Small nucleic acid molecules for the treatment of skin melanoma

Abstract

The present invention discloses a small interfering nucleic acid molecule, which is composed of a sense strand and an antisense strand of the following sequence: the sense strand is GAUUGUGGUUACUGUUCUANn, and the antisense strand is UAGAACAGUAACCACAAUCNn; or the sense strand is CGAGUGACACUGAGACCUANn, and the antisense strand is UAGGUCUCAGUGUCACUCGNn, wherein N is dT; n represents the number of N, and n is 0 or 2. The small interfering nucleic acid molecule of the present invention can be used to prepare a drug for treating skin melanoma.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a small interfering nucleic acid molecule and use thereof in preparing a drug for treating skin melanoma. Background Art

[0002] Skin melanoma is a highly malignant tumor originating from melanocytes in the basal layer of the epidermis, and its mortality rate ranks first among all types of skin malignancies. Studies have shown that the incidence and mortality of skin melanoma have been increasing year by year worldwide in recent years, and its age of death is lower than that of other solid tumors. The pathogenesis of skin melanoma is complex, involving multiple cell signaling pathways, and its specific molecular mechanism needs further study. Early surgical resection is the most effective means of treating skin melanoma, but due to its high invasiveness, many patients have already developed tumor metastasis when they seek medical treatment, which brings great difficulties to treatment. In recent years, although emerging treatments such as targeted therapy and immunotherapy have been increasingly used in the treatment of metastatic skin melanoma, they still have many limitations. The treatment of advanced skin melanoma is still very difficult to a large extent, and the 5-year survival rate of patients is low. Therefore, further in-depth research on the pathogenesis of skin melanoma and exploration of new therapeutic targets are particularly important for improving the efficacy of advanced skin melanoma and prolonging the life of patients.

[0003] The ubiquitin-proteasome pathway (UPP) is an important protein degradation regulatory system in cells. It can affect or regulate a variety of cellular activities, including gene transcription, cell cycle regulation, immune response, cell receptor function, tumor growth, inflammatory process, etc., by polyubiquitinating substrate proteins and degrading them through proteasomes. The UPP pathway is also a dynamic protein bidirectional modification regulatory system. In vivo, the substrate is ubiquitinated by the ubiquitin ligase system (E1-E2-E3). The deubiquitinating enzymes (DUB) family is responsible for hydrolyzing the ester bond, peptide bond or isopeptide bond at the carboxyl end of ubiquitin, specifically hydrolyzing the ubiquitin molecule from the protein or precursor protein linked to ubiquitin, playing a role in deubiquitination, reversely regulating protein degradation, and thus affecting the function of the protein. DUB is a large family of proteases. The human genome encodes about 100 deubiquitinases, which are mainly divided into five families, namely, the ubiquitin carboxy terminal hydrolases (UCH) family, the ubiquitin specific protease (UBP) family, the Otubaim (OTU) family, the Josephin domain protein family and the JAMM family. The UBP family is the most diverse and structurally diverse family of deubiquitinases known to date. UBPs can not only reverse the ubiquitination of proteins, but also have many different functions, such as protein trafficking, apoptosis, chromatin remodeling, DNA damage repair and cell cycle regulation, and are particularly involved in cancer-related cell signaling. Since components of the ubiquitin machinery are involved in different types of cancer, targeting UBP family proteins can be used for anti-tumor therapy.

[0004] Ubiquitin Specific Peptidase 41 (UBP41) is an important member of the UBP family, but its function in tissues and cells has been relatively less studied. The latest studies show that UBP41 is also abnormally highly expressed in human tumors. Its high expression promotes the proliferation and migration of cancer cells and induces apoptosis, which is associated with the poor prognosis of some malignant tumors, suggesting that targeting UBP41 may become a tumor treatment strategy, but UBP41 has not been reported in skin melanoma. Summary of the invention

[0005] In order to obtain RNAi drugs that can effectively treat skin melanoma, the inventors tried to design and screen a series of small interfering nucleic acid molecules (siRNA) with the UBP41 gene as the target. Experiments have found that some of the siRNA molecules can specifically inhibit the expression of UBP41, and two groups of small interfering nucleic acid molecules can not only effectively inhibit the UBP41 gene, but also achieve the effect of treating skin melanoma. Based on this research finding, the technical solution of the present invention is as follows.

[0006] The first aspect of the present invention provides a small interfering nucleic acid molecule for treating skin melanoma, which consists of a sense strand and an antisense strand of the following sequence:

[0007] Sense strand: 5'-GAUUGUGGUUACUGUUCUANn-3',

[0008] Antisense strand: 5'-UAGAACAGUAACCACAAUCNn-3'; or

[0009] Sense strand: 5'-CGAGUGACACUGAGACCUANn-3',

[0010] Antisense strand: 5′-UAGGUCUCAGUGUCACUCGNn-3′.

[0011] Wherein, N is deoxycytosine dC, deoxyguanine dG, deoxyadenine dA or deoxythymine dT; n represents the number of N, and n is 0 or 2.

[0012] In one embodiment, n is 0, that is, the small interfering nucleic acid molecule consists of a sense strand and an antisense strand of the following sequence:

[0013] Sense strand: 5'-GAUUGUGGUUACUGUUCUA-3' (SEQ ID NO: 1),

[0014] Antisense strand: 5'-UAGAACAGUAACCACAAUC-3' (SEQ ID NO: 2); or

[0015] Sense strand: 5'-CGAGUGACACUGAGACCUA-3' (SEQ ID NO: 3),

[0016] Antisense strand: 5'-UAGGUCUCAGUGUCACUCG-3' (SEQ ID NO: 4).

