and normal skin and nevus cells in the dataset.

[0087] 2. Experimental methods

[0088] This example first analyzed the expression of ARMC7 and ARMC12 in melanoma tissues. Secondly, in order to study the prognostic value of ARMC7 and ARMC12 in CMM, a survival analysis was performed. The survival and survminer packages were used to draw Kaplan-Meier plots, and log-rank tests were performed. The pROC and survival packages were used to analyze the diagnostic ability and prognostic judgment of ARMC7 and ARMC12 for CMM, with p < 0.05 being considered statistically significant.

[0089] In addition, this embodiment uses an estimation algorithm to evaluate the matrix score, immune score and ESTIMATE score between the two groups. The matrix score is an indicator for evaluating the content and composition of the extracellular matrix in tumor tissue. A high matrix score may indicate that it is difficult for immune cells to enter the tumor, resulting in a low potential response to immunotherapy. The immune score is an indicator for evaluating the level of immune cell infiltration in tumor tissue. The higher the immune score, the more immune cells enter the tumor tissue, and the better the immune response and prognosis. The ESTIMATE score takes into account the matrix score and the immune score, providing a comprehensive indicator for evaluating immune infiltration. By estimating the matrix score, immune score and estimated score of the two groups, the relationship between ARMC7 gene expression and melanoma immune infiltration can be suggested, and its potential role in tumor treatment can be further explored. p <0.05 is statistically significant.

[0090] The infiltration of various immune cells in the tumor microenvironment is an important factor affecting the prognosis of CMM. This example further studied the infiltration of multiple immune cells in the ARMC12 high and low expression groups.

[0091] This example uses the UCSC Xena database (https: / / http: / / xena.ucsc.edu / ) to analyze the DNA methylation level of the ARMC7 gene in CMM in the TCGA database. In addition, this example also evaluates the prognostic value of ARMC7 CpG methylation.

[0092] 3. Statistical analysis methods

[0093] This example uses the R (v.3.6.3) platform to perform statistical analysis. The difference between the two groups was compared using the nonparametric rank sum test (Wilcox Test); the correlation analysis was performed using the Person or Spearman correlation test; the survival analysis was expressed using the Kaplan-Meier curve, and the inter-group survival rate was tested using the log-rank test. All of the above were tested at α = 0.05, and p < 0.05 was considered statistically significant.

[0094] 4. Experimental results

[0095] The results showed that ARMC7 was highly expressed in melanoma compared with normal tissues in the TCGA-CKCM-GTEx dataset ( Figure 1 ), in the GSE3189 dataset, ARMC7 is highly expressed in melanoma compared with nevus cell tissue (normal melanocytes) ( Figure 2 ), in the TCGA-CKCM-GTEx dataset, ARMC12 is less expressed in melanoma than in normal tissues ( Figure 3 ).

[0096] The results showed that ARMC7 had a good diagnostic value for melanoma (AUC=0.712) ( Figure 4 ), ARMC12 has good diagnostic value for melanoma (AUC=0.910) ( Figure 5 The above results indicate that ARMC7 and ARMC12 can be used for effective diagnosis of melanoma.

[0097] The results showed that ARMC7 has a good prognostic value for melanoma ( Figure 6 ), ARMC12 has good prognostic value for melanoma ( Figure 7 ). The above results indicate that ARMC7 and ARMC12 can be used for accurate prognosis prediction of melanoma.

[0098] The results showed that the immune infiltration score level was low in the ARMC7 high expression group ( Figure 8 ), suggesting that ARMC7 gene expression is significantly correlated with melanoma immune infiltration. The results of evaluating the prognostic value of ARMC7 CpG methylation showed that ARMC7 was methylated in melanoma at high levels at cg00850747, cg02186444, cg10590861, cg25749101, cg23179678, and cg19875472 sites ( Fig. 9 ), ARMC7 methylation is associated with the prognosis of melanoma patients: ARMC7 low methylation group has a poor prognosis ( Fig.10 The results of multiple immune cell infiltration in the ARMC12 high and low expression groups are as follows Fig.11 shown.

[0099] Example 2 Application of ARMC7 and ARMC12 in the treatment of melanoma

[0100] 1. Knockdown of ARMC7 in melanoma cell lines can significantly inhibit the growth of tumor cells

[0101] (1) Cell culture and treatment

[0102] A375 melanoma cell line was purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Cells were cultured in DMEM medium (PromoCell) supplemented with 10% fetal bovine serum (FBS, Sijiqing) at 37°C with 5% CO 2 Cultured in a humidified incubator. Using Lipo8000 TM Transfection reagent (Beyotime) was used for cell transfection according to the manufacturer's protocol. Briefly, cells were seeded in Corning six-well plates and cultured to a cell density of 50%, then transfected and cultured at 37°C for 48 h and harvested for quantitative reverse transcription polymerase chain reaction (qRT-PCR) and other experiments.

[0103] In this example, A375 cells were transfected with siRNA expressing ARMC7 to knock down ARMC7, wherein the sequence information of the siRNA is as follows:

[0104] Sense strand: 5′-ACCCUGGUGGAGUUUGCUAUUTT-3′ (SEQ ID NO: 1);

[0105] Antisense strand: 5′-AAUAGCAAACUCCACCAGGGUTT-3′ (SEQ ID NO: 2).