[0017] In another embodiment, N is dT, and n is 2, that is, the small interfering nucleic acid molecule consists of a sense strand and an antisense strand of the following sequence:

[0018] Sense strand: 5'-GAUUGUGGUUACUGUUCUANN-3' (SEQ ID NO: 5),

[0019] It can also be expressed as 5'-GAUUGUGGUUACUGUUCUAdTdT-3' (SEQ ID NO: 5),

[0020] Antisense strand: 5'-UAGAACAGUAACCACAAUCNN-3' (SEQ ID NO: 6),

[0021] It can also be expressed as 5'-UAGAACAGUAACCACAAUCdTdT-3' (SEQ ID NO: 6).

[0022] Sense strand: 5'-CGAGUGACACUGAGACCUANN-3' (SEQ ID NO: 7),

[0023] It can also be expressed as 5'-CGAGUGACACUGAGACCUAdTdT-3' (SEQ ID NO: 7),

[0024] Antisense strand: 5'-UAGGUCUCAGUGUCACUCGNN-3' (SEQ ID NO: 8),

[0025] It can also be expressed as 5'-UAGGUCUCAGUGUCACUCGdTdT-3' (SEQ ID NO: 8).

[0026] The second aspect of the present invention provides use of the above-mentioned small interfering nucleic acid molecule in the preparation of a drug for inhibiting the expression of UBP41.

[0027] Preferably, the above-mentioned drug is used to inhibit the proliferation, metastasis, invasion and promote the apoptosis of skin melanoma cells.

[0028] The third aspect of the present invention provides a drug for treating skin melanoma, which comprises the above-mentioned small interfering nucleic acid molecule as a drug active ingredient.

[0029] Optionally, the above-mentioned medicine is a pharmaceutical composition, which, in addition to comprising a therapeutically effective amount of the above-mentioned small interfering nucleic acid molecule as an active ingredient, also comprises a pharmaceutically acceptable carrier.

[0030] In one embodiment, the above-mentioned drug is a pharmaceutical composition, which, in addition to a therapeutically effective amount of the above-mentioned small interfering nucleic acid molecule, further comprises one or more other pharmaceutical ingredients for inhibiting the expression of UBP41.

[0031] In another embodiment, the above-mentioned drug is a pharmaceutical composition, which, in addition to a therapeutically effective amount of the above-mentioned small interfering nucleic acid molecule, further comprises one or more other pharmaceutical ingredients for treating cutaneous melanoma.

[0032] The above-mentioned medicine can be in the form of an injection, suitable for subcutaneous injection, intramuscular injection, intravenous injection or intravenous drip.

[0033] In vitro experiments have shown that the above-mentioned small interfering nucleic acid molecules SEQ ID NO: 1-2 and SEQ ID NO: 3-4 screened by the present invention can specifically downregulate UBP41 gene expression, and have the technical effect of inhibiting the proliferation, metastasis, invasion or promoting apoptosis of skin melanoma cells, indicating that these small interfering nucleic acid molecules can be used as siRNA molecules for the treatment of skin melanoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The expression of UBP41 involved in the present invention in skin melanoma tissue and adjacent tissue, where *P<0.05.

[0035] Figure 2 The expression of UBP41 in the skin melanoma cell lines A375 and A875 involved in the present invention is compared with that of normal human skin cells HaCaT, wherein *P<0.05, represents the expression of UBP41 in A375 cells compared with HaCaT cells; #P<0.05, represents the expression of UBP41 in A875 cells compared with HaCaT cells.

[0036] Figure 3 The present invention relates to the inhibition of UBP41 mRNA expression in A375 cells by some of the siRNA molecules targeting UBP41, wherein *P<0.05, compared with the normal group.

[0037] Figure 4 The present invention relates to the inhibition of UBP41 mRNA expression in A875 cells by some of the siRNA molecules targeting UBP41, wherein *P<0.05, compared with the normal group.

[0038] Figure 5 The results are as follows: The results are as follows: The results are as follows: The results are as follows: The results are as follows: The results are as follows: The results are as follows: The results are as follows: The results are as follows: The results are as follows: The results are as follows: The results are as follows: The results are as follows: The results are as follows: The results are as follows: The results are as follows: The results are as follows: The results are as follows: The results are as follows: The results are as follows: The results are as follows: The results are as follows:

[0039] Figure 6The figures are the inhibition of UBP41 mRNA expression in A875 cells by the siRNA molecules siUBP41-1 and siUBP41-2 screened out by the present invention, where *P<0.05, compared with the normal group.

[0040] Figure 7 The results are as follows: The results are as follows: The results are as follows: The results are as follows: The results are as follows: The results are as follows: The results are as follows: The results are as follows: The results are as follows:

[0041] Figure 8 This is the curve of the inhibitory effect of siUBP41-1 and siUBP41-2 screened out in the present invention on A375 cell proliferation over time, where *P<0.05, compared with the normal group.

[0042] Fig. 9 This is the curve of the inhibitory effect of siUBP41-1 and siUBP41-2 screened out in the present invention on A875 cell proliferation over time, where *P<0.05, compared with the normal group.

[0043] Fig.10 It is a statistical bar chart of the inhibitory effect of siUBP41-1 and siUBP41-2 screened out in the present invention on the migration of A375 and A875 cells, wherein *P<0.05, compared with the normal group.

[0044] Fig.11 It is a statistical bar chart of the inhibitory effect of siUBP41-1 and siUBP41-2 screened out in the present invention on the invasion of A375 and A875 cells, where *P<0.05, compared with the normal group.

[0045] Fig.12 It is a statistical bar chart of the promoting effect of siUBP41-1 and siUBP41-2 screened out in the present invention on apoptosis of A375 and A875 cells, where *P<0.05, compared with the normal group. DETAILED DESCRIPTION

[0046] The present invention is further described in detail below in conjunction with specific examples. It should be understood that the following examples are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0047] The present inventors have discovered a small interfering nucleic acid molecule, which is a double-stranded siRNA molecule consisting of a sense chain and an antisense chain (SEQ ID NO: 1-2 and SEQ ID NO: 3-4), respectively named siUBP41-1 and siUBP41-2 in the examples, which can specifically target the UBP41 gene, block the mRNA transcription of the UBP41 gene, and prevent the translation of the gene, thereby fundamentally inhibiting the expression of UBP41, and can achieve the purpose of inhibiting skin melanoma, and can be used to improve the therapeutic effect of skin melanoma.

[0048] In this article, the terms "small interfering nucleic acid", "small interfering nucleic acid", "siRNA", "siRNA sequence", "siRNA molecule", "double-stranded siRNA", or "RNAi molecule" are interchangeable, and they have the same meaning and scope. Among them, siRNA is a double-stranded structure formed by annealing of the sense strand and the antisense strand.

[0049] In this article, for the sake of simplicity, the names of UBP41 protein and its encoding gene (DNA) are sometimes used interchangeably. Those skilled in the art should understand that they represent different substances in different description occasions. For example, UBP41 gene refers to the gene DNA encoding UBP41 protein.

[0050] Similarly, for the sake of simplicity of description, RNA, such as siRNA, is sometimes used interchangeably with the names of its encoding genes, and those skilled in the art should understand that they represent different substances in different description occasions. Those skilled in the art can easily understand their meanings based on the context and background.

[0051] For example, sometimes for the purpose of convenience of description or making sequence lists, the sense and antisense strands of the RNA molecules SEQ ID NOs: 1-8 may be expressed as deoxyribonucleic acid DNA:

[0052] SEQ ID NO:1: GATTGTGGTTACTGTTCTA,

[0053] SEQ ID NO:2: TAGAACAGTAACCACAATC,

[0054] SEQ ID NO:3:CGAGTGACACTGAGACCTA,

[0055] SEQ ID NO:4: TAGGTCTCAGTGTCACTCG.

[0056] SEQ ID NO:5: GATTGTGGTTACTGTTCTANN (wherein N=dT),

[0057] It can also be expressed as GATTGTGGTTACTGTTCTAdTdT,

[0058] SEQ ID NO:6:TAGAACAGTAACCACAATCNN (wherein N=dT),

[0059] It can also be expressed as TAGAACAGTAACCACAATCdTdT,

[0060] SEQ ID NO:7: CGAGTGACACTGAGACCTANN (wherein N=dT),

[0061] It can also be expressed as CGAGTGACACTGAGACCTAdTdT,

[0062] SEQ ID NO:8:TAGGTCTCAGTGTCACTCGNN (wherein N=dT),

[0063] It can also be expressed as TAGGTCTCAGTGTCACTCGdTdT.

[0064] The above-mentioned siRNA molecules can be prepared by a variety of methods, such as chemical synthesis, in vitro transcription, enzyme cleavage of long-chain dsRNA, vector expression of RNA, PCR synthesis of RNA expression elements, etc. The emergence of these methods provides researchers with room for choice and can better obtain gene silencing efficiency.

[0065] The above siRNA molecules can be used as active pharmaceutical ingredients in the treatment of skin melanoma, and can be made into RNAi drugs.

[0066] Optionally, in addition to a therapeutically effective amount of the RNAi molecule siUBP41-1 or siUBP41-2, the RNAi drug may also contain other UBP41 expression inhibitors as auxiliary active ingredients to enhance the therapeutic effect of the siRNA molecule on skin melanoma.

[0067] The term "(therapeutic effect) enhancement" or "improvement" is used herein to indicate a statistically significant amount of improvement. In some embodiments, "enhancement" or "increase" or "improvement" generally refers to an improvement of at least 10% compared to a reference level (e.g., there is no given treatment or medicament), and can include, for example, an improvement of at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or more.

[0068] In another embodiment, the above-mentioned medicine contains, in addition to the above-mentioned RNAi molecule in a therapeutically effective amount, one or more UBP41 gene inhibitors and / or drug components for treating skin melanoma. Such a pharmaceutical composition may have the effect of treating skin melanoma.