[0106] (2) RNA extraction and qRT-PCR

[0107] TRIzol reagent (Pufer, Shanghai, China) was used according to the manufacturer's protocol from approximately 1 × 10 6 Total RNA was isolated from cells. Primers used for qRT-PCR, including ARMC and ACTB primers, were from Hippo Biotechnology. The specific primer sequences are as follows:

[0108] ARMC7 forward primer: 5′-AAGACGCCAAGGAGCAAGTC-3′ (SEQ ID NO: 3);

[0109] ARMC7 reverse primer: 5′- CCGACAGCGAATCGAGAAATAA-3′ (SEQ ID NO: 4);

[0110] ACTB internal reference forward primer: 5'-GGATGCAGAAGGAGTCACTG-3' (SEQ ID NO: 5);

[0111] ACTB internal reference reverse primer: 5'-GGAGATTACTGCCCTGGCTCCTA-3' (SEQ ID NO: 6).

[0112] The QRT-PCR parameters were: 95°C for 3 min; (95°C for 10 s, 55°C for 30 s, 72°C for 32 s) × 40 amplification cycles. The relative expression was normalized to 2 △△CT Method calculation.

[0113] (3) Experimental results

[0114] The experimental results are as follows Fig.12 As shown, the results showed that in the A375 melanoma cell line, knocking down ARMC7 could significantly inhibit the growth of tumor cells, indicating that knocking down ARMC7 can be used for the effective treatment of melanoma.

[0115] 2. Overexpression of ARMC12 in melanoma cell lines can significantly inhibit the growth of tumor cells

[0116] (1) Cell culture and treatment

[0117] A375 melanoma cell line was purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Cells were cultured in DMEM medium (PromoCell) supplemented with 10% fetal bovine serum (FBS, Sijiqing) at 37°C with 5% CO 2 Cultured in a humidified incubator. Using Lipo8000 TM Transfection reagent (Beyotime) was used for cell transfection according to the manufacturer's protocol. Briefly, cells were seeded in Corning six-well plates and cultured to a cell density of 50%, then transfected and cultured at 37°C for 48 h and harvested for quantitative reverse transcription polymerase chain reaction (qRT-PCR) and other experiments.

[0118] In this example, ARMC12 knockdown was performed using transfected A375 cells expressing ARMC12 siRNA, wherein the sequence information of the siRNA is as follows:

[0119] Sense strand: 5′-GCUACAUGCUGGAUGACAATT-3′ (SEQ ID NO: 7);

[0120] Antisense strand: 5′-UUGUCAUCCAGCAUGUAGUAGCTT-3′ (SEQ ID NO: 8).

[0121] (2) RNA extraction and qRT-PCR

[0122] TRIzol reagent (Pufer, Shanghai, China) was used according to the manufacturer's protocol from approximately 1 × 10 6 Total RNA was isolated from cells. Primers used for qRT-PCR, including ARMC and ACTB primers, were from Hippo Biotechnology. The specific primer sequences are as follows:

[0123] ARMC12 forward primer: 5′-CAAAAGCGTAGTCAGCCTGG-3′ (SEQ ID NO: 9);

[0124] ARMC12 reverse primer: 5′-GATGCAGATGGGTTAGTTGC-3′ (SEQ ID NO: 10);

[0125] ACTB internal reference forward primer: 5'-GGATGCAGAAGGAGTCACTG-3' (SEQ ID NO: 5);

[0126] ACTB internal reference reverse primer: 5'-GGAGATTACTGCCCTGGCTCCTA-3' (SEQ ID NO: 6).

[0127] The QRT-PCR parameters were: 95°C for 3 min; (95°C for 10 s, 55°C for 30 s, 72°C for 32 s) × 40 amplification cycles. The relative expression was normalized to 2 △△CT Method calculation.

[0128] (3) Experimental results

[0129] The experimental results are as follows Fig.13 As shown, the results showed that in the A375 melanoma cell line, knocking down ARMC12 can significantly promote the growth of tumor cells. In addition, the present invention also used cell experiments to verify the effect of the vector overexpressing ARMC12 on the melanoma cell line. The results showed that overexpression of ARMC12 can significantly inhibit the growth of melanoma cells, indicating that overexpression of ARMC7 can be used for the effective treatment of melanoma.

Claims

1. Use of an agent that inhibits ARMC7 expression in the preparation of a drug for treating melanoma; The reagent for inhibiting ARMC7 expression is siRNA targeting ARMC7; The sequence of the siRNA targeting ARMC7 is shown in SEQ ID NO: 1-2.

2. A pharmaceutical composition for treating melanoma, characterized in that: The pharmaceutical composition comprises the agent for inhibiting ARMC7 expression as claimed in claim 1.

3. A pharmaceutical preparation for treating melanoma, characterized in that: The pharmaceutical preparation comprises the pharmaceutical composition according to claim 2.

4. The pharmaceutical preparation according to claim 3, characterized in that The dosage form of the pharmaceutical preparation is solution, lyophilized powder, tablet, capsule, granule, suspension or microemulsion.

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