[0069] It should be understood that the term "or" as used herein sometimes means "and / or", and the term "or" sometimes also means "and / or". The term "and / or" as used in phrases such as "A and / or B" herein is intended to include both A and B; A or B; A (alone); and B (alone). Similarly, the term "and / or" as used in phrases such as "A, B and / or C" is intended to cover each of the following embodiments: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0070] As used herein, the term "effective amount" refers to the therapeutic amount required to alleviate at least one or more symptoms of a disease or condition, and relates to a sufficient amount of a drug that provides the desired effect. Therefore, the term "therapeutically effective amount" refers to a therapeutic amount sufficient to cause a specific effect when applied to a typical subject. In various contexts, the effective amount as used herein also includes an amount sufficient to delay the development of a disease condition, change the course of a disease condition (for example, but not limited to, slowing the progression of a disease condition), or reverse a disease condition. It should be understood that there are many ways known in the art to determine the effective amount for a given application. For example, pharmacological methods for dose determination can be used in a therapeutic context. In the context of therapeutic or preventive applications, the amount of the composition applied to the subject will depend on the type and severity of the disease and the characteristics of the individual, such as overall health, age, sex, weight, and tolerance to the drug. It also depends on the extent, severity, and type of the disease. Those skilled in the art will be able to determine the appropriate dose based on these and other factors. For example, the therapeutically effective amount of an RNAi molecule can be determined by clinical investigation. The appropriate effective dosage also takes into account therapeutic factors such as the dosage form, the constitution of the individual being administered, weight, age, course of the condition, and site of administration.

[0071] The dosage range of the agent depends on the efficacy, including an amount sufficient to produce the desired effect, such as slowing the development of skin melanoma or even causing reversal of the condition. The dosage should not be so large as to cause unacceptable adverse side effects. Generally, the dosage will vary with the age, condition and sex of the patient and can be determined by a person skilled in the art. The dosage can also be adjusted by the individual physician in the event of any complications.

[0072] The dosage form of the drug of the present invention can be in various forms, as long as it is suitable for administration to the corresponding disease and properly maintains the activity of the RNA molecule. For example, for an injection administration system, the dosage form can be a lyophilized powder.

[0073] Optionally, the above-mentioned pharmaceutical dosage form may contain any pharmaceutically acceptable carrier and adjuvant, as long as they are suitable for the corresponding administration system and properly maintain the activity of the RNAi molecule.

[0074] The phrase "pharmaceutically acceptable" as used herein refers to compounds, materials, compositions and / or dosage forms that are suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions or other problems or complications, and that are commensurate with a reasonable benefit / risk ratio, within the scope of reasonable medical judgment. Pharmaceutically acceptable carriers are well known in the art, and these carriers include liquid or solid fillers, diluents, excipients, solvents or encapsulating materials. Each carrier must be "acceptable" in the sense that it is compatible with the other ingredients of the formulation and harmless to the patient, including, for example, aqueous solutions (such as water or physiologically buffered saline) or other solvents or vehicles (such as glycols, glycerol, oils (such as olive oil) or injectable organic esters). Excipients can be selected, for example, to achieve delayed release of the medicament or to selectively target one or more cells, tissues or organs. For the purposes of the present invention, the pharmaceutical composition can be in dosage unit form, such as powder injections, solutions, injections, etc.

[0075] The preparation of pharmacological compositions comprising active ingredients dissolved or dispersed therein is well known in the art and does not need to be limited based on the formulation. Typically, such compositions are prepared as injectable liquid solutions or suspensions, however, solid forms of solutions or suspensions suitable for being placed in liquids before use can also be prepared. The preparation can also be emulsified or present in the form of liposome compositions. The active ingredient can be mixed with a pharmaceutically acceptable and compatible excipient for the active ingredient, and in an amount suitable for the treatment methods described herein. Suitable excipients include, for example, water, saline, glucose, glycerol, ethanol, etc. and combinations thereof. In addition, if necessary, the composition may contain a small amount of auxiliary substances, such as wetting agents or emulsifiers, pH buffers, etc., which enhance or maintain the effectiveness of the active ingredient. The therapeutic composition as described herein may include pharmaceutically acceptable salts of the components thereof. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of polypeptides), which are formed by inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, tartaric acid, mandelic acid, etc. Salts formed by free carboxyl groups can also be derived from inorganic bases, for example, sodium, potassium, ammonium, calcium or iron hydroxides, and organic bases such as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, etc. Physiologically tolerable carriers are well known in the art. Exemplary liquid carriers are sterile aqueous solutions that do not contain any substance except active ingredients and water, or contain buffers such as sodium phosphate, saline or both at physiological pH, such as phosphate buffered saline. Further, aqueous carriers can contain more than one buffer salt, as well as salts such as sodium chloride and potassium chloride, glucose, polyethylene glycol and other solutes. In addition to water, liquid compositions can also include a liquid phase. Examples of such other liquid phases are glycerol, vegetable oils (e.g., cottonseed oil) and water-oil emulsions. The amount of the active agent used in the present invention to effectively treat a specific disease or condition such as DCM will depend on the nature of the disease or condition, and can be determined by standard clinical techniques.

[0076] As used herein, the terms "treat," "therapy," "treatment," or "improvement," when used in reference to a disease, disorder, or medical disorder such as cutaneous melanoma, refer to the therapeutic treatment of a disorder, wherein the objective is to reverse, alleviate, ameliorate, inhibit, slow, or stop the progression or severity of a symptom or disorder. The term "treat" includes reducing or alleviating at least one adverse reaction or symptom of a disorder. A treatment is generally "effective" if one or more symptoms or clinical markers are reduced. Alternatively, a treatment is "effective" if the progression of a disorder is reduced or stopped. That is, "treatment" includes not only the improvement of symptoms or markers, but also the stopping or at least slowing of the progression or worsening of symptoms that would be expected in the absence of treatment. Beneficial or desired clinical outcomes include, but are not limited to, relief of one or more symptoms, reduction in the extent of a defect, stabilization (i.e., no worsening) or a decreased state of UBP41 gene expression levels, a delay or slowing of cardiomyocyte apoptosis or pyroptosis, and an increased lifespan compared to the lifespan expected in the absence of treatment.

[0077] A therapeutically effective amount is an amount of the agent sufficient to produce a statistically significant, measurable change in inhibiting UBP41 gene expression, promoting cardiomyocyte growth, etc. Such an effective amount can be measured in clinical trials and animal studies.

[0078] The efficacy of a given treatment for (e.g., inhibiting UBP41 gene expression and / or treating skin melanoma) can be determined by a skilled clinician. However, if, for example, any or all signs or symptoms of inhibiting UBP41 gene expression and / or treating skin melanoma are altered in a beneficial manner, or other clinically acceptable symptoms are improved or even alleviated, for example, by at least 10% after treatment with an agent described herein, the treatment is considered to be "effective treatment," as the term is used herein. Efficacy can also be measured by the failure of an individual to worsen, as assessed by the need for hospitalization or medical intervention (i.e., cessation of progression of the disease). Methods for measuring these indicators are known to those skilled in the art and / or described herein.

[0079] An effective amount for treating a disease is an amount sufficient to result in effective treatment of the disease as that term is defined herein when administered to a mammal in need thereof. The efficacy of an agent can be determined by assessing physical indicators such as inhibition of UBP41 gene expression and / or treatment of skin melanoma.

[0080] The above-mentioned agents can be administered by subcutaneous injection, intramuscular injection, intravenous administration by injection, or intravenous administration by gradual infusion over time. Given an appropriate formulation for a given route, for example, the agents that can be used in the methods and compositions described herein can be administered intravenously, intranasally, by inhalation, intraperitoneally, intramuscularly, subcutaneously, intracavitary, and if necessary, can be delivered by means of a peristaltic pump, a venous port, or by other means known to those skilled in the art. Preferably, the patient with skin melanoma to be treated is administered intravenously or intramuscularly. Local administration directly to sites of high expression of the UBP41 gene, such as skin tissue, is also particularly contemplated.

[0081] For example, therapeutic compositions comprising at least one pharmaceutical agent may be conventionally administered in unit doses. When used in relation to therapeutic compositions, the term "unit dose" refers to physically discrete units suitable as unit dosage forms for administration to a subject, each unit containing a predetermined quantity of active material, in association with the required physiologically acceptable diluent (i.e., carrier or vehicle), calculated to produce the desired therapeutic effect.

[0082] The compositions are administered in a therapeutically effective amount in a manner compatible with the dosage formulation. The amount and timing of administration will depend upon the subject to be treated, the subject's body's ability to utilize the active ingredients, and the extent of the therapeutic effect desired.

[0083] The exact amount of active ingredient, such as RNAi, that needs to be administered depends on the physician's judgment and varies from individual to individual. However, suitable dosage ranges for systemic administration are disclosed herein and depend on the route of administration. Suitable dosing regimens are also variable, but are represented by an initial administration, followed by repeated administration at intervals of one or more hours by subsequent injections or other administrations. Alternatively, it is contemplated that continuous intravenous infusion is sufficient to maintain the concentration in the blood within the range specified for in vivo treatment.

[0084] The present invention has verified at the cellular level the inhibitory effect of individual siRNA molecules such as siUBP41-1 and siUBP41-2 on the UBP41 gene and the inhibitory effect on the proliferation, metastasis and invasion of skin melanoma cells, or the promoting effect on the apoptosis of skin melanoma cells.

[0085] To make the present invention more clearly understandable, preferred embodiments are described in detail below with reference to the accompanying drawings. Those skilled in the art should understand that the following embodiments are only used to illustrate the present invention and are not intended to limit the present invention.

[0086] Example

[0087] The siRNA and PCR primers in this article were synthesized by Biomimetic Biotechnology Co., Ltd.

[0088] In the embodiments of the present invention, if there is no specific description on the experimental operation temperature, the temperature generally refers to room temperature (10-30° C.).

[0089] This article involves the addition amount, content and concentration of various substances, and the percentages mentioned here refer to the mass percentages unless otherwise specified.

[0090] Statistical analysis: In this study, all numerical variables were expressed as mean ± standard error. The two-group comparison was performed using the two-tailed Student's t test, and the three-group comparison was performed using the ANOVA test. When P < 0.05, it was considered statistically significant.

[0091] Example 1

[0092] 1. siRNA design:

[0093] According to the coding region of the UBP41 gene sequence in the NCBI database (Gene Bank No. AF079564), a specific siRNA sequence was designed, and sequence homology analysis showed that it was 100% homologous to the UBP41 gene.

[0094] 2. siRNA sequence:

[0095] The siRNA sequences obtained according to the design are shown in Table 1.

[0096] 3. siRNA structure:

[0097] All siRNA molecules are double-stranded, consisting of a sense strand and an antisense strand with reverse complementary sequences, and may also contain 1-2 base overhanging structures dC, dG, dA, dT, dCdC, dGdG, dAdA or dTdT at the 3' end of the sense strand and the 5' end of the antisense strand.

[0098] Table 1: List of designed siRNA sequences

[0099] Molecular number Sense strand (5'-3') Antisense strand (5'-3') siUBP41-1 GAUUGUGGUUACUGUUCUA UAGAACAGUAACCACAAUC siUBP41-2 CGAGUGACACUGAGACCUA UAGGUCUCAGUGUCACUCG siUBP41-3 GAGUGACACUGAGACCUAA UUAGGUCUCAGUGUCACUC siUBP41-4 GGAUGUGCUUGAUGGAGAU AUCUCCAUCAAGCACAUCC siUBP41-5 GCAUGAGGCUCUUCACCAA UUGGUGAAGAGCCUCAUGC siUBP41-6 CACUGAGACCUAAGUCCAA UUGGACUUAGGUCUCAGUG siUBP41-7 GGCCGACAGAUGUGGAGA UUCUCCACAUCUGUCGGCC siUBP41-8 GGAACACGUGCUUCAUGA UUCAUGAAGCACGUGUUCC siUBP41-9 GCCGACAGAUGUGGAGAAA UUUCUCCACAUCUGUCGGC siUBP41-10 UGUGCUUGAUGGAGAUGAA UUCAUCUCCAUCAAGCACA siUBP41-11 CAAAGAUCUUGGUGCUCCA UGGAGCACCAAGAUCUUUG siUBP41-12 AGACCUGGACUUAAGAGAA UUCUCUUAAGUCCAGGUCU siUBP41-13 CCAGGAUUCGAACCAGCAA UUGCUGGUUCGAAUCCUGG siUBP41-14 CGUGCUUCAUGAACUCAAU AUUGAGUUCAUGAAGCACG siUBP41-15 CGUGUACAGAUUGUGGUUA UAACCACAAUCUGUACACG siUBP41-16 CGAGGUGAACCGAGUGACA UGUCACUCGGUUCACCUCG siUBP41-17 GGCUGGUCUUCGAAACCUU AAGGUUUCGAAGACCAGCC siUBP41-18 AGACCCAGAUCCAGAGAUA UAUCUCUGGAUCUGGGUCU

[0100] The siRNA molecules A, U, C, and G in Table 1 above may contain various chemical modifications that can improve the stability and activity of the siRNA.

[0101] Example 2

[0102] 1. Cell culture:

[0103] Human skin melanoma cells A375 and A875 and normal skin cells HaCaT were cultured in DMEM medium (ThermoFisher Scientific) containing 10% fetal bovine serum (Lonsera) and 1% penicillin-streptomycin (ThermoFisher Scientific) at 37°C with 5% CO 2 cultured in an incubator.

[0104] 2. siRNA synthesis:

[0105] The siRNAs in Table 1 (including siRNA molecules containing 1-2 base overhanging structures) were synthesized, and the sense strand and the corresponding antisense strand were annealed into siRNA double strands, respectively, and the concentration was configured to 20 μM before transfection.

[0106] In addition, siRNA that is not homologous to the human UBP41 gene was designed and synthesized as a negative control siNC, which has a sense strand (UUCUCCGAACGUGUCACGUdTdT) and an antisense strand (ACGUGACACGUUCGGAGAAdTdT). The sense strand and the corresponding antisense strand were annealed into siNC double strands, and the concentration was configured to 20 μM before transfection.

[0107] 3. siRNA transfection:

[0108] The cells obtained in step 1 were plated on a cell culture plate and incubated in DMEM medium containing 10% fetal bovine serum at 37°C and 5% CO 2 Culture in an incubator overnight. 3000 (Thermo Fisher Scientific), and siRNA and siNC were transfected into cells according to its instructions.

[0109] Example 3

[0110] Real-time quantitative PCR was used to screen siRNA with high inhibition efficiency.

[0111] 1. Cell culture and transfection:

[0112] Skin melanoma cells A375 and A875 were cultured according to the method in Example 2, and A375 and A875 cells in the logarithmic growth phase were inoculated into 96-well plates. After the cells grew for 24 hours and reached a confluence of about 75%, the siRNA molecules in Table 1 (including siRNA molecules containing 1-2 base overhanging structures) were transfected into the cells respectively, and the cells were placed at 37°C with 5% CO 2 cultured in an incubator.

[0113] 2. RNA extraction and RT-qPCR reaction:

[0114] 48 hours after the cells were transfected with siRNA, the cells were collected and total cellular RNA was extracted using RNA extraction reagent Trizol (Thermo Fisher Scientific) according to its instructions.

[0115] Gene-specific primers were used to detect the mRNA expression level of the UBP41 gene in the samples, and the housekeeping gene GAPDH was used as an internal reference. Quantitative PCR reaction was performed using SYBR Green RT-qPCR kit (ThermoFisher Scientific) according to the manufacturer's instructions.

[0116] The primer sequences used are as follows:

[0117] UBP41:

[0118] Upstream: 5′-GTTCCTTCGCTTTCTTCT-3′;

[0119] Downstream: 5′-TCCTACTGTCTTCCCGTT-3′.

[0120] GAPDH:

[0121] Upstream: 5′-GGAGCGAGATCCCTCCAAAAT-3′;

[0122] Downstream: 5′-GGCTGTTGTCATACTTCTCATGG-3′.

[0123] After the reaction is completed, export the data using 2 -ΔΔCt The experimental results were analyzed by the method, and the siRNA inhibition rate was calculated as follows: (1-2 -ΔΔCt value)×100%.

[0124] like Figure 1 As shown, UBP41 was significantly overexpressed in skin melanoma tissues compared with adjacent adjacent tissues.

[0125] like Figure 2 As shown, UBP41 was significantly overexpressed in melanoma cell lines A375 and A875 compared with normal human skin cells HaCaT.

[0126] like Figure 3 As shown, compared with the normal group and negative control siNC, the designed siRNA targeting UBP41 can inhibit the mRNA expression of UBP41 in A375 cells to a certain extent, especially siUBP41-1 and siUBP41-2 have the most significant inhibitory effects.

[0127] like Figure 4 As shown, compared with the normal group and negative control siNC, the designed siRNA targeting UBP41 can inhibit the mRNA expression of UBP41 in A875 cells to a certain extent, especially siUBP41-1 and siUBP41-2 have the most significant inhibitory effects.

[0128] like Figure 5 As shown, compared with the normal group and the negative control siNC, siUBP41-1 and siUBP41-2 can significantly inhibit the mRNA expression of UBP41 in A375 cells. Among them, the normal group refers to the group that has not been treated with siRNA molecules, and siNC is the negative control, the same below.

[0129] like Figure 6 As shown, compared with the normal group and the negative control siNC, siUBP41-1 and siUBP41-2 could significantly inhibit the mRNA expression of UBP41 in A875 cells.

[0130] The results showed that siUBP41-1 and siUBP41-2 had a highly effective inhibitory effect on UBP41 in skin melanoma cells, suggesting that siUBP41-1 and siUBP41-2, which effectively inhibit the expression of UBP41, can be used as potential drugs for the treatment of skin melanoma.

[0131] Example 4

[0132] Western blot was used to detect the effect of siRNA molecules on the expression level of UBP41 protein in skin melanoma cells.

[0133] Skin melanoma cells A375 and A875 were cultured according to the method in Example 2, and A375 and A875 cells in the logarithmic growth phase were inoculated into 6-well plates. After the cells grew for 24 hours and reached a confluence of about 75%, the siRNA molecule siUBP41 screened in Example 3 was transfected into the cells and placed at 37°C with 5% CO 2 cultured in an incubator.

[0134] 48 hours after cell transfection, cells were lysed with RIPA protein lysis buffer (Beyotime), and protein quantification was performed using the BCA method. About 30 μg of protein was separated by SDS-polyacrylamide gel electrophoresis, and then the protein was transferred to a PVDF membrane (Merck Millipore). It was blocked with 5% skim milk at room temperature for 2 hours, and then the primary antibodies: mouse anti-human UBP41 antibody (Proteintech, 1:1,000 dilution); GAPDH antibody (Proteintech, 1:20,000 dilution) were used as internal references. After washing with TBST, the secondary antibody coupled with horseradish peroxidase was incubated, and washed with TBST for 5 min × 3 times. BeyoECL Plus (Beyotime) was used for protein blotting detection, and the gray value of the protein band was scanned with Image J software, and the statistical data was used to make a bar chart analysis result.

[0135] like Figure 7 As shown, compared with A375 and A875 cells treated with negative control siNC, siUBP41-1 and siUBP41-2 significantly inhibited the expression of UBP41 protein in cells.

[0136] Example 5

[0137] The CCK-8 method was used to detect the effect of siRNA inhibiting UBP41 expression on the proliferation of skin melanoma cells.

[0138] Skin melanoma cells A375 and A875 were cultured according to the method in Example 2, and A375 and A875 cells in the logarithmic growth phase were inoculated into 96-well plates. After the cells grew for 24 hours and reached a confluence of about 75%, the siRNA molecule siUBP41 screened in Example 3 was transfected into the cells and placed at 37°C with 5% CO 2 cultured in an incubator.

[0139] Cells from each experimental group were taken for cell proliferation assay 24h, 48h, 72h and 96h after transfection. The assay method was as follows: 10 μL CCK-8 (Beyotime) was added to each well, incubated in an incubator for 4h, OD value was measured at a wavelength of 450nm using an enzyme marker, and growth curve was prepared.

[0140] like Figure 8 and Fig. 9 As shown in the figure, compared with A375 and A875 cells treated with negative control siNC, siUBP41-1 and siUBP41-2 significantly inhibited cell proliferation. The cell proliferation rates of A375 and A875 decreased more and more over time.

[0141] Example 6

[0142] The cell scratch assay was used to detect the effect of siRNA inhibition of UBP41 expression on the migration of skin melanoma cells.

[0143] Skin melanoma cells A375 and A875 were cultured according to the method in Example 2, and A375 and A875 cells in the logarithmic growth phase were inoculated into a 3.5 cm culture dish at a cell density of 5×10 5 After the cells grew for 24 h, when the cell fusion rate reached 90%, a 200 μL pipette tip was used to scratch the cell monolayer until the cells were wounded. After washing with PBS, the siRNA molecules siUBP41-1 and siUBP41-2 selected in Example 3 were transfected into the cells and placed at 37°C with 5% CO 2 At 0 h or 48 h, the cells were observed under a microscope and photographed.

[0144] like Fig.10 As shown, siUBP41-1 and siUBP41-2 significantly inhibited the migration of A375 and A875 cells compared with A375 and A875 cells treated with negative control siNC.

[0145] Example 7

[0146] The Transwell assay was used to detect the effect of siRNA inhibition of UBP41 expression on the invasion of skin melanoma cells.

[0147] Skin melanoma cells A375 and A875 were cultured according to the method in Example 2, and A375 and A875 cells in the logarithmic growth phase were collected and centrifuged at 1,000 rpm for 5 min. 5 The cells were resuspended in serum-free DMEM at a concentration of 1 mg / mL. Matrigel (BD) was preheated at 37°C and adjusted to a concentration of 1 mg / mL with serum-free DMEM. After adding 300 μL of precooled DMEM containing 10% FBS to a 24-well plate, a precooled Transwell was placed in the 24-well plate and 100 μL of Matrigel (1 mg / mL) was added to the upper chamber of each Transwell. After incubation at 37°C for 4-5 hours, the above 200 μL of resuspended cells was added to the upper chamber of each Transwell and incubated at 37°C with 5% CO 2 The cells were cultured in an incubator at 37°C with 5% CO. 2Cultured in an incubator. 48 hours after transfection, the Transwell was washed with PBS and fixed in 70% pre-cooled ethanol for 1 hour, and then stained with 0.5% crystal violet for 20 minutes at room temperature. After washing with PBS, the non-invasive cells on one side of the upper chamber were wiped with a clean cotton swab, and the invasive cells were observed and photographed under a microscope.

[0148] like Fig.11 As shown, compared with A375 and A875 cells treated with negative control siNC, siUBP41-1 and siUBP41-2 significantly inhibited the invasion of A375 and A875 cells.

[0149] Example 8

[0150] Annexin V-FITC / PI and flow cytometry were used to detect the effect of siRNA inhibition of UBP41 expression on apoptosis of skin melanoma cells.

[0151] Skin melanoma cells A375 and A875 were cultured according to the method in Example 2, and A375 and A875 cells in the logarithmic growth phase were collected and inoculated into 6-well plates at a cell density of 5×10 5 cells / well and then incubate at 37°C in 5% CO 2 The cells were cultured in an incubator for 24 h. The siRNA molecules siUBP41-1 and siUBP41-2 screened in Example 3 were transfected into the cells and placed in a 37°C incubator containing 5% CO 2 Incubate the cells in a 5% PBS incubator. 48 h after transfection, wash the cells with PBS, resuspend the cells with 100 μL PBS, and add 5 μL PI and Annexin V-FITC to each well. After incubation at 37°C in the dark for 48 h, wash the cells with PBS, resuspend the cells with 100 μL PBS, and finally analyze them using a flow cytometer (BD Biosciences).

[0152] like Fig.12 As shown, compared with A375 and A875 cells treated with negative control siNC, siUBP41-1 and siUBP41-2 significantly promoted the apoptosis of A375 and A875 cells.

[0153] From the above experiments, it can be seen that the siRNA molecules siUBP41-1 and siUBP41-2 targeting UBP41 show high efficiency in inhibiting the expression of UBP41; they can inhibit the proliferation, migration and invasion of skin melanoma cells and promote the growth of skin melanoma cells, and thus have the potential to be used in the treatment of skin melanoma.

Claims

1. A small interfering nucleic acid molecule for treating skin melanoma, characterized in that: The sense strand and antisense strand of the following sequence composition: Sense strand: 5'-CGAGUGACACUGAGACCUANn-3', Antisense strand: 5'-UAGGUCUCAGUGUCACUCGNn-3', Wherein, N is deoxycytosine dC, deoxyguanine dG, deoxyadenine dA or deoxythymine dT; n represents the number of N, and n is 0 or 2.

2. The small interfering nucleic acid molecule according to claim 1, characterized in that The n is 0, that is, Sense strand: 5'-CGAGUGACACUGAGACCUA-3' (SEQ ID NO: 3), Antisense strand: 5'-UAGGUCUCAGUGUCACUCG-3' (SEQ ID NO: 4).

3. The small interfering nucleic acid molecule according to claim 1, characterized in that The N is dT, n is 2, i.e. the positive strand: 5'-CGAGUGACACUGAGACCUANN-3' (SEQ ID NO: 7), Antisense strand: 5'-UAGGUCUCAGUGUCACUCGNN-3' (SEQ ID NO: 8).

4. Use of the small interfering nucleic acid molecule according to any one of claims 1 to 3 in the preparation of a drug for inhibiting the proliferation and metastasis of skin melanoma cells, or promoting the apoptosis of skin melanoma cells.

5. A drug for treating skin melanoma, characterized in that: The method comprises the small interfering nucleic acid molecule according to any one of claims 1 to 3 as a pharmaceutical active ingredient.

6. The drug according to claim 5, characterized in that The pharmaceutical composition comprises, in addition to a therapeutically effective amount of the small interfering nucleic acid molecule according to any one of claims 1 to 3 as an active ingredient, a pharmaceutically acceptable carrier.

7. The drug according to claim 5, characterized in that The pharmaceutical composition comprises, in addition to a therapeutically effective amount of the small interfering nucleic acid molecule according to any one of claims 1 to 3, one or more other pharmaceutical ingredients for treating skin melanoma.

8. The drug according to any one of claims 5 to 7, characterized in that The medicine is in the form of an injection, suitable for subcutaneous injection, intramuscular injection, intravenous injection or intravenous drip.

